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US20100216212A1 - Anti-obesity agent and anti-obesity food - Google Patents

Anti-obesity agent and anti-obesity food Download PDF

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US20100216212A1
US20100216212A1 US11/997,034 US99703406A US2010216212A1 US 20100216212 A1 US20100216212 A1 US 20100216212A1 US 99703406 A US99703406 A US 99703406A US 2010216212 A1 US2010216212 A1 US 2010216212A1
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amino acid
acid sequence
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Hidetoshi Morita
Toshio Masaoka
Takehito Suzuki
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SCHOOL Corp AZABU VETERINARY MEDICINE EDUCATIONAL INSTITUTION
Nestec SA
SCHOOL CORP AZABU VETERIN MED EDUCA'L INSTITU
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SCHOOL CORP AZABU VETERIN MED EDUCA'L INSTITU
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/335—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Lactobacillus (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/173—Reuteri
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present invention relates to an anti-obesity agent and an anti-obesity food, and in more detail, the invention relates to an anti-obesity agent and an anti-obesity food each capable of inhibiting a lipid from being taken from a digestive tract to prevent obesity from occurring.
  • the obesity is a disease in a weight control system which is characterized by an excess of body fat.
  • a neutral fat is accumulated in the body, and the number of persons who are judged to be obese continues to increase, resulting in a serious problem.
  • the present inventors paid attention to a mechanism where a lipid is absorbed into the body and made studies regarding a method of inhibiting obesity. As a result, it was found that when a microorganism flora within intestine ingests and degrades a lipid, the lipid to be ingested by a person reduces as a result, whereby the obesity can be spontaneously prevented. Then, the present inventors have made extensive and intensive investigations regarding microorganisms capable of ingesting and degrading such a lipid and as a result, found out a microorganism having such an action among those belonging to lactic acid bacteria, leading to accomplishment of the invention.
  • an anti-obesity agent comprising, as an active ingredient, a microorganism belonging to the species Lactobacillus reuteri and capable of producing lipases respectively depicted in the following amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3).
  • the “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping lipase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • the invention is concerned with an anti-obesity food comprising, as an active ingredient, a microorganism belonging to the species Lactobacillus reuteri and capable of producing lipases respectively depicted in the foregoing amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3).
  • the invention is concerned with a glycerol-degrading enzyme composed of subunits respectively depicted in the following amino acid sequences (5) to (7) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7).
  • the “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-degrading activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • amino acid sequence (8) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8).
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping activity for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • the anti-obesity agent and anti-obesity food and drink of the invention are able to prevent the absorption of a lipid from the intestinal tract by ingesting them while taking a normal meal. Since lactic acid bacteria are used from old in producing fermented foods such as yogurt and fermented milk and have extremely high safety, they can be ingested without anxiety.
  • the microorganism which is an active ingredient of the anti-obesity agent and anti-obesity food and drink of the invention is one belonging to the species Lactobacillus reuteri which is a lactic acid bacterium and capable of producing lipases respectively depicted in the foregoing amino acid sequences (1) to (3) (corresponding to SEQ ID NO: 1, 3 and 5, respectively) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3) (these lipases will be hereinafter referred to as “lipases of the invention”) (this microorganism will be hereinafter referred to as “lactic acid bacterium of the invention”).
  • the lipases of the invention degrade a lipid (triacylglycerol) present in the vicinity of intestinal mucosa cells into a fatty acid and glycerol, which are then taken into the lactic acid bacterium of the invention. As shown in FIG. 1 , the thus taken fatty acid is utilized as a fungus constituent, and the glycerol is converted into carbon dioxide, acetone, ethanol, lactate or reuterin through various metabolic pathways.
  • a lipid triacylglycerol
  • the lactic acid bacterium of the invention has ability for producing the foregoing lipases, namely has the nucleotide sequences (genes) for encoding the foregoing lipases and besides, has a nucleotide sequence as a gene encoding a transporter and depicted in the following (4) (corresponding to SEQ ID NO: 8).
  • the invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence depicted in (4) and encoding a protein having glycerol transporter activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence depicted in (4) under a stringent condition and encoding a protein having glycerol transporter activity.
  • This gene is one encoding a transporter for taking glycerol into the cell of the lactic acid bacterium of the invention, and, as shown in FIG. 1 , the thus taken glycerol is subjected to metabolism by the enzymes within the lactic acid bacterium.
  • the lactic acid bacterium of the invention is one having a glycerol-degrading enzyme composed of the subunits respectively depicted in the foregoing amino acid sequences (5) to (7) (corresponding to SEQ ID NO: 9, 11 and 13, respectively) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7).
  • This glycerol-degrading enzyme composed of the subunits (5) to (7) is a glycerol dehydratase which functions in a pdu (propanediol utilization) operon and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • nucleotide sequences (9) to (11) corresponding to SED ID NO: 10, 12 and 14, respectively
  • the invention also includes nucleotide sequences having homology of 80% or more with the nucleotide sequences (9) to (11) and encoding a protein having glycerol-degrading activity; and nucleotide sequences for achieving hybridization with the nucleotide sequences (9) to (12) under a stringent condition and encoding a protein having glycerol-degrading activity.
  • the lactic acid bacterium of the invention is one holding an enteroadherent protein depicted in the following amino acid sequence (8) (corresponding to SEQ ID NO: 15) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8).
  • This enteroadherent protein has an action for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells, and the lactic acid bacterium of the invention having this is able to exist in the vicinity of an intestinal mucosa for a certain period of time and stably take a lipid thereinto. It becomes possible to obtain a lactic acid bacterium with a long intestinal residence time by incorporating a gene encoding this protein and depicted in the following (12) (corresponding to SEQ ID NO: 16) into other lactic acid bacterium by a known technique.
  • the invention also involves a nucleotide sequence having homology of 80% or more with the nucleotide sequence (12) and encoding a protein having activity for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (12) under a stringent condition and encoding a protein having activity for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells.
  • the lactic acid bacterium of the invention is one having a glycerol-degrading enzyme depicted in any of the following amino acid sequences (16) to (20) (corresponding to SEQ ID NO: 17, 19, 21, 23 and 25, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (16) to (20).
  • This glycerol-degrading enzyme is an alcohol dehydrogenase (ADH (8) in FIG. 1 ) and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-degrading activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (32) to (36) (corresponding to SED ID NO: 18, 20, 22, 24 and 26, respectively) encoding the glycerol-degrading enzymes (16) to (20), respectively into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with any of the nucleotide sequences (32) to (36) and encoding a protein having glycerol-degrading activity; and a nucleotide sequence for achieving hybridization with any of the nucleotide sequences (32) to (36) under a stringent condition and encoding a protein having glycerol-degrading activity.
  • the lactic acid bacterium of the invention is one having a glycerol-degrading enzyme depicted in the following amino acid sequence (21) or (22) (corresponding to SEQ ID NO: 27 or 29, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (21) or (22).
  • This glycerol-degrading enzyme is an alcohol dehydrogenase (ADH (8) in FIG. 1 ) and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-degrading activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequence (37) or (38) (corresponding to SED ID NO: 28 or 30, respectively) encoding the glycerol-degrading enzyme (21) or (22), respectively into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (37) or (38) and encoding a protein having glycerol-degrading activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (37) or (38) under a stringent condition and encoding a protein having glycerol-degrading activity.
  • the lactic acid bacterium of the invention is one having an aldehyde dehydrogenase depicted in any of the following amino acid sequences (23) to (25) (corresponding to SEQ ID NO: 31, 33 and 35, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (23) to (25).
  • This aldehyde dehydrogenase is an aldehyde dehydrogenase (9) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping aldehyde dehydrogenase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (39) to (41) (corresponding to SED ID NO: 32, 34 and 36, respectively) encoding the aldehyde dehydrogenases (23) to (25), respectively into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with any of the nucleotide sequences (39) to (41) and encoding a protein having aldehyde dehydrogenase activity; and a nucleotide sequence for achieving hybridization with any of the nucleotide sequences (39) to (41) under a stringent condition and encoding a protein having aldehyde dehydrogenase activity.
  • the lactic acid bacterium of the invention is one having a glycerate kinase depicted in the following amino acid sequence (26) (corresponding to SEQ ID NO: 37) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (26).
  • This glycerate kinase is Glycerate kinase (10) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerate kinase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • nucleotide sequence (42) (corresponding to SED ID NO: 38) encoding the glycerate kinase (26) into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (42) and encoding a protein having glycerate kinase activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (42) under a stringent condition and encoding a protein having glycerate kinase activity.
  • the lactic acid bacterium of the invention is one having a glycerol kinase depicted in the following amino acid sequence (27) (corresponding to SEQ ID NO: 39) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (27).
  • This glycerol kinase is GK (5) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • the “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol kinase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • nucleotide sequence (43) (corresponding to SED ID NO: 40) encoding the glycerol kinase (27) into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (43) and encoding a protein having glycerol kinase activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (43) under a stringent condition and encoding a protein having glycerol kinase activity.
  • the lactic acid bacterium of the invention is one having a glycerol-3-phosphate dehydrogenase depicted in the following amino acid sequence (28) (corresponding to SEQ ID NO: 41) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (28).
  • This glycerol-3-phosphate dehydrogenase is GPD (6) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-3-phosphate dehydrogenase activity.
  • amino acid sequence include those exhibiting homology of 80% or more.
  • a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequence (44) (corresponding to SED ID NO: 42) encoding the glycerol-3-phosphate dehydrogenase (28) into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (44) and encoding a protein having glycerol-3-phosphate dehydrogenase activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (44) under a stringent condition and encoding a protein having glycerol-3-phosphate dehydrogenase activity.
  • the lactic acid bacterium of the invention is one having triosephosphate isomerase depicted in any of the following amino acid sequences (29) to (32) (corresponding to SEQ ID NO: 43, 45 and 47, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (29) to (32).
  • This triosephosphate isomerase is Triosephosphate isomerase (15) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping triosephosphate isomerase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (45) to (47) (corresponding to SED ID NO: 44, 46 and 48, respectively) encoding the triosephosphate isomerases (29) to (31), respectively into other lactic acid bacterium or the like by a genetic engineering technique.
  • the invention also includes a nucleotide sequence having homology of 80% or more with any of the nucleotide sequences (45) to (47) and encoding a protein having triosephosphate isomerase activity; and a nucleotide sequence for achieving hybridization with any of the nucleotide sequences (45) to (47) under a stringent condition and encoding a protein having triosephosphate isomerase activity.
  • the above-described lactic acid bacteria of the invention can be obtained by subjecting a microorganism belonging to the species Lactobacillus reuteri to genetic analysis by the ordinary method.
  • a microorganism belonging to the species Lactobacillus reuteri it is possible to obtain the targeted lactic acid bacterium of the invention by examining whether or not there are nucleotide sequences having high homology with the following genes (13) to (15) (corresponding to SEQ ID NO: 2, and 6, respectively) encoding the lipases (1) to (3), respectively.
  • Examples of the “stringent condition” as referred to in the invention include a condition under which hybridization is carried out by preserving in a solution containing 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15 M of NaCl, 0.015 M of sodium citrate, pH 7.0), 0.5% SDS, 5 ⁇ Denhardt and 100 ⁇ g/mL of thermally denatured herring sperm DNA together with a probe at a temperature of from 50 to 65° C. overnight.
  • 6 ⁇ SSC composition of 1 ⁇ SSC: 0.15 M of NaCl, 0.015 M of sodium citrate, pH 7.0
  • SDS sodium citrate
  • 5 ⁇ Denhardt a condition under which hybridization is carried out by preserving in a solution containing 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15 M of NaCl, 0.015 M of sodium citrate, pH 7.0), 0.5% SDS, 5 ⁇ Denhardt and 100 ⁇ g/mL of thermally denatured herring sperm DNA together with
  • the lactic acid bacterium of the invention having the transport gene (4) and the lactic acid bacteria of the invention having each of the genes (9) to (11) encoding a subunit of glycerol-degrading enzyme, the genes (32) to (38) encoding a glycerol-degrading enzyme, the genes (39) to (41) encoding an aldehyde dehydrogenase, the gene (42) encoding a glycerate kinase, the gene (43) encoding a glycerol kinase, the gene (44) encoding a glycerol-3-phosphate dehydrogenase, the genes (45) to (47) encoding triosephosphate isomerase and the gene (12) encoding an enteroadherent protein can also be obtained in the same manner as described above.
  • lactic acid bacterium of the invention include Lactobacillus reuteri JCM1112T which is a standard strain of RIKEN, Japan.
  • the anti-obesity agent of the invention is prepared by processing the foregoing lactic acid bacterium of the invention into a live bacterial agent which can be orally administered and made to arrive at the intestinal tract in a live state as it is.
  • the formulation is not particularly limited and may be, for example, a solid such as a powder, a granule, a tablet and a capsule, a semi-solid such as a jelly and a paste or a liquid such as a suspension and a syrup. These respective formulations can be produced by a known method in the pharmaceutical field.
  • the lactic acid bacterium of the invention which is blended in the foregoing anti-obesity agent can be cultured by applying a known culture method of lactic acid bacteria.
  • a culture obtained by liquid culturing the lactic acid bacterium of the invention by the ordinary method may be utilized as it is; bacterial cells collected from this culture by means of centrifugation or the like may be used; or a powder obtained by freeze-drying a culture may be used.
  • the anti-obesity agent of the invention which is a solid
  • a method in which the lactic acid bacterium of the invention is blended together with a carrier such as water, starch, microcrystalline cellulose, wheat flour and sugar and processed into a desired form is also known and can be properly chosen and used in conformity with the use form.
  • powder may be prepared by freeze-drying a bacterial cell of the lactic acid bacterium of the invention as obtained by culturing by the ordinary method to form a powder and mixing it with sugar.
  • a tablet can be obtained by mixing a bacterial cell of the lactic acid bacterium of the invention together with an adequate carrier for tablet and subjecting to tablet making by the ordinary method. Furthermore, a wet bacterial cell of the lactic acid bacterium of the invention may be suspended in a syrup to form a syrup formulation.
  • other components for example, other microorganisms and active ingredients, sweeteners, flavors and coloring agents may be contained as the need arises.
  • the dose of the thus obtained anti-obesity agent can be properly determined while taking into consideration the physical state of a subject, for example, state of health, weight, age, medical history and other components to be used. In general, it is from about 10 8 to 10 9 CFU/day per an adult in terms of a bacterial number of the lactic acid bacterium of the invention.
  • an orally ingestible fermented food may be prepared by utilizing a conventionally known culture method of lactic acid bacteria.
  • fermented milk such as yogurt, lactic acid bacteria beverage and fermented sausage can be prepared, and the production of such a food can be achieved by processing apart or the whole of used lactic acid bacteria into the lactic acid bacterium of the invention.
  • the lactic acid bacterium of the invention can be processed into a form containing a larger amount thereof to prepare a healthy food or functional good.
  • other lactic acid bacteria may be contained instead of single use of the lactic acid bacterium of the invention, and food additives or seasonings or the like may be added.
  • the lactic acid bacterium of the invention shows a significant body weight gain-inhibiting effect (slimming effect) as described later in Examples, and the reasons for this are thought as follows.
  • the lactic acid bacterium of the invention degrades a fat in a digestive tract into glycerol and a fatty acid by the action of three lipases (lipases (1) to (3)).
  • the degraded glycerol is then taken into a bacterial cell by a transporter (PduF; encoded by the nucleotide (4)) of the lactic acid bacterium and metabolized by a glycerol-degrading enzyme gene in the bacterial cell (PduCDE; composed of the subunits (5) to (7)) to produce reuterin, or converted into an energy source of the bacterium per se.
  • the fatty acid is utilized as a bacterial cell component of the present bacterium but not absorbed in a living body.
  • the peptide (8) has such a function to fix the lactic acid bacterium of the invention to the intestinal tract of a human being or a mammal and enables the lactic acid bacterium of the invention to stably exist in the intestinal tract for a fixed period of time.
  • the lactic acid bacterium of the invention since the lactic acid bacterium of the invention stably exists in the intestinal tract, positively degrades a fat and utilizes its metabolites or further metabolizes them, it inhibits the absorption of a lipid from the intestinal tract into the body and even when a normal meal is ingested, is able to prevent obesity from occurring and bring maintenance and improvement of a slimming effect.
  • an embodiment of the invention includes the use of the lactic acid bacterium of the invention for the prevention or therapy of obesity and further includes the use of the lactic acid bacterium of the invention for the production of an anti-obesity agent.
  • another embodiment of the invention includes a method for therapy of obesity, which is characterized by administering a patient suffering from obesity with the lactic acid bacterium of the invention and also a method for therapy of obesity, which is characterized by administering the anti-obesity agent of the invention.
  • Wistar rats of SPF grade males of 8-week-old; Japan SLC, Inc. having a body weight of from about 180 to 200 g were used, an acclimatization period of 7 days from the day for the sending in a laboratory was provided, and the experiment was then started.
  • the breeding circumstance was set up at a temperature of 22 ⁇ 1° C., a humidity of 55 ⁇ 5% and a lighting time of 12 hours (from 8:00 to 20:00); and the rats were caged individually and provided with free access to sterile distilled water through a watering bottle and a radiation-sterilized solid diet* for rat (CE-2, CLEA Japan, Inc.) by a feeder, respectively. All of the breeding instruments to be used were ones sterilized by a high-pressure steam sterilizer.
  • test bacteria L. reuteri JCM1112T (a standard strain of RIKEN, Japan, which was received from the same) and L. rhamnosus ATCC53103 (GG strain) were used. These test bacteria were inoculated in an MRS liquid medium (Oxid) and cultured at 37° C. overnight to prepare pre-culture solutions. An MRS liquid medium was newly added such that the concentration of this pre-culture solution was 1% and cultured at 37° C. for 18 hours to prepare a test bacterial solution.
  • MRS liquid medium Oxid
  • test bacterial groups were divided into two test bacterial groups and a control group (five animals per group), and the foregoing test bacterial solutions were orally administered in the test bacterial groups respectively.
  • the test bacterial solution was forcibly administered via probe.
  • the bacterial solutions were prepared at the time of use, and the bacterial dose was set up at 10 9 CFU per rat.
  • the control group was administered with the same volume of PBS.
  • the body weight of the experimental animal was measured every day by a scale. General observation of symptoms was made every day. The symptoms were recorded for every individual, and symptom items at which a remarkable change was observed were expressed in terms of number of the animals.
  • FIG. 2 shows the body weight gain with time in the rats of the groups administered either of the foregoing Lactobacillus bacteria and the control group.
  • the body weight gain was significantly inhibited as compared with the control group, and the degree of the inhibition was larger than that in the L. rhamnosus ATCC53103 group.
  • DNA was obtained from L. reuteri JCM1112T by using the following chemicals in the following method, thereby achieving genome analysis.
  • L. reuteri JCM1112T is cultured at 37° C. for 24 hours under static conditions, and the obtained culture solution (50 mL) is centrifuged at 3,500 r.p.m. for 15 minutes and then suspended in physiological saline. 2.
  • the suspension as obtained in 1 is centrifuged at 3,500 r.p.m. for 15 minutes, a supernatant is removed, and the residue is suspended in 5 mL of 50 mM EDTA.
  • 200 ⁇ L of a 50 mg/mL lysozyme solution is added and incubated at 37° C. for 60 minutes.
  • the incubation time is set up at from 2 to 3 hours.
  • the resultant is centrifuged at 3,500 r.p.m. for 15 hours, and a supernatant is removed. 5.
  • 5 mL of a Nuclei Lysis solution is added and incubated at 80° C. for 10 minutes.
  • 6 ⁇ L of 2 mg/mL RNase A is added and incubated at 37° C. for 45 minutes.
  • 10 mL of PCI is added and mixed, and the mixture is centrifuged at 3,500 r.p.m. for 15 minutes. 8.
  • a supernatant is transferred into a new tube, and 10 mL of PCI is further added and mixed. 9. The same operations are repeated 3 times in total. (The operations are carried out until no protein layer is identified.) 10. 10 mL of CIA is added and gently mixed, and the mixture is then centrifuged at 3,500 r.p.m for 15 minutes (removal of phenol). 11. A supernatant is transferred into a new tube, followed by precipitation with ethanol. 12. A precipitate as obtained in 11 is dissolved in 1 mL of a TE buffer solution to obtain a lactic acid bacterium DNA.
  • the DNA thus obtained was subjected to structural gene prediction and annotation.
  • the structural gene prediction and the like were carried out by combining the results of GENOMEGAMBLER (Sakiyama, T., Takami, H., Ogasawara, N., Kuhara, S., Kozuki, T., Doga, K., Ohyama, A., Horikoshi, K., “An automated system for genome analysis to support microbial whole-genome shotgun sequencing”, Biosci. Biotechnol. Biochem., 64: 670 to 673 2000), GLIMMER 2.0 (Salzberg, SL., Delcher, A L., Kasif, S., and White, O., “Microbial gene identification using interpolated Markov models”, Nucleic. Acid.
  • INTERPRO Mulder, N J., Apweiler, R., Attwood, T K., Bairoch, A., Barrell, D., Bateman, A., Binns, D., Biswas, M., Bradley, P., Bork, P., Bucher, P., Copley, R R., Courcelle, E., Das, U., Durbin, R., Falquet, L., Fleischmann, W., Griffiths-Jones, S., Haft, D., Harte, N., Hulo, N., Kahn, D., Kanapin, A., Krestyaninova, M., Lopez, R., Letunic, I., Lonsdale, D., Silventoinen, V., Orchard, S E., Pagni, M., Peyruc, D., Ponting, CP., Selengut, J D., Servant, F., Sigrist, CJ., Vaughan,
  • the genes depicted in (13) to (15) were identified as encoding the lipases; the gene depicted in (4) as encoding a transporter gene; the genes depicted in (9), (10) and (11) as encoding a glycerol-degrading enzyme; and the gene depicted in (12) as an adhesive gene, respectively.
  • genes depicted in (32) to (38) were identified as encoding a glycerol-degrading enzyme; the genes depicted in (39) to (41) as encoding an aldehyde dehydrogenase; the gene depicted in (42) as encoding a glycerate kinase; the gene depicted in (43) as encoding a glycerol kinase; the gene depicted in (44) as encoding a glycerol-3-phosphate dehydrogenase; and the genes depicted in (45) to (47) as encoding triosephosphate isomerase, respectively.
  • PCR was carried out by using DNA as purified in Example 2 as a template and the following nucleotide sequences as primers and using the following reaction solutions. The PCR condition is also shown below.
  • pduCDE(F) CACCATGAAACGTCAAAAACGATTT
  • pduCDE(R) AAAAGCTTAGTTATCGCCCTTTAGC
  • PCR was carried out in the same manner as in Example 3, except using the following sequences as primers, thereby amplifying the lipase gene and adhesive gene.
  • the anti-obesity agent or anti-obesity food and drink utilizing the lactic acid bacterium of the invention is able to prevent obesity and to bring a slimming effect without requiring particular therapy or treatment other than intake of the agent, or the food or drink per se.
  • FIG. 1 is a drawing showing a metabolism map of L. reuteri JCM1112T.
  • FIG. 2 is a drawing showing the body weight gain with time in the rats administered with L. reuteri JCM1112T in comparison with the comparative group and the control group.

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Abstract

An anti-obesity agent containing, as an active ingredient, a microorganism which belongs to the species Lactobacillus reuteri and is capable of producing lipases having the amino acid sequences respectively depicted in SEQ ID NO: 1, 3 or 5 or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences respectively depicted in SEQ ID NO: 1, 3 or 5. The anti-obesity agent enables a patient to take a normal meal yet preventing the absorption of a fat into the body which is the primary cause of obesity.

Description

    TECHNICAL FIELD
  • The present invention relates to an anti-obesity agent and an anti-obesity food, and in more detail, the invention relates to an anti-obesity agent and an anti-obesity food each capable of inhibiting a lipid from being taken from a digestive tract to prevent obesity from occurring.
    • BACKGROUND ART
  • The obesity is a disease in a weight control system which is characterized by an excess of body fat. In the modern society, as the result of lack of exercise and meals with excessive calories, a neutral fat is accumulated in the body, and the number of persons who are judged to be obese continues to increase, resulting in a serious problem.
  • In order to prevent this obesity, though it is the best to perform exercise for consuming a fat to be ingested, it is actually difficult to perform exercise, and a reduction in the ingestion of a fat is demanded.
  • However, when it was intended to inhibit the obesity through unreasonable dietary restrictions, an intake of other necessary nutrients was insufficient, or the balance was upset, resulting in possibly adversely affecting the body. It may be said that the same is also applicable to the case where a food which makes a person feel full in spite of less nutrients and which is called a diet food is ingested.
    • DISCLOSURE OF THE INVENTION Problems to be solved by the invention
  • Accordingly, it has been eagerly demanded to develop a measure capable of preventing a lipid (triacylglycerol) as a primary cause of obesity from being absorbed into the body while taking a normal meal.
    • Means for Solving the Problems
  • The present inventors paid attention to a mechanism where a lipid is absorbed into the body and made studies regarding a method of inhibiting obesity. As a result, it was found that when a microorganism flora within intestine ingests and degrades a lipid, the lipid to be ingested by a person reduces as a result, whereby the obesity can be spontaneously prevented. Then, the present inventors have made extensive and intensive investigations regarding microorganisms capable of ingesting and degrading such a lipid and as a result, found out a microorganism having such an action among those belonging to lactic acid bacteria, leading to accomplishment of the invention.
  • Specifically, the invention is concerned with an anti-obesity agent comprising, as an active ingredient, a microorganism belonging to the species Lactobacillus reuteri and capable of producing lipases respectively depicted in the following amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3). The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping lipase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (1)
    MVKLMTIHELANNPTLSGQVRLIENIVYGA
    MDGEALHMSILAPWTQRFPKQYQTEPRPLI
    VFVQGSSWRTPKMGEEIPQLVQFVRAGYIV
    ATVQHRSSIDSHPFPAFLQDVKTAIRFLRA
    NAQKYAIDPQQVAIWGTSSGANAAMLVGLT
    GDDPRYKVDLYQDESDAVDAVVSCFAPMDV
    EKTFEYDANVPGNKLLQYCLLGPDVSKWPE
    IEKQMSPLYQVKDGQNYPPFLLFHGDADKV
    VPYEQMEKMYMRLKDNGNSVEAYRVKGANH
    ERDFWSPTIYNIVQKFLGDQFK
    (2)
    LIYVLKDLCNTIAEVYGKSILKGVFIMKHT
    LKVDQVRDGLWLDSDITYTQVPGWLGNTTR
    DLKLSVIRHFQTNDDTRYPVIFWFAGGGWM
    DTDHNVHLPNLVDFARHGYIVVGVEYRDSN
    KVQFPGQLEDAKAAIRYMRANAKRFQADPN
    RFIVMGESAGGHMASMLGVTNGLNQFDKGA
    NLDYSSDVQVAVPFYGVVDPLTAKTGSASN
    DFDFVYRNLLGAEPENAPELDSAANPLTYV
    NSNSTPFLIFHGTEDVVVPIKDSEKLYDAL
    VENNVPAELYEIEGASHMDVKFLQPQVFKI
    VMDFLDKYLTRS
    (3)
    MEIKSVNLDQPYSSLDIYHSNTDKALPGLV
    ILPGGSYNQIMERDSERVALTFATHAWQTF
    VVRYPVVEHKNYEEAKIAVHQAFEYIVNHA
    AELDVDADRLGIIGFSAGGQIAAAYSNEKL
    THARFAALGYPVIQPLIDERMGVTTENVAK
    LVNPQTPPTFMWGSAKDELTPFVDHLQVYA
    DALIKNDIPYELHEFGTGGHGIALANEYTG
    IVNNDRVDNHMGKWFPLFLEWLTELNLI
  • Also, the invention is concerned with an anti-obesity food comprising, as an active ingredient, a microorganism belonging to the species Lactobacillus reuteri and capable of producing lipases respectively depicted in the foregoing amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3).
  • Furthermore, the invention is concerned with a glycerol-degrading enzyme composed of subunits respectively depicted in the following amino acid sequences (5) to (7) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7). The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-degrading activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (5)
    MKRQKRFEELEKRPIHQDTFVKEWPEEGFV
    AMMGPNDPKPSVKVENGKIVEMDGKKLEDF
    DLIDLYIAKYGINIDNVEKVMNMDSTKIAR
    MLVDPNVSRDEIIEITSALTPAKAEEIISK
    LDFGEMIMAVKKMRPRRKPDNQCHVTNTVD
    NPVQIAADAADAALRGFPEQETTTAVARYA
    PFNAISILIGAQTGRPGVLTQCSVEEATEL
    QLGMRGFTAYAETISVYGTDRVFTDGDDTP
    WSKGFLASCYASRGLKMRFTSGAGSEVLMG
    YPEGKSMLYLEARCILLTKASGVQGLQNGA
    VSCIEIPGAVPNGIREVLGENLLCMMCDIE
    CASGCDQAYSHSDMRRTERFIGQFIAGTDY
    INSGYSSTPNYDNTFAGSNTDAMDYDDMYV
    MERDLGQYYGIHPVKEETIIKARNKAAKAL
    QAVFEDLGLPKITDEEVEAATYANTHDDMP
    KRDMVADMKAAQDMMDRGITAIDIIKALYN
    HGFKDVAEAILNLQKQKVVGDYLQTSSIFD
    KDWNVTSAVNDGNDYQGPGTGYRLYEDKEE
    WDRIKDLPFALDPEHLEL
    (6)
    MADIDENLLRKIVKEVLSETNQIDTKIDFD
    KSNDSTATATQEVQQPNSKAVPEKKLDWFQ
    PVGEAKPGYSKDEVVIAVGPAFATVLDKTE
    TGIPHKEVLRQVIAGIEEEGLKARVVKVYR
    SSDVAFCAVQGDHLSGSGIAIGIQSKGTTV
    IHQKDQDPLGNLELFPQAPVLTPETYRAIG
    KNAAMYAKGESPEPVPAKNDQLARIHYQAI
    SAIMHIRETHQVVVGKPEEEIKVTFD
    (7)
    MSEVDDLVAKIMAQMGNSSSANSSTGTSTA
    STSKEMTADDYPLYQKHRDLVKTPKGHNLD
    DINLQKVVNNQVDPKELRITPEALKLQGEI
    AANAGRPAIQKNLQRAAELTRVPDERVLEM
    YDALRPFRSTKQELLNIAKELRDKYDANVC
    AAWFEEAADYYESRKKLKGDN
  • Moreover, the invention is concerned with an enteroadherent protein depicted in the following amino acid sequence (8) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8). The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping activity for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (8)
    MFGHDGRIVTKVYQWAGTYYYFDPNTYLRV
    DNDYRQSQWGDWYMFGPDGRIVTGLKEWYG
    SYYYFDPTTYLKVTNKWIDNKYFGPAGQQA
    ISRFERLDNKYYYFDANGAVLNIHDQFKNI
    DNHTYYFGADGACYTSQFLNKDGKQYYFDN
    DGIMLTDQEKIIDGKFYHFNVNGEAIQVND
    PSEI
    • EFFECT OF THE INVENTION
  • The anti-obesity agent and anti-obesity food and drink of the invention are able to prevent the absorption of a lipid from the intestinal tract by ingesting them while taking a normal meal. Since lactic acid bacteria are used from old in producing fermented foods such as yogurt and fermented milk and have extremely high safety, they can be ingested without anxiety.
    • BEST MODES FOR CARRYING OUT THE INVENTION
  • The microorganism which is an active ingredient of the anti-obesity agent and anti-obesity food and drink of the invention (hereinafter often referred to as “anti-obesity agent and the like”) is one belonging to the species Lactobacillus reuteri which is a lactic acid bacterium and capable of producing lipases respectively depicted in the foregoing amino acid sequences (1) to (3) (corresponding to SEQ ID NO: 1, 3 and 5, respectively) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3) (these lipases will be hereinafter referred to as “lipases of the invention”) (this microorganism will be hereinafter referred to as “lactic acid bacterium of the invention”).
  • The lipases of the invention degrade a lipid (triacylglycerol) present in the vicinity of intestinal mucosa cells into a fatty acid and glycerol, which are then taken into the lactic acid bacterium of the invention. As shown in FIG. 1, the thus taken fatty acid is utilized as a fungus constituent, and the glycerol is converted into carbon dioxide, acetone, ethanol, lactate or reuterin through various metabolic pathways.
  • It is preferable that the lactic acid bacterium of the invention has ability for producing the foregoing lipases, namely has the nucleotide sequences (genes) for encoding the foregoing lipases and besides, has a nucleotide sequence as a gene encoding a transporter and depicted in the following (4) (corresponding to SEQ ID NO: 8). The invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence depicted in (4) and encoding a protein having glycerol transporter activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence depicted in (4) under a stringent condition and encoding a protein having glycerol transporter activity.
  • (4)
    ATGCATGGATTTATTGGCGAATTTTTTGGCACCATGGTTTTAATCCTATTAGGAGCAGGA
    TGTTGTGCTGGTAATAGTTTGAATAAAACATATGGGAAACAAAGTGGCTGGTGGTTTATC
    TGTATTTTCATGGGGCTTAGCAGTTACAATGGGAGTTTATGTTGCAGGATTTCTGGGTTCA
    TTAGGGCACTTAAATCCCGCTGTAACAATTCCTTTTGCTATTTTTGGCTTATTCCCATGG
    AGTAACGTTATACCTTACTTACTTGGTCAATTTCTTGGTGCGTTTGTTGGTGCAGTATTA
    GTAATTATTCAATTCTATCCACAATTTAAAGCAACCCCAAATGAAGAAGAAGGAAATAAT
    GTTGGTATTTTTGCTACTCGTCCAGCGATAAATAGTCCAATTTTTAACTTTTTCTCAGAA
    GTGATTGCGACCTTTGCATTTATTTTCATCTTATTAAATCTTGGCAACTTTACACAGGGA
    TTGAAGCCATTTATCGTAGGAATGGTTATTGCAGTTGTTGGTACATGTCTCGGGACAACT
    ACTGGCTTTGCATTAAACCCAGCTCGTGATTGGTCACCACGTTTAGCATATACTATTTTG
    CCAATTCCTAATAAGGGTGTTTCAGAATGGTGGTATGCATGGGTTCCAATGTGTGGCCCA
    ATTGTTGGGGGCCTTCTTGCTTGTGCTTTACAAACGGCACTAGTTTAG
  • This gene is one encoding a transporter for taking glycerol into the cell of the lactic acid bacterium of the invention, and, as shown in FIG. 1, the thus taken glycerol is subjected to metabolism by the enzymes within the lactic acid bacterium.
  • Furthermore, it is preferable that the lactic acid bacterium of the invention is one having a glycerol-degrading enzyme composed of the subunits respectively depicted in the foregoing amino acid sequences (5) to (7) (corresponding to SEQ ID NO: 9, 11 and 13, respectively) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7). This glycerol-degrading enzyme composed of the subunits (5) to (7) is a glycerol dehydratase which functions in a pdu (propanediol utilization) operon and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention.
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (9) to (11) (corresponding to SED ID NO: 10, 12 and 14, respectively) encoding the subunits (5) to (7) into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes nucleotide sequences having homology of 80% or more with the nucleotide sequences (9) to (11) and encoding a protein having glycerol-degrading activity; and nucleotide sequences for achieving hybridization with the nucleotide sequences (9) to (12) under a stringent condition and encoding a protein having glycerol-degrading activity.
  • (9)
    ATGAAACGTCAAAAACGATTTGAAGAACTAGAAAAACGGCCAATTCATCAAGATACATTT
    GTTAAAGAATGGCCAGAAGAAGGTTTCGTTGCAATGATGGGGCCTAATGACCCTAAGCCT
    AGTGTAAAAGTTGAAAATGGCAAGATCGTAGAGATGGATGGTAAAAAGCTCGAAGATTTT
    GATTTGATTGACTTGTACATTGCTAAGTATGGAATCAATATTGACAACGTTGAAAAAGTT
    ATGAATATGGATTCTACCAAGATTGCACGGATGCTTGTTGATCCTAATGTTTCTCGTGAT
    GAAATTATTGAAATTACATCAGCTTTGACTCCTGCTAAGGCTGAAGAGATCATCAGTAAG
    CTTGATTTTGGTGAAATGATTATGGCTGTCAAGAAGATGCGCCCACGTCGTAAGCCTGAC
    AACCAGTGTCACGTTACCAATACTGTTGATAACCCAGTTCAAATTGCTGCTGATGCTGCT
    GATGCCGCTCTTCGTGGATTTCCAGAACAAGAAACCACGACAGCTGTGGCACGTTATGCA
    CCATTCAATGCTATTTCAATTTTAATTGGTGCACAAACAGGTCGCCCTGGTGTATTGACA
    CAATGTTCTGTTGAAGAAGCTACTGAATTGCAATTAGGTATGCGTGGTTTTACCGCATAT
    GCTGAAACCATTTCAGTTTACGGTACTGATCGTGTATTTACCGATGGTGATGATACTCCA
    TGGTCTAAAGGCTTCTTGGCATCTTGTTATGCATCACGTGGTTTGAAGATGCGATTTACT
    TCAGGTGCCGGTTCAGAAGTTTTGATGGGTTATCCAGAAGGTAAGTCAATGCTTTACCTT
    GAAGCGCGTTGTATTTTACTTACTAAGGCTTCAGGTGTTCAAGGACTTCAAAATGGTGCC
    GTAAGTTGTATTGAAATTCCTGGTGCTGTTCCTAATGGTATTCGTGAAGTTCTCGGTGAA
    AACTTGTTATGTATGATGTGTGACATCGAATGTGCTTCTGGTTGTGACCAAGCATACTCA
    CACTCCGATATGCGGCGGACTGAACGGTTTATTGGTCAATTTATTGCCGGTACTGATTAT
    ATTAACTCTGGTTACTCATCAACTCCTAACTACGATAATACCTTCGCTGGTTCAAACACT
    GATGCTATGGACTACGATGATATGTATGTTATGGAACGTGACTTGGGTCAATATTATGGT
    ATTCACCCTGTTAAGGAAGAAACCATTATTAAGGCACGTAATAAGGCCGCTAAAGCCCTT
    CAAGCAGTATTTGAAGATCTTGGATTACCAAAGATTACTGATGAAGAGGTCGAAGCAGCA
    ACGTATGCTAACACCCATGATGACATGCCAAAGCGGGATATGGTTGCAGATATGAAGGCT
    GCTCAAGATATGATGGATCGTGGAATTACTGCTATTGATATTATCAAGGCATTGTACAAC
    CACGGATTTAAAGATGTCGCTGAAGCAATTTTGAACCTTCAAAAACAAAAAGTTGTTGGT
    GATTACCTTCAAACATCTTCTATTTTTGATAAAGATTGGAACGTCACTTCTGCTGTTAAC
    GACGGAAATGATTATCAAGGACCAGGTACTGGATACCGTCTATATGAAGACAAGGAAGAA
    TGGGATCGGATTAAAGACTTACCATTCGCCCTTGATCCAGAACATTTGGAACTGTAG
    (10)
    ATGGCTGATATTGATGAAAACTTATTACGTAAAATCGTTAAAGAAGTTTTAAGCGAAACT
    AATCAAATCGATACTAAGATTGACTTTGATAAAAGTAATGATAGTACTGCAACAGCAACT
    CAAGAGGTGCAACAACCAAATAGTAAAGCTGTTCCAGAAAAGAAACTTGACTGGTTCCAA
    CCAGTTGGAGAAGCAAAACCTGGATATTCTAAGGATGAAGTTGTAATTGCAGTCGGTCCT
    GCATTCGCAACTGTTCTTGATAAGACAGAAACTGGTATTCCTCATAAAGAAGTGCTTCGT
    CAAGTTATTGCTGGTATTGAAGAAGAAGGGCTTAAGGCGCGGGTAGTTAAAGTTTACCGG
    AGTTCAGATGTAGCATTCTGTGCTGTCCAAGGTGATCACCTTTCTGGTTCAGGAATTGCT
    ATTGGTATCCAATCAAAAGGGACGACAGTTATTCACCAAAAGGATCAAGACCCTCTTGGT
    AACCTTGAGTTATTCCCACAAGCGCCAGTACTTACTCCCGAAACTTATCGTGCAATTGGT
    AAGAATGCCGCTATGTATGCTAAGGGTGAATCTCCAGAACCAGTTCCAGCTAAAAACGAT
    CAACTTGCTCGTATTCACTATCAAGCTATTTCAGCAATTATGCATATTCGTGAAACTCAC
    CAAGTTGTTGTTGGTAAGCCTGAAGAAGAAATTAAGGTTACGTTTGATTAA
    (11)
    ATGAGTGAAGTTGATGATTTAGTAGCAAAGATCATGGCTCAGATGGGAAACAGTTCATCT
    GCTAATAGCTCTACAGGTACTTCAACTGCAAGTACTAGTAAGGAAATGACAGCAGATGAT
    TACCCACTTTATCAAAAGCACCGTGATTTAGTAAAAACACCAAAAGGACACAATCTTGAT
    GACATCAATTTACAAAAAGTAGTAAATAATCAAGTTGATCCTAAGGAATTACGGATTACA
    CCAGAAGCATTGAAACTTCAAGGTGAAATTGCAGCTAATGCTGGCCGTCCAGCTATTCAA
    AAGAATCTTCAACGAGCTGCAGAATTAACACGAGTACCTGACGAACGGGTTCTTGAAATG
    TATGATGCATTGCGTCCTTTCCGTTCAACTAAGCAAGAATTATTGAACATTGCAAAGGAA
    TTACGGGACAAGTATGACGCTAATGTTTGCGCAGCATGGTTTGAAGAAGCTGCTGATTAT
    TATGAAAGTCGTAAGAAGCTAAAGGGCGATAACTAA
  • Moreover, it is more preferable that the lactic acid bacterium of the invention is one holding an enteroadherent protein depicted in the following amino acid sequence (8) (corresponding to SEQ ID NO: 15) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8).
  • (8)
    MFGHDGRIVTKVYQWAGTYYYFDPNTYLRV
    DNDYRQSQWGDWYMFGPDGRIVTGLKEWYG
    SYYYFDPTTYLKVTNKWIDNKYFGPAGQQA
    ISRFERLDNKYYYFDANGAVLNIHDQFKNI
    DNHTYYFGADGACYTSQFLNKDGKQYYFDN
    DGIMLTDQEKIIDGKFYHFNVNGEAIQVND
    PSEI
  • This enteroadherent protein has an action for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells, and the lactic acid bacterium of the invention having this is able to exist in the vicinity of an intestinal mucosa for a certain period of time and stably take a lipid thereinto. It becomes possible to obtain a lactic acid bacterium with a long intestinal residence time by incorporating a gene encoding this protein and depicted in the following (12) (corresponding to SEQ ID NO: 16) into other lactic acid bacterium by a known technique. The invention also involves a nucleotide sequence having homology of 80% or more with the nucleotide sequence (12) and encoding a protein having activity for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (12) under a stringent condition and encoding a protein having activity for making a lactic acid bacterium adhere to the vicinity of intestinal mucosa cells.
  • (12)
    ATGTTCGGTCACGATGGCCGCATTGTTACTAAAGTTTACCAATGGGCTGGCACGTATTAC
    TACTTTGATCCGAATACTTATTTGCGAGTAGATAATGATTACCGTCAATCTCAGTGGGGC
    GATTGGTATATGTTTGGCCCAGATGGTCGTATCGTTACAGGGTTAAAGGAATGGTACGGT
    AGTTATTATTACTTTGATCCGACGACTTACTTAAAAGTAACTAATAAGTGGATAGATAAT
    AAGTACTTTGGTCCAGCTGGTCAGCAAGCTATTTCACGCTTTGAGAGACTTGATAATAAG
    TATTACTATTTCGATGCTAATGGGGCAGTTCTTAATATCCATGATCAATTTAAGAATATT
    GATAACCACACTTATTACTTTGGAGCTGATGGTGCTTGTTATACCAGTCAATTCTTAAAT
    AAGGATGGTAAACAGTATTATTTCGATAATGATGGAATTATGCTCACTGATCAAGAGAAG
    ATCATTGACGGTAAATTCTATCATTTCAATGTTAATGGTGAAGCAATCCAAGTAAATGAT
    CCTTCTGAAATTTGA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having a glycerol-degrading enzyme depicted in any of the following amino acid sequences (16) to (20) (corresponding to SEQ ID NO: 17, 19, 21, 23 and 25, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (16) to (20). This glycerol-degrading enzyme is an alcohol dehydrogenase (ADH (8) in FIG. 1) and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-degrading activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (16)
    MKAAVINDPVDGFVTVKDVQLRDLKPGEAL
    VDMEYCGLCHTDLHVAAGDFGKKPGRIIGH
    EGVGRVSKVAPGVTSLKVGDRVSIAWFFKG
    CGHCEYCLTGRETLCRNVLNAGYTADGAMA
    EQCIVPADYAVKVPEGLDPVEATSLTCAGV
    TMYKALKVADIKPGQWVSIVGAGGLGNLGI
    QLAHNVFGAHVIAVDGNPDKLEAAKKNGAE
    ILINRHDGDVDKQIQEKVGGVHAAVVTAVS
    ASAFDQAVDSLRPDGKLVAVALPQGDMKLN
    IAKTVLDGIIVAGSLVGTRQDLAECFQFGA
    EGKVHPIVKTRKLSEINDMIQELKDNKVVG
    RNVVDFVHNDND
    (17)
    MEKRENAIPKTMKAWAVTTPGPIDGKESPI
    EFTEKPVPTPKRGEVLVKVITCGVCHTDLH
    VTEGDLPVHHEHVTPGHEIVGKVVGFGPET
    QRFKFGERIGIPWFRHACGVCKFCRSGHEN
    LCPHSLYTGWDHDGGYAEYVTVPEGFAYRL
    PEKFDSLEAAPLLCAGIIGYRAFERANVPA
    GGRLGLYGFGGSAHITAQIALAQGIEVHVF
    TRGEDAKKFALELGCASVQGSYDPAPVPLD
    SSIIFAPVGDMVLPALASLVPGGTLALAGI
    HMTDIPTMNYQKEIFHEKTLTSVESNTRRD
    GEEFLTLADRLNIHPEVHEYPLAKADEALR
    YVKHGDIKGACVLRVSED
    (18)
    MQIKAALATKPNADLEIQTVELDEPKENEV
    LIKIASTGFCHTDIVGRSGATTPLPVVLGH
    EGAGVVQKVGANVTDVKPGDHVVLSFSYCG
    HCYNCTHNHQGLCENFNQLNFEGKTYDGTH
    RLHLDDGTPVSVFFGQSSFATYVTANVHNI
    VKVDQDVDLNLLGPLGCGMQTGAGTVLNYI
    KPAPEDAIAVFGAGAVGLAAIMAAKIAGVK
    HIIAINRNGNHLDLAKELGATETINNTAED
    PVKAIKEIVPRGVTYAIDTTGNTGVIKSAI
    DSLATAGECVLLGVGGDITLDLMNDILSES
    KKISGVVEGDSNPQEFIPQLVKYYKQSKFP
    LDKLVKYYDFADINQVIADSTNGKVIKPII
    KIDPELAKLPLTNDGSNVQKMVAEAGLADQ
    ITIDSAGTSNIAEGSPADSRTKAILDKYHI
    KDDGMIARQLQDRDYYDADYIIAMDQMNVR
    DAKDMAPAGLENKVHGIFEATPGKENCYIV
    DPWITH
    (19)
    MKKAIFEKAGQMKIVDVDRPTIEKPDDVII
    KVVRTCVCGSDLWNFRGINPVEKDSENSGH
    EAIGIVEEVGEDITTVKPGDFVIAPFTHGC
    GHCAACRAGFDGSCQSHNDNFSSGVQAQYV
    RFQHGQWALVKVPGKPSDYSEGMLKSLLTL
    ADVMATGYHAARVANVSDGDTVVVMGDGAV
    GLCAIIAAKMRGAKKIISTSRHADRQALAK
    EFGATDNVAERSDEAVQKIMELTNGAGADA
    VLECVGTEQSTDTAMKVGRPGTIVGRVGLP
    HTPKMDMTVLFYNNTIVGGGPASVTTYDKD
    VLLKAVLDGDINPGKVFTKSFDLDQIQEAY
    EAMDKREAIKSYIIMDGFERD
    (20)
    MGRLDNKVAIITGGSKGIGAAVAKKFIEEG
    AKVVLTARKMDEGQKVADQLGDNAIFIQQD
    VARKGDWDRVIRQTVQVFGKLNIVVNNAGI
    AEYADVEKTDAEIWDKTIAVNLTGTMWGTK
    LGIEAMKNNGEKNSIINMSSIEGLIGDPDL
    FAYNASKGGVRLLTKSAALDCARKGYDIRV
    NTIHPGYISTPLVDNLVKDDPKAEGHLESL
    HPLGRLGKPEEIANLALYLASDESSFSTGS
    EFVADGGYTAQ
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (32) to (36) (corresponding to SED ID NO: 18, 20, 22, 24 and 26, respectively) encoding the glycerol-degrading enzymes (16) to (20), respectively into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with any of the nucleotide sequences (32) to (36) and encoding a protein having glycerol-degrading activity; and a nucleotide sequence for achieving hybridization with any of the nucleotide sequences (32) to (36) under a stringent condition and encoding a protein having glycerol-degrading activity.
  • (32)
    ATGAAAGCTGCTGTTATTAATGATCCAGTAGACGGTTTTGTTACTGTTAAAGATGTTCAA
    CTTCGGGATTTGAAGCCCGGTGAAGCTTTAGTTGACATGGAATATTGTGGTCTTTGTCAC
    ACTGATCTACACGTTGCTGCTGGGGACTTTGGTAAGAAGCCCGGTCGTATTATCGGTCAC
    GAAGGGGTTGGTCGTGTATCTAAGGTTGCCCCTGGCGTTACTTCCTTGAAAGTTGGCGAC
    CGTGTATCAATTGCATGGTTCTTCAAGGGCTGTGGACACTGTGAATATTGTTTAACTGGT
    CGTGAAACTCTTTGTCGGAACGTTCTTAATGCGGGTTACACTGCTGACGGTGCAATGGCT
    GAACAATGTATCGTACCAGCTGACTACGCTGTTAAGGTTCCAGAAGGTCTTGATCCTGTT
    GAAGCTACTTCATTAACTTGTGCTGGTGTTACGATGTACAAGGCATTAAAGGTTGCTGAC
    ATCAAGCCAGGTCAATGGGTATCAATCGTTGGTGCTGGTGGTTTAGGTAACTTGGGTATT
    CAACTTGCTCACAACGTATTTGGTGCTCATGTTATCGCTGTTGATGGTAATCCTGATAAG
    CTTGAAGCCGCTAAGAAGAATGGTGCTGAAATTTTAATTAACCGTCATGACGGTGATGTT
    GATAAGCAAATTCAAGAAAAGGTTGGCGGTGTTCACGCTGCTGTAGTAACAGCTGTTTCT
    GCCTCTGCATTCGACCAAGCAGTTGATTCACTTCGCCCAGATGGTAAGCTTGTTGCCGTT
    GCGCTTCCACAAGGTGACATGAAGCTTAACATTGCTAAGACTGTTCTTGATGGTATCATT
    GTTGCTGGTTCATTAGTTGGTACCCGTCAAGACTTAGCTGAATGTTTCCAATTTGGTGCA
    GAAGGTAAGGTTCACCCAATTGTTAAGACTCGTAAGTTAAGCGAAATTAATGATATGATC
    CAAGAACTTAAGGATAACAAGGTTGTTGGTCGGAATGTTGTTGATTTTGTTCACAACGAT
    AACGACTAA
    (33)
    ATGGAAAAACGCGAAAATGCTATTCCGAAAACAATGAAGGCTTGGGCAGTCACAACTCCT
    GGGCCGATTGATGGTAAGGAATCACCAATCGAATTTACCGAAAAGCCTGTGCCGACTCCT
    AAACGGGGAGAAGTCCTTGTTAAGGTAATAACGTGTGGAGTATGTCATACGGACTTGCAC
    GTGACTGAAGGAGACTTGCCGGTTCACCACGAACACGTTACTCCTGGTCATGAAATTGTT
    GGTAAAGTTGTCGGCTTTGGACCAGAGACACAACGATTTAAGTTTGGTGAGCGAATTGGG
    ATTCCATGGTTTCGGCATGCTTGTGGTGTATGCAAGTTTTGCCGATCAGGTCATGAGAAT
    CTCTGTCCTCATTCACTTTATACCGGTTGGGATCATGATGGCGGTTATGCAGAATATGTC
    ACAGTTCCAGAAGGATTTGCATATCGGCTTCCAGAAAAGTTTGATTCCCTAGAGGCAGCT
    CCGTTATTATGTGCAGGGATTATTGGTTATCGGGCCTTTGAACGTGCCAATGTTCCGGCT
    GGCGGTCGCCTAGGATTATATGGCTTCGGTGGTTCAGCTCATATTACAGCTCAAATTGCA
    CTTGCTCAGGGAATTGAAGTGCATGTCTTTACGCGTGGTGAGGATGCCAAGAAATTCGCC
    CTAGAATTAGGTTGTGCTTCTGTTCAGGGCTCCTATGACCCAGCACCAGTTCCTTTGGAT
    TCATCAATCATTTTTGCGCCGGTTGGTGATATGGTCTTGCCGGCTTTAGCTAGTTTAGTT
    CCAGGGGGGACATTAGCATTAGCCGGTATTCATATGACTGATATTCCAACAATGAATTAC
    CAAAAAGAAATATTCCACGAAAAGACATTAACGAGTGTTGAGAGTAATACTCGTCGTGAT
    GGGGAAGAATTCTTAACATTAGCTGATCGTCTTAATATCCATCCTGAAGTCCACGAATAT
    CCCCTAGCAAAGGCTGACGAAGCATTACGCTATGTTAAGCACGGTGATATTAAGGGAGCT
    TGTGTATTACGTGTTAGTGAGGACTAA
    (34)
    ATGCAAATTAAAGCTGCTCTTGCAACCAAACCTAACGCTGATTTAGAGATTCAAACCGTC
    GAATTGGATGAACCAAAAGAAAATGAAGTATTAATAAAAATTGCTTCAACAGGTTTTTGT
    CATACAGATATTGTTGGTCGAAGCGGTGCCACTACCCCTCTCCCCGTTGTCCTCGGGCAT
    GAAGGTGCGGGCGTCGTCCAAAAAGTAGGAGCTAACGTTACGGACGTTAAACCCGGCGAC
    CATGTTGTTCTATCATTTAGCTACTGTGGCCATTGCTATAACTGTACTCATAATCATCAA
    GGCTTATGCGAAAACTTCAATCAGCTAAACTTTGAAGGAAAAACCTATGATGGTACTCAC
    CGCCTGCACTTAGATGATGGCACGCCAGTCAGTGTCTTTTTTGGTCAGTCTTCCTTTGCG
    ACCTATGTAACAGCCAATGTCCATAATATTGTTAAAGTTGATCAAGATGTTGATCTTAAC
    TTATTAGGGCCACTCGGTTGTGGAATGCAAACAGGTGCTGGAACCGTTCTAAATTATATT
    AAACCTGCTCCTGAAGATGCAATTGCCGTTTTCGGTGCTGGTGCTGTTGGCTTAGCCGCA
    ATTATGGCTGCTAAAATTGCTGGAGTTAAACATATTATTGCGATTAATCGTAACGGTAAC
    CACCTTGACCTGGCGAAGGAATTGGGCGCTACTGAAACGATTAATAATACGGCTGAAGAT
    CCCGTCAAAGCAATTAAAGAAATCGTTCCGCGTGGTGTAACTTATGCAATCGATACTACC
    GGAAACACCGGTGTAATTAAATCAGCAATTGATAGTCTTGCCACCGCTGGAGAATGTGTC
    CTCTTAGGAGTTGGCGGCGATATTACCTTAGACTTAATGAATGATATCTTATCAGAATCT
    AAGAAAATCTCTGGGGTTGTCGAAGGAGATAGCAATCCCCAAGAGTTTATTCCTCAACTA
    GTTAAGTACTACAAGCAAAGCAAGTTCCCCCTTGATAAGCTTGTTAAGTACTACGATTTT
    GCTGATATTAACCAAGTTATCGCTGACTCAACAAACGGAAAGGTTATTAAGCCAATCATC
    AAAATTGATCCTGAATTAGCTAAATAATTGCCGCTCACCAATGACGGAAGCAATGTTCAA
    AAAATGGTTGCAGAAGCTGGCCTTGCTGATCAAATTACTATTGATTCAGCCGGAACAAGT
    AACATTGCAGAAGGTTCACCTGCTGATAGTCGAACAAAAGCCATTCTCGATAAATATCAC
    ATTAAAGACGACGGAATGATTGCCCGTCAATTGCAGGACAGGGATTATTATGATGCCGAT
    TATATTATCGCAATGGATCAGATGAATGTCCGGGACGCAAAAGATATGGCACCAGCTGGG
    TTAGAAAATAAGGTTCATGGAATCTTTGAAGCTACCCCAGGAAAAGAAAATTGCTATATC
    GTTGACCCCTGGATCACTCACTGA
    (35)
    ATGAAAAAAGCTATTTTTGAAAAGGCGGGTCAAATGAAGATTGTTGATGTTGACCGTCCA
    ACAATTGAAAAGCCTGATGACGTAATTATTAAGGTAGTGCGGACCTGTGTTTGTGGTTCT
    GACCTATGGAACTTCCGAGGAATTAATCCGGTTGAAAAAGATTCTGAAAACTCTGGCCAT
    GAAGCAATTGGAATTGTTGAAGAAGTTGGTGAAGATATCACTACTGTCAAACCTGGGGAC
    TTTGTGATTGCTCCATTTACTCATGGATGTGGGCACTGTGCTGCTTGTCGCGCGGGCTTC
    GATGGTTCTTGCCAAAGTCACAACGATAACTTTAGCTCTGGTGTGCAAGCTCAATACGTT
    CGGTTCCAACACGGTCAATGGGCGCTTGTTAAAGTTCCGGGCAAGCCAAGTGACTACAGT
    GAAGGAATGCTTAAGTCCCTCTTAACCCTTGCTGATGTTATGGCTACTGGTTACCACGCT
    GCACGAGTTGCTAACGTTAGTGATGGTGATACAGTTGTTGTAATGGGTGACGGTGCTGTT
    GGCCTTTGTGCGATTATTGCTGCTAAGATGCGGGGCGCTAAGAAGATCATTTCTACTAGT
    CGCCATGCTGACCGTCAAGCCCTTGCTAAGGAATTTGGTGCTACTGACAATGTTGCTGAA
    CGTAGTGACGAAGCGGTTCAAAAGATCATGGAACTCACTAACGGTGCCGGTGCTGATGCT
    GTCCTTGAATGCGTTGGTACTGAACAATCAACTGATACTGCCATGAAAGTTGGCCGTCCA
    GGTACCATCGTTGGTCGGGTTGGCTTACCTCATACCCCAAAGATGGACATGACGGTGCTA
    TTCTACAACAACACTATTGTCGGCGGTGGTCCAGCATCAGTAACCACTTACGACAAGGAC
    GTATTGTTGAAGGCTGTTCTTGATGGTGACATTAACCCTGGTAAGGTCTTTACTAAGAGC
    TTCGACCTTGACCAAATTCAAGAAGCTTATGAAGCAATGGATAAGCGTGAAGCAATCAAG
    TCTTACATTATTATGGATGGCTTTGAACGCGATTAA
    (36)
    ATGGGTCGTTTAGATAATAAAGTTGCAATTATTACTGGTGGTTCTAAAGGAATTGGAGCT
    GCTGTCGCAAAAAAGTTTATCGAAGAAGGCGCAAAGGTTGTTTTAACCGCTCGGAAGATG
    GATGAGGGACAAAAAGTCGCTGACCAACTAGGTGACAATGCGATCTTTATCCAACAAGAC
    GTTGCTCGGAAAGGAGACTGGGACCGGGTAATCCGCCAAACTGTCCAAGTCTTTGGGAAG
    CTCAATATTGTGGTTAACAATGCGGGAATTGCCGAATACGCCGATGTTGAGAAGACGGAC
    GCTGAAATTTGGGATAAAACAATTGCCGTTAACCTTACCGGTACGATGTGGGGAACTAAG
    CTCGGTATTGAAGCAATGAAGAACAACGGGGAAAAGAATTCAATCATCAATATGTCATCC
    ATTGAAGGACTAATTGGTGATCCTGATCTCTTTGCATACAATGCTTCTAAGGGTGGTGTC
    CGCCTCTTAACTAAGTCCGCTGCGCTTGATTGTGCCCGGAAAGGCTATGACATCCGTGTA
    AATACAATTCATCCTGGTTATATCTCAACTCCACTAGTTGATAATTTGGTCAAGGATGAT
    CCAAAAGCAGAAGGACACCTAGAAAGCCTTCATCCCCTTGGCCGTCTTGGAAAGCCAGAA
    GAGATTGCTAACCTCGCTTTATACCTTGCTTCAGATGAATCAAGCTTTAGTACTGGTTCG
    GAATTTGTCGCTGATGGTGGCTATACGGCTCAATAA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having a glycerol-degrading enzyme depicted in the following amino acid sequence (21) or (22) (corresponding to SEQ ID NO: 27 or 29, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (21) or (22). This glycerol-degrading enzyme is an alcohol dehydrogenase (ADH (8) in FIG. 1) and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-degrading activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (21)
    MTNVPTVKLNNGVEMPTLGFEVFQVPDLSQ
    AEQAVTDALEVGYRLIDTAAAYQNEEAVGK
    AIKNSSVNREDVFVTSKLWVSDFNYKRAKA
    GIDASLQKLGLDYMDLYLLHQPYGDTMGAW
    RALQEAQKEGKIRAIGVSNFYADQLKDLEL
    TMPVKPAVNQIEVNPWYQQDQEVKFAQSED
    IRVEAWAPFAEGKHDIFTNEIIAEIAAKYG
    KSNGQVILRWLLQRGITVIPKSVHKNRMEE
    NIDVFDFELSNDDMKKIASLNKKESQFFDH
    RDPVTIEQIFGSSLKMVQDDEK
    (22)
    MILDETITLNSGVKIPKFALGTWMIDDDQA
    AEAVRNAIKMGYRHIDTAQAYDNERGVGEG
    VRTAGIDRDKIFVTSKIAAEHKDYDVTKKS
    IDETLEKMGLDYIDMMLIHSPQPWKEVNQS
    DNRYLEGNLAAWRAMEDAVNEGKIRTIGVS
    NFKKADLENIIKNSDTVPAVDQVLAHIGHT
    PFNLLSFTHEHDIAVEAYSPVAHGAALDNP
    VIEKMAKKYNVSVPQLCIRYDWQIGMIVLP
    KTTNPEHMKENTEIDFEISEADMDLLRRVK
    PLDYGDFDIYPVYGGKM
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequence (37) or (38) (corresponding to SED ID NO: 28 or 30, respectively) encoding the glycerol-degrading enzyme (21) or (22), respectively into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (37) or (38) and encoding a protein having glycerol-degrading activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (37) or (38) under a stringent condition and encoding a protein having glycerol-degrading activity.
  • (37)
    ATGACAAATGTACCAACAGTAAAATTAAATAACGGAGTAGAAATGCCAACCCTTGGATTT
    GAAGTATTCCAAGTTCCAGACTTAAGCCAAGCTGAACAAGCAGTTACCGATGCTCTTGAA
    GTCGGCTATCGTTTAATCGATACTGCTGCTGCTTACCAAAATGAAGAAGCAGTTGGAAAG
    GCAATTAAGAATAGTAGTGTAAACCGTGAAGATGTCTTTGTAACTTCTAAGTTATGGGTG
    TCTGATTTTAACTATAAGCGGGCTAAAGCAGGGATTGACGCTTCACTGCAAAAACTTGGC
    CTTGATTACATGGATCTTTACCTTCTCCATCAACCATATGGCGATACAATGGGGGCTTGG
    CGAGCATTACAAGAAGCACAGAAAGAAGGTAAGATTCGCGCAATCGGTGTATCGAACTTC
    TACGCTGATCAACTAAAGGATCTTGAATTAACAATGCCTGTTAAGCCAGCGGTCAACCAA
    ATTGAAGTTAACCCTTGGTACCAGCAAGATCAAGAGGTTAAGTTTGCGCAAAGTGAAGAT
    ATTCGTGTTGAAGCATGGGCACCATTTGCGGAAGGTAAGCATGATATTTTTACCAACGAA
    ATAATTGCGGAAATTGCTGCCAAGTATGGCAAGAGCAATGGTCAAGTAATTCTTCGCTGG
    CTTTTACAACGGGGTATTACTGTCATTCCAAAGTCAGTCCACAAGAACCGGATGGAAGAA
    AATATCGATGTCTTTGATTTTGAACTTTCCAATGATGATATGAAAAAGATAGCTAGTCTT
    AACAAGAAGGAAAGCCAATTCTTTGACCACCGTGATCCGGTTACGATTGAACAAATCTTT
    GGCTCCAGCTTAAAGATGGTTCAAGATGACGAAAAATAA
    (38)
    ATGATTTTAGATGAGACAATTACTCTTAATAGTGGTGTGAAAATTCCAAAGTTTGCATTA
    GGAACCTGGATGATTGATGATGACCAAGCAGCCGAAGCAGTTCGGAATGCGATTAAGATG
    GGATATCGGCACATCGATACAGCTCAGGCTTATGATAATGAGCGGGGAGTCGGTGAAGGT
    GTACGAACAGCCGGTATTGATCGGGATAAAATCTTTGTTACTTCAAAGATCGCTGCTGAA
    CACAAAGATTATGATGTAACTAAAAAGTCGATTGACGAGACTCTTGAAAAGATGGGTCTT
    GATTATATCGACATGATGCTTATTCATAGTCCTCAACCATGGAAAGAAGTAAATCAATCT
    GATAATCGTTACCTTGAAGGAAATCTCGCTGCTTGGCGAGCCATGGAAGATGCCGTTAAC
    GAAGGTAAGATTCGAACAATTGGCGTTTCTAATTTCAAAAAAGCCGATCTTGAAAATATT
    ATTAAGAATAGCGATACCGTTCCCGCTGTTGATCAAGTTTTAGCTCATATTGGTCATACT
    CCATTCAATCTTTTATCATTTACTCATGAACATGACATTGCGGTTGAAGCATATTCACCA
    GTTGCTCACGGCGCTGCTTTAGACAACCCCGTAATTGAAAAGATGGCTAAAAAGTACAAC
    GTTTCAGTCCCACAATTGTGCATTCGGTATGATTGGCAAATAGGAATGATCGTCTTACCA
    AAGACTACTAATCCAGAACACATGAAGGAAAACACTGAAATTGATTTTGAAATTTCTGAA
    GCTGATATGGACCTATTGCGGCGAGTAAAGCCATTAGACTATGGCGATTTTGATATCTAC
    CCTGTTTACGGTGGAAAAATGTAA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having an aldehyde dehydrogenase depicted in any of the following amino acid sequences (23) to (25) (corresponding to SEQ ID NO: 31, 33 and 35, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (23) to (25). This aldehyde dehydrogenase is an aldehyde dehydrogenase (9) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping aldehyde dehydrogenase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (23)
    MPANNKKQVEKKELTAEEKKQNAQKLVDDL
    MTKSQAAFEKLRYYSQEQVDKICQAMALAA
    EEHHMDLAVDAANETGRGVAEDKAIKNIYA
    SEYIWNNIRHDKTVGIIEDNDEDQTIKIAD
    PLGVIAGIVPVTNPTSTTIFKSIISAKTRN
    TIIFSFHRQAMKSSIKTAKILQEAAEKAGA
    PKNMIQWLPESTRENTTALLQHPNTATILA
    TGGPSLVKAAYSSGNPALGVGPGNGPAYIE
    KTANIERSVYDIVLSKTFDNGMICATENSV
    VVDEEIYDKVKEEFQKWNCYFLKPNEIDKF
    TDGFIDPDRHQVRGPIAGRSANAIADMCGI
    KVPDNTKVIIAEYEGVGDKYPLSAEKLSPV
    LTMYKATSHENAFDICAQLLHYGGEGHTAA
    IHTLDDDLATKYGLEMRASRIIVNSPSGIG
    GIGNIYNNMTPSLTLGTGSYGSNSISHNVT
    DWDLLNIKTIAKRRENRQWVKIPPKVYFQR
    NSLKELQDIPNINRAFIVTGPGMSKRGYVQ
    RVIDQLRQRQNNTAFLVFDDVEEDPSTNTV
    EKGVAMMNDFKPDTIIALGGGSPMDAAKAM
    WMFYEHPETSWYGVMQKYLDIRKRAYQIKK
    PTKSQLIGIPTTSGTGSEVTPFAVITDSKT
    HVKYPLADYALTPNIAIVDSQFVETVPAKT
    TAWTGLDVLCHATESYVSVMATDYTRGWSL
    QTIKGVMENLPKSVQGDKLARRKMHDFSTM
    AGMAFGQAFLGINHSLAHKMGGAFGLPHGL
    LIAIAMPQVIRFNAKRPQKLALWPHYETYH
    ATKDYADIARFIGLKGNTDEELAEAYAKKV
    IELAHECGVKLSLKDNGVTREEFDKAVDDL
    ARLAYEDQCTTTNPVEPLVSQLKELLERCY
    DGTGVEEK
    (24)
    MAYQSINPFTNQVEKTFENTTDEELEQTLT
    TAHQLYLDWRKYNDLEERKRQILKLGQILR
    ERRVEYATVMSKEMGKLISEAEGEVDLCAS
    FCDYYAAHADEFLQPKIIATTSGRAKVLKQ
    SLGILVAVEPWNFPFYQIARVFIPNFIAGN
    PMILKDASNCPASAQAFNDAVKEAGAPAGS
    LTNLFLSYDQVNKAIADKRVAGVCLTGSER
    GGATVAKEAGANLKKSTLELGGNDAFIILD
    DADWDLVEKVAPAARLYNAGQVCTSSKRFI
    VLEKDYDRFLKMMKDAFSKVKMGDPLDPLT
    TLAPLSSKKAKEKLQQQVATAVENGAKVYY
    GNKPVDMEGQFFMPTILTDITPDNPIFDTE
    MFGPVASVYKVSSEEEAIELANNSSYGLGN
    TIFSNDSEHAERVAAKIETGMSWINAGWAS
    LPELPFGGVKNSGYGRELSSYGIDEFTNKH
    LIYEARQ
    (25)
    MQINDIESAVRKILAEELDNASSSSANVAA
    TTDNGHRGIFTNVNDAIAAAKAAQEIYRDK
    PIAVRQQVIDAIKEGFRPYIEKMAKDIKEE
    TGMGTVEAKIAKLNNALYNTPGPEILEPVV
    ENGDGGMVMYERLPYGVIGAVGPSTNPSET
    VIANAIMMLAGGNTLYFGAHPGAKNVTRWT
    IEKMNDFIADATGLHNLVVSIETPTIESVQ
    QMMKHPDIAMLAVTGGPAVVHQAMTSGKKA
    VGAGPGNPPAMVDATADIDLAAHNIITSAS
    FDNDILCTAEKEVVAESSIKDELIRKMQDE
    GAFVVNREQADKLADMCIQENGAPDRKFVG
    KDATYILDQANIPYTGHPVEIICELPKEHP
    LVMTEMLMPILPVVSCPTFDDVLKTAVEVE
    KGNHHTATIHSNNLKHINNAAHRMQCSIFV
    VNGPSYVGTGVADNGAHSGASALTIATPTG
    EGTCTARTFTRRVRLNSPQGFSVRNWY
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (39) to (41) (corresponding to SED ID NO: 32, 34 and 36, respectively) encoding the aldehyde dehydrogenases (23) to (25), respectively into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with any of the nucleotide sequences (39) to (41) and encoding a protein having aldehyde dehydrogenase activity; and a nucleotide sequence for achieving hybridization with any of the nucleotide sequences (39) to (41) under a stringent condition and encoding a protein having aldehyde dehydrogenase activity.
  • (39)
    ATGCCTGCTAACAACAAGAAACAAGTTGAAAAGAAAGAATTAACTGCTGAAGAAAAAAAG
    CAAAACGCCCAAAAGCTAGTTGACGATTTAATGACTAAGAGTCAAGCTGCTTTTGAAAAG
    TTACGTTACTATTCACAAGAACAAGTTGACAAGATTTGTCAGGCAATGGCTCTCGCTGCC
    GAAGAACACCACATGGACTTAGCTGTTGATGCTGCTAACGAAACTGGTCGTGGGGTTGCT
    GAAGATAAGGCTATCAAGAACATCTACGCAAGTGAATACATTTGGAACAACATCCGTCAC
    GATAAGACTGTTGGTATTATCGAAGACAATGATGAAGACCAAACTATCAAAATTGCTGAT
    CCACTTGGTGTCATTGCCGGAATTGTTCCAGTTACTAACCCTACTTCAACAACGATCTTC
    AAATCAATCATTAGTGCTAAGACACGGAATACAATCATCTTTTCTTTCCACCGTCAAGCA
    ATGAAGTCATCTATCAAGACTGCAAAGATTCTCCAAGAAGCTGCTGAAAAAGCCGGTGCG
    CCAAAGAACATGATTCAATGGCTCCCTGAAAGTACCCGCGAAAACACTACCGCATTACTC
    CAACACCCTAATACTGCTACTATTTTAGCAACCGGTGGTCCTTCATTAGTTAAGGCTGCC
    TACAGTTCTGGTAACCCTGCTCTTGGTGTTGGTCCTGGTAACGGTCCTGCTTACATCGAA
    AAAACTGCCAACATCGAACGTTCTGTTTACGACATCGTTCTTTCTAAGACATTCGATAAC
    GGTATGATTTGTGCCACTGAAAACTCAGTTGTTGTTGATGAAGAAATCTACGACAAGGTT
    AAAGAAGAATTCCAAAAGTGGAACTGTTACTTCTTGAAGCCAAACGAAATTGATAAATTT
    ACTGATGGCTTTATTGACCCAGATCGTCATCAAGTTCGTGGTCCAATCGCTGGTCGTTCA
    GCTAATGCTATTGCTGACATGTGTGGTATTAAAGTACCTGACAACACTAAGGTTATCATT
    GCTGAATACGAAGGTGTTGGTGACAAGTACCCACTTTCAGCTGAAAAGCTTTCACCAGTA
    TTAACAATGTACAAGGCAACCTCTCACGAAAATGCCTTTGATATCTGTGCTCAATTATTA
    CACTACGGTGGTGAAGGTCACACTGCTGCTATTCACACCCTTGATGATGATTTAGCTACT
    AAGTACGGTCTTGAAATGCGTGCTTCACGGATCATTGTTAACTCCCCATCTGGTATTGGT
    GGTATTGGTAACATCTACAACAACATGACTCCATCCCTTACTTTAGGTACTGGTTCATAC
    GGTACTAACTCAATTTCTCACAACGTTACTGATTGGGACCTCTTAAACATCAAAACAATT
    GCAAAGCGGCGTGAAAACCGTCAATGGGTTAAGATTCCCCCAAAAGTATACTTTCAACGC
    AACTCACTAAAAGAATTGCAAGATATTCCAAACATTAACCGGGCATTCATCGTTACTGGT
    CCTGGAATGAGCAAGCGTGGTTACGTTCAACGTGTTATCGATCAATTGCGTCAACGCCAA
    AACAACACTGCTTTCTTAGTATTTGATGACGTTGAAGAAGATCCATCAACAAACACTGTT
    GAAAAAGGTGTTGCCATGATGAATGACTTCAAACCTGATACAATTATTGCTCTTGGTGGT
    GGTTCACCAATGGATGCTGCTAAGGCTATGTGGATGTTCTATGAGCACCCAGAAACTTCA
    TGGTATGGGGTTATGCAAAAGTACCTTGATATTCGGAAGCGTGCTTACCAAATCAAGAAG
    CCTACTAAGTCTCAACTTATTGGTATCCCTACTACATCAGGTACTGGTTCAGAAGTTACT
    CCATTTGCGGTTATTACCGATTCAAAAACTCATGTTAAGTACCCACTTGCTGACTACGCC
    TTAACACCAAACATTGCAATCGTTGACTCACAATTCGTTGAAACTGTTCCAGCAAAAACT
    ACTGCTTGGACTGGACTAGATGTTTTATGTCACGCTACTGAATCATATGTTTCTGTTATG
    GCAACTGACTACACTCGTGGTTGGTCACTACAAACCATCAAGGGTGTTATGGAAAACCTT
    CCTAAGTCAGTTCAAGGTGATAAGTTAGCTCGTCGTAAGATGCACGACTTCTCAACAATG
    GCCGGGATGGCATTTGGTCAAGCCTTCTTAGGAATTAACCACTCCCTTGCCCACAAGATG
    GGTGGAGCATTCGGTCTTCCTCACGGTTTGCTTATCGCTATTGCAATGCCACAAGTAATT
    CGCTTTAACGCAAAACGTCCACAAAAGCTTGCTCTCTGGCCTCACTATGAGACTTACCAT
    GCAACTAAGGACTACGCTGACATTGCACGGTTCATTGGTTTGAAAGGCAACACTGATGAA
    GAATTAGCTGAAGCATATGCTAAGAAAGTTATCGAACTTGCTCACGAATGTGGTGTTAAG
    CTTAGTCTTAAGGACAATGGTGTTACACGTGAAGAATTTGATAAGGCGGTTGACGATCTT
    GCTCGCTTAGCTTACGAAGATCAATGTACTACTACTAACCCAGTTGAACCACTTGTTAGC
    CAACTCAAGGAATTACTTGAACGTTGCTACGATGGTACTGGCGTTGAAGAAAAATAA
    (40)
    ATGGCATATCAAAGTATCAATCCATTTACGAACCAAGTAGAAAAAACGTTTGAAAATACA
    ACTGATGAAGAATTAGAACAAACATTAACTAGGGCGCATCAATTATATTTAGATTGGCGG
    AAGTATAATGACCTTGAAGAACGGAAACGGCAAATTTTAAAGTTAGGTCAAATATTACGT
    GAACGGCGTGTTGAATATGCGACAGTTATGAGTAAGGAAATGGGAAAATTAATTAGCGAA
    GCAGAAGGCGAGGTTGACCTTTGTGCTTCTTTCTGTGATTATTATGCAGCCCATGCAGAT
    GAATTTCTGCAACCAAAAATTATTGCGACAACGAGTGGACGCGCCAAAGTTTTGAAGCAA
    TCATTAGGAATTTTAGTTGCAGTTGAACCTTGGAATTTCCCATTCTATCAAATTGCCCGG
    GTATTTATTCCCAACTTTATTGCAGGAAACCCCATGATCTTGAAGGATGCGTCGAATTGT
    CCAGCATCCGCCCAAGCATTTAACGATGCCGTTAAGGAAGCTGGTGCGCCAGCCGGCAGT
    TTAACTAATTTATTCCTTTCATATGACCAAGTAAATAAGGCAATTGCTGATAAGCGGGTA
    GCCGGCGTTTGTCTTACTGGTTCTGAACGTGGTGGTGCAACCGTTGCTAAAGAGGCTGGT
    GCTAATTTGAAGAAGAGCACTTTGGAACTTGGTGGTAATGATGCCTTTATTATCTTAGAC
    GATGCAGATTGGGATCTTGTCGAAAAAGTTGCCCCGGCAGCCCGTCTGTATAATGCTGGA
    CAAGTATGTACATCATCAAAACGTTTTATTGTCCTTGAAAAGGATTATGATCGTTTCTTA
    AAGATGATGAAAGATGCGTTCTCGAAAGTTAAAATGGGTGATCCCCTTGATCCATTAACA
    ACTCTGGCACCATTATCATCTAAGAAAGCAAAAGAAAAGCTCCAACAGCAAGTCGCAACA
    GCAGTAGAAAATGGGGCCAAAGTTTACTATGGTAATAAGCCGGTTGACATGGAAGGTCAA
    TTCTTTATGCCAACGATCTTAACTGATATCACTCCAGATAACCCAATATTTGATACGGAA
    ATGTTTGGGCCAGTGGCTTCGGTTTATAAGGTTAGTTCCGAAGAGGAAGCAATCGAACTG
    GCTAATAATTCAAGCTATGGGTTAGGAAACACTATCTTTAGCAATGATTCCGAACATGCG
    GAACGAGTAGCAGCGAAGATCGAAACTGGAATGAGTTGGATTAATGCCGGCTGGGCTTCA
    TTACCAGAATTACCATTTGGTGGTGTTAAGAATTCAGGTTACGGTCGTGAACTCAGCAGT
    TACGGAATTGATGAATTTACTAACAAACATCTAATTTACGAAGCACGACAATAA
    (41)
    ATGCAGATTAATGATATTGAAAGTGCTGTACGCAAAATTCTTGCCGAAGAACTAGATAAT
    GCCAGCTCTTCAAGTGCAAACGTTGCAGCTACTACTGATAATGGTCATCGCGGAATTTTC
    ACTAATGTCAATGATGCAATTGCTGCTGCAAAAGCTGCTCAAGAAATATATCGGGATAAG
    CCAATTGCTGTTCGCCAACAAGTGATTGATGCCATTAAGGAAGGATTCCGCCCATATATT
    GAAAAAATGGCTAAAGATATCAAAGAAGAAACAGGAATGGGAACAGTAGAGGCCAAAATT
    GCTAAGTTAAACAATGCCTTGTACAACACTCCTGGTCCCGAGATTCTTGAACCAGTTGTA
    GAAAACGGTGACGGTGGGATGGTTATGTATGAACGGTTACCATATGGTGTTATTGGTGCG
    GTTGGCCCAAGTACAAACCCTTCAGAAACTGTAATTGCTAATGCGATCATGATGCTTGCC
    GGTGGTAATACTCTTTACTTTGGTGCTCACCCTGGCGCAAAGAATGTTACTCGCTGGACA
    ATTGAAAAGATGAACGATTTTATTGCAGATGCAACAGGCGTTCATAATTTAGTTGTAAGT
    ATTGAAACACCAACAATTGAATCAGTTCAACAAATGATGAAGCACCCCGACATTGCAATG
    TTAGCAGTAACTGGTGGCCCAGCTGTTGTTCACCAAGCAATGACCAGTGGTAAGAAAGCG
    GTTGGTGCTGGTCCTGGTAATCCTCCTGCAATGGTTGATGCTACTGCTGATATTGATTTA
    GCTGCTCATAATATCATTACATCTGCTTCATTTGATAATGATATTTTATGTACTGCTGAA
    AAGGAAGTAGTTGCAGAAAGTAGCATTAAAGATGAATTAATTCGTAAGATGCAAGATGAA
    GGTGCCTTTGTAGTTAACCGTGAACAAGCCGATAAATTAGCTGATATGTGTATCCAAGAA
    AATGGTGCTCCTGATCGTAAATTTGTTGGTAAGGATGCAACTTATATCTTAGACCAAGCT
    AATATTCCTTACACAGGCCACCCAGTTGAAATTATTTGTGAACTTCCTAAGGAACATCCA
    TTAGTAATGACTGAAATGTTAATGCCAATTTTACCAGTTGTTTCTTGTCCAACATTTGAT
    GATGTTTTGAAGACTGCTGTTGAAGTTGAAAAAGGTAACCATCACACAGCTACTATTCAT
    TCCAATAACCTTAAGCATATTAATAATGCTGCTCACCGGATGCAATGTTCAATCTTTGTT
    GTTAATGGCCCATCCTATGTTGGTACAGGTGTTGCAGATAATGGAGCTCACTCAGGTGCT
    TCAGCATTAACAATTGCTACGCCAACTGGTGAAGGAACATGTACTGCACGAACATTTACT
    CGTCGGGTTCGTTTGAACTCACCACAAGGATTCTCAGTACGTAACTGGTATTAA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having a glycerate kinase depicted in the following amino acid sequence (26) (corresponding to SEQ ID NO: 37) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (26). This glycerate kinase is Glycerate kinase (10) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerate kinase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (26)
    MKFVIAPDSFKGGLTAKEAANVMAEGIKRV
    FPNAEYALVPMADGGEGTVQSLVDATNGQK
    MIAKVHNPLNKLVNAEYGILGDGETAVIEM
    AAASGLQFVNKETANPLITTTYGTGELIKD
    ALDHNIKKIIIGIGGSATVDGGAGMAQALG
    ARLLDADNHEIGLGGGELASLEQVDFGGLD
    PRLKNVDIQIASDVTNPLTGKNGAAPVFGP
    QKGADEEMVNILDKNLHHYARKIVAAGGPD
    VEQTAGAGAAGGLGAGLIAFTGATMKRGVE
    LVIEATQLQKKAVGADYVFTGEGGIDFQTK
    FGKTPYGVAKATKEVAPTAPVIVLAGNIGK
    GVNDLYSSTAIDAIFATPEGAKPLKTALAD
    APIDIAQTAENVARLIKVSHVSN
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequence (42) (corresponding to SED ID NO: 38) encoding the glycerate kinase (26) into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (42) and encoding a protein having glycerate kinase activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (42) under a stringent condition and encoding a protein having glycerate kinase activity.
  • (42)
    ATGAAATTTGTAATTGCTCCAGATTCATTTAAAGGCGGATTAACAGCAAAAGAAGCAGCA
    AATGTGATGGCAGAAGGAATCAAAAGAGTGTTTCCGAATGCCGAGTATGCTTTAGTTCCA
    ATGGCTGATGGAGGAGAGGGGACTGTTCAATCCTTAGTTGATGCGACTAACGGTCAAAAA
    ATGATTGCTAAAGTCCACAACCCATTAAATAAATTAGTTAATGCTGAGTACGGAATATTA
    GGTGATGGGGAAACGGCAGTGATTGAGATGGCGGCGGCAAGTGGCCTTCAATTTGTTAAT
    AAGGAGACTGCGAACCCGCTTATTACAACTACATATGGTACCGGCGAGTTAATTAAGGAT
    GCTCTTGACCATAACATTAAAAAAATAATTATTGGAATTGGTGGAAGTGCAACCGTTGAT
    GGCGGAGCGGGGATGGCCCAAGCACTTGGAGCACGTTTATTGGATGCTGATAATCATGAA
    ATTGGTTTAGGCGGTGGTGAGTTAGCAAGTTTAGAGCAAGTAGATTTTGGAGGATTAGAT
    CCTCGCTTAAAAAATGTAGATATTCAGATTGCATCAGACGTAACCAACCCATTAACAGGA
    AAAAATGGGGCAGCCCCAGTATTTGGCCCGCAAAAAGGAGCTGATGAAGAAATGGTGAAC
    ATCTTGGACAAAAATCTTCATCATTATGCCCGAAAAATAGTTGCAGCTGGTGGGCCAGAC
    GTTGAACAAACGGCAGGTGCAGGGGCAGCCGGTGGTTTAGGAGCCGGGTTGATAGCATTT
    ACCGGTGCGACAATGAAGCGAGGAGTAGAATTAGTGATTGAAGCAACTCAACTACAAAAA
    AAGGCAGTTGGCGCTGATTATGTTTTTACTGGTGAAGGAGGAATTGATTTCCAGACTAAA
    TTTGGTAAAACGCCATATGGAGTCGCTAAGGCAACTAAAGAGGTGGCTCCAACTGCTCCG
    GTAATTGTGTTGGCTGGAAATATTGGTAAAGGCGTAAATGATCTATATTCATCCACGGCC
    ATTGATGCAATTTTTGCAACTCCTGAAGGGGCTAAACCATTAAAAACAGCATTAGCAGAT
    GCACCTATTGATATTGCTCAAACAGCGGAAAACGTTGCACGTTTAATTAAAGTGAGTCAT
    GTTAGTAATTAA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having a glycerol kinase depicted in the following amino acid sequence (27) (corresponding to SEQ ID NO: 39) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (27). This glycerol kinase is GK (5) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol kinase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (27)
    LSEQQYIMAIDQGTTSSRAIIFDHDGNKVA
    ISQQEFPQYFPQPGWVEHDPLEIWDSVQSV
    ISNVMIKSQIKPYKIAAIGITNQRETTVIW
    DRHTGKPIYNAIVWQSKQTSDIAEQLIKDG
    YKDMIHQKTGLVIDSYFAATKIKWILDHVP
    GAREKAAKGDLMFGTIDTWLLWNLSGRRVH
    ATDVTNASRTMLFNIHTLDWDQDILDLLDI
    PQSLLPVVKPSSAIYGYTGDYHFYGVQIPI
    AGIAGDQQAALFGQAAYDKGSIKNTYGTGA
    FIVMNTGLKPTLSDNGLLTTIAYGLDGQTH
    YALEGSIFVAGSAVQWLRDGLKMFDKASES
    EQMAVDAKTTGGVYVVPAFTGLGAPYWDQE
    VRGAMFGLTRGTERGHIIRATLEAIAYQTK
    DVVDTMVKDTQLPLTALTVNGGASRNNFMM
    QFQADILQTPIKRAAMEETTALGAAFLAGL
    AVDFWEDQDELRKLSRIGDQFDPQMDPQKA
    ADLYRGWQRAIAAAQFYGKD
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequence (43) (corresponding to SED ID NO: 40) encoding the glycerol kinase (27) into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (43) and encoding a protein having glycerol kinase activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (43) under a stringent condition and encoding a protein having glycerol kinase activity.
  • (43)
    TTGAGTGAACAACAATATATCATGGCGATTGACCAGGGAACGACGAGCTCACGGGCGATT
    ATCTTTGACCATGACGGAAATAAGGTTGCGATCAGTCAGCAGGAATTTCCCCAATACTTC
    CCGCAGCCGGGGTGGGTTGAACATGATCCTCTAGAGATTTGGGATAGCGTTCAATCAGTG
    ATTTCAAATGTAATGATTAAGTCCCAGATCAAGCCCTATAAGATTGCGGCAATTGGGATT
    ACTAACCAACGGGAGACGACGGTTATTTGGGATCGCCATACCGGTAAGCCGATTTATAAC
    GCAATTGTCTGGCAATCGAAGCAAACGAGCGACATCGCCGAACAATTGATTAAAGATGGT
    TATAAGGATATGATCCACCAGAAGACTGGCTTGGTGATTGATTCGTATTTCGCGGCCACT
    AAGATCAAGTGGATCCTTGACCATGTTCCTGGTGCCCGGGAAAAAGCAGCAAAGGGAGAC
    TTGATGTTTGGGACTATCGATACTTGGTTACTATGGAATTTATCGGGACGGCGGGTCCAC
    GCAACGGATGTGACCAATGCCAGCCGGACGATGCTTTTTAATATCCATACCCTCGACTGG
    GATCAAGATATCCTTGACCTGCTTGATATTCCCCAGTCGCTTTTGCCAGTAGTAAAGCCA
    AGTTCAGCCATTTACGGTTATACTGGCGACTACCACTTCTATGGGGTGCAGATTCCAATT
    GCCGGGATTGCAGGTGACCAACAAGCAGCCCTCTTTGGTCAAGCAGCCTATGATAAAGGT
    TCAATCAAGAACACCTATGGGACTGGAGCCTTCATCGTCATGAATACGGGACTAAAACCC
    ACGCTTTCGGATAACGGCTTGTTGACGACGATTGCGTATGGCCTGGACGGGCAAACTCAT
    TACGCGCTTGAAGGAAGTATCTTTGTGGCCGGTTCTGCCGTTCAATGGTTGCGGGATGGT
    CTCAAGATGTTTGATAAGGCAAGCGAGTCCGAACAAATGGCTGTCGATGCCAAGACAACT
    GGCGGCGTTTATGTCGTCCCCGCCTTTACAGGATTAGGCGCACCGTACTGGGATCAAGAA
    GTGCGGGGCGCAATGTTTGGCCTTACCCGTGGAACTGAACGGGGACATATCATCCGTGCA
    ACTTTGGAAGCCATTGCCTACCAGACCAAAGATGTTGTCGATACGATGGTCAAGGACACC
    CAATTACCACTAACAGCACTAACGGTTAACGGGGGCGCTTCACGGAACAACTTCATGATG
    CAGTTCCAGGCCGATATCTTACAAACGCCAATCAAGCGGGCAGCAATGGAAGAGACAACC
    GCGCTGGGAGCAGCCTTTCTCGCTGGATTGGCCGTTGATTTCTGGGAAGACCAGGATGAG
    TTACGGAAGCTATCACGGATTGGCGACCAGTTTGATCCACAAATGGATCCGCAAAAGGCA
    GCTGACTTGTATCGGGGATGGCAACGGGCCATTGCAGCTGCGCAGTTTTATGGCAAAGAT
    TAA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having a glycerol-3-phosphate dehydrogenase depicted in the following amino acid sequence (28) (corresponding to SEQ ID NO: 41) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (28). This glycerol-3-phosphate dehydrogenase is GPD (6) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping glycerol-3-phosphate dehydrogenase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (28)
    MAEKIAVLGAGSWGSVLANMLTENGHDVTL
    WSRNEEQVKQLNTEHTNPRYMKDFVYSTNL
    TATTDMKKAVKGASVVLIVIPTKGLREVAK
    QLNAILTELHQKPLVIHATKGLEQNTYKRP
    SEMLSEDISPENRQAIVVLSGPSHAEDVAI
    KDMTAVTAACEDLASAKKAQKLFSNSYFRV
    YTNDDVIGAEFGAALKNIIAIGAGAIQGLG
    YHDNARAALITRGLAEIRRLGVAFGANPMT
    FIGLSGVGDLVVTATSKNSRNWRAGYQLGQ
    GKKLQDVIDNMGMVIEGVYTTKAAYELSRK
    RQVQMPITEALYRVLYEGEDIKTAISQLMD
    RDLTSENE
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequence (44) (corresponding to SED ID NO: 42) encoding the glycerol-3-phosphate dehydrogenase (28) into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with the nucleotide sequence (44) and encoding a protein having glycerol-3-phosphate dehydrogenase activity; and a nucleotide sequence for achieving hybridization with the nucleotide sequence (44) under a stringent condition and encoding a protein having glycerol-3-phosphate dehydrogenase activity.
  • (44)
    ATGGCAGAAAAAATTGCTGTTTTAGGTGCTGGTTCGTGGGGCAGTGTTTTAGCAAACATG
    CTTACAGAAAATGGCCACGATGTAACATTATGGTCTCGTAATGAGGAACAAGTTAAGCAA
    TTAAATACTGAACATACAAATCCTCGCTATATGAAAGATTTTGTTTATTCTACTAACTTA
    ACAGCAACAACGGACATGAAAAAAGCTGTTAAGGGTGCCAGTGTGGTCCTGATTGTAATT
    CCAACAAAGGGTCTTCGTGAAGTTGCTAAGCAATTAAATGCAATTTTGACTGAATTACAT
    CAAAAACCGCTAGTTATTCACGCAACGAAAGGCTTAGAACAAAATACTTATAAGCGGCCA
    TCGGAAATGCTTAGCGAAGATATTTCTCCTGAAAACCGTCAGGCAATTGTTGTTTTATCA
    GGTCCGAGTCATGCTGAAGATGTGGCGATTAAAGATATGACAGCTGTAACCGCAGCTTGT
    GAGGACCTGGCCAGTGCTAAAAAGGCGCAGAAGTTATTTAGTAATTCTTATTTCCGTGTG
    TACACTAATGACGATGTAATTGGTGCCGAATTTGGCGCAGCCTTAAAGAACATTATTGCA
    ATTGGTGCTGGAGCTATTCAGGGACTTGGTTATCATGATAATGCTCGGGCAGCGTTAATT
    ACTCGTGGACTTGCAGAAATTCGCCGATTGGGAGTTGCTTTTGGTGCCAACCCGATGACT
    TTTATTGGTCTTTCTGGGGTTGGTGACCTTGTTGTTACTGCTACCAGTAAAAATTCTCGA
    AATTGGCGTGCTGGCTATCAATTGGGGCAAGGAAAAAAGCTTCAAGATGTAATTGATAAT
    ATGGGAATGGTTATCGAAGGTGTCTATACTACCAAAGCCGCTTATGAATTAAGTCGTAAA
    CGACAAGTACAGATGCCAATTACCGAAGCTCTTTACCGTGTTTTGTATGAAGGCGAAGAT
    ATTAAAACTGCAATTTCTCAATTAATGGACCGAGATCTTACTTCAGAAAACGAATAA
  • Also, it is preferable that the lactic acid bacterium of the invention is one having triosephosphate isomerase depicted in any of the following amino acid sequences (29) to (32) (corresponding to SEQ ID NO: 43, 45 and 47, respectively) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (29) to (32). This triosephosphate isomerase is Triosephosphate isomerase (15) in FIG. 1 and is able to efficiently metabolize glycerol produced by the action of the lipases of the invention. The “amino acid sequence having deletion, substitution, inversion, addition or insertion of one or more amino acids” as referred to herein means a sequence equivalent to the original sequence thereof and refers to a sequence still keeping triosephosphate isomerase activity. Examples of such an amino acid sequence include those exhibiting homology of 80% or more.
  • (29)
    MRKPFIAANWKMHKNVQESVEFVDAIKGKL
    PDPQEVEVGIAAQAFALPSMVQAADDSGLK
    IIAQNAAAEYSGAFTGEISLRGLADAGVSY
    VMLGHIERRHLFHEDNELVNRKVLAALQMG
    VTPIICTDETMVQKEVNGEIHYVFQQLMSV
    LRGVSLDQIKNVVVSYEPSWAVGYGQHANP
    VLAEEGCRQIRRTIADNYTYEIADKIRILY
    GGSVNPDNIGMIMNKPDVDGVLIGRASLDV
    DNFLRMVNYLKNDQEK
    (30)
    MRKPFIIANWKMNKNVHESVAFVKAIKEKL
    PADKEIGIAAQAVSLYNMKKVASSSNLQII
    AQNASAELEGPYTGEISMRSLADAGVTYVM
    LGHLERRRLFNESNDSINQKVLAALNAGII
    PIICTDEEMVQTEVNGQIHYVFRQLKSVLK
    GVPANKLSQIVISYEPSWAVGSTHQANPDI
    AEEGCQAIRQSLVEMYGNEIGEQVRILYGG
    SVNPENIGQIMSKPNVDGALIGRASLEIES
    FLQMINYIELASKQKLQVI
    (31)
    MRVPIIAGNWKMHKDVQEAVSFIEKVKNQL
    PPADQLETAIAAPTLCLVPMVKAAEESPLK
    IMAENCYYKNEGAYTGETSPYALYQAGIHH
    VILGHSERRTYFNETDELINKKVKAALVNG
    LCPIVCCDDTMRRRVAGKKVHWVVSRILAD
    LHGLTNDEICHVTVAYEPSWAIGTGESADP
    EQAAEGCYLIRQTISDMYGDEVANNVRILY
    GGSVTTSNINALMAKNDIDGVLVGAASLNP
    ETFLQLVHH
  • It is also possible to obtain a lactic acid bacterium with high glycerol-degrading properties by incorporating the following nucleotide sequences (45) to (47) (corresponding to SED ID NO: 44, 46 and 48, respectively) encoding the triosephosphate isomerases (29) to (31), respectively into other lactic acid bacterium or the like by a genetic engineering technique. The invention also includes a nucleotide sequence having homology of 80% or more with any of the nucleotide sequences (45) to (47) and encoding a protein having triosephosphate isomerase activity; and a nucleotide sequence for achieving hybridization with any of the nucleotide sequences (45) to (47) under a stringent condition and encoding a protein having triosephosphate isomerase activity.
  • (45)
    ATGCGCAAACCCTTTATTGCTGCTAATTGGAAGATGCATAAGAATGTCCAAGAATCGGTT
    GAATTTGTGGATGCAATTAAAGGAAAGCTACCAGATCCGCAAGAAGTTGAAGTCGGAATT
    GCAGCCCAAGCTTTTGCATTACCCAGTATGGTTCAAGCCGCTGATGATTCAGGATTAAAG
    ATAATCGCGCAAAACGCGGCGGCTGAATATTCGGGAGCTTTCACTGGTGAAATTAGCTTA
    CGAGGTTTAGCTGACGCCGGTGTTTCATATGTAATGTTAGGACATATTGAACGGCGCCAT
    TTATTCCACGAGGATAATGAGTTGGTTAATCGGAAAGTGTTGGCAGCCCTTCAAATGGGA
    GTTACCCCGATAATTTGTACGGATGAAACGATGGTCCAGAAAGAAGTTAATGGTGAAATT
    CACTACGTTTTCCAGCAATTGATGAGCGTATTGAGGGGCGTTTCTCTTGATCAAATTAAA
    AATGTAGTTGTTTCCTATGAACCAAGTTGGGCAGTTGGATATGGTCAGCATGCTAATCCA
    GTTCTTGCTGAAGAAGGATGCCGTCAAATTCGGCGAACGATTGCTGATAACTACACTTAT
    GAGATTGCTGATAAGATCAGGATTCTTTATGGGGGCAGTGTCAATCCAGATAATATCGGA
    ATGATTATGAACAAGCCAGATGTAGATGGGGTATTAATCGGTCGGGCAAGTTTAGATGTT
    GATAATTTTTTGCGAATGGTCAATTATTTAAAAAATGATCAAGAAAAATAA
    (46)
    ATGCGCAAACCGTTTATTATTGCGAACTGGAAAATGAATAAAAACGTTCATGAATCTGTT
    GCGTTTGTTAAAGCAATTAAAGAAAAGCTCCCGGCAGATAAAGAAATTGGGATCGCCGCG
    CAAGCAGTTTCGCTATATAACATGAAAAAAGTGGCGAGCTCTTCCAACTTACAAATTATT
    GCTCAAAATGCATCTGCTGAGTTAGAGGGACCATATACTGGAGAAATTAGCATGCGAAGT
    TTAGCAGATGCGGGCGTGACATACGTGATGCTAGGCCATTTAGAGCGCCGACGCCTTTTT
    AACGAGAGTAATGATTCAATTAATCAAAAAGTTTTAGCAGCCCTCAATGCTGGTATTATT
    CCAATCATTTGTACGGATGAAGAGATGGTCCAAACAGAAGTTAACGGACAAATTCATTAT
    GTATTTCGCCAACTAAAAAGCGTCCTTAAAGGGGTACCAGCTAATAAACTATCACAGATT
    GTTATTTCGTATGAACCAAGTTGGGCCGTTGGGAGCACGCATCAAGCAAATCCAGACATT
    GCGGAAGAGGGATGTCAGGCAATTCGTCAAAGCCTGGTTGAAATGTATGGTAATGAGATT
    GGCGAGCAAGTCCGAATACTCTATGGTGGCAGCGTTAATCCCGAGAACATTGGTCAAATT
    ATGAGTAAACCAAATGTTGATGGGGCGCTAATCGGTCGCGCAAGTCTCGAGATTGAAAGT
    TTCTTACAAATGATTAATTATATCGAATTAGCGAGCAAGCAGAAGTTACAGGTAATTTAG
    (47)
    ATGAGAGTACCGATTATTGCTGGTAATTGGAAAATGCATAAGGATGTACAAGAAGCTGTC
    TCTTTTATCGAAAAAGTAAAAAATCAGCTTCCGCCTGCCGACCAACTTGAAACAGCAATT
    GCTGCTCCTACTCTTTGTTTAGTACCAATGGTTAAAGCAGCTGAAGAATCCCCGTTAAAA
    ATAATGGCAGAAAACTGCTACTATAAGAATGAGGGAGCTTATACTGGTGAAACAAGTCCA
    TATGCTTTATACCAAGCAGGAATCCATCATGTGATTTTAGGCCATTCTGAACGCCGAACT
    TACTTTAATGAAACTGATGAATTAATTAATAAAAAAGTGAAGGCAGCATTAGTAAATGGG
    TTATGTCCGATTGTTTGTTGTGATGATACTATGCGTCGACGAGTTGCTGGAAAGAAAGTT
    CATTGGGTGGTGAGCCGAATTCTCGCTGACCTTCATGGATTGACCAATGACGAAATTTGT
    CATGTTACGGTTGCTTATGAACCAAGTTGGGCGATTGGAACAGGCGAGAGTGCTGATCCA
    GAACAAGCGGCGGAAGGTTGTTACCTTATTCGGCAAACGATTAGTGATATGTATGGCGAT
    GAAGTTGCAAATAACGTTCGAATTCTCTATGGCGGAAGTGTGACAACTTCTAATATCAAT
    GCACTAATGGCAAAAAATGATATTGATGGTGTTTTAGTCGGAGCGGCGAGCTTAAATCCA
    GAAACATTTTTACAATTAGTTCACCATTAG
  • The above-described lactic acid bacteria of the invention can be obtained by subjecting a microorganism belonging to the species Lactobacillus reuteri to genetic analysis by the ordinary method. For example, with respect to many microorganisms belonging to the species Lactobacillus reuteri, it is possible to obtain the targeted lactic acid bacterium of the invention by examining whether or not there are nucleotide sequences having high homology with the following genes (13) to (15) (corresponding to SEQ ID NO: 2, and 6, respectively) encoding the lipases (1) to (3), respectively.
  • (13)
    ATGGTGAAATTGATGACAATACACGAATTAGCAAATAACCCAACGTTAAGCGGCCAAGTA
    CGCTTGATTGAAAATATTGTTTATGGTGCGATGGATGGTGAGGCATTACATATGTCGATC
    TTAGCACCGTGGACGCAACGTTTCCCGAAACAATATCAAACTGAACCTCGACCATTGATT
    GTCTTTGTTCAAGGAAGCTCGTGGCGAACACCAAAAATGGGAGAAGAAATTCCACAACTG
    GTTCAATTTGTTCGGGCCGGTTATATTGTAGCGACTGTTCAACACCGTAGTTCAATTGAT
    AGCCACCCATTTCCTGCCTTTTTGCAAGATGTTAAGACTGCCATTCGTTTCTTACGGGCC
    AATGCGCAAAAATATGCAATTGATCCGCAACAGGTTGCAATTTGGGGGACTTCCTCTGGA
    GCCAATGCGGCAATGCTAGTCGGCTTAACGGGTGATGATCCGCGCTATAAAGTTGACCTT
    TATCAAGACGAATCGGATGCAGTAGATGCTGTGGTTAGTTGTTTTGCCCCAATGGACGTG
    GAGAAGACGTTTGAGTATGATGCTAATGTTCCAGGAAATAAGTTACTGCAATATTGCTTA
    TTAGGGCCTGATGTATCAAAGTGGCCAGAAATTGAAAAGCAAATGAGTCCCTTATATCAA
    GTCAAAGATGGGCAAAACTACCCACCATTCTTATTGTTCCACGGAGATGCTGATAAAGTT
    GTTCCATATGAACAGATGGAAAAAATGTATATGCGGTTGAAGGATAATGGAAATTCTGTT
    GAAGCGTACCGGGTTAAGGGTGCGAACCATGAACGAGATTTCTGGAGTCCAACAATTTAT
    AATATTGTGCAGAAGTTTCTTGGCGATCAATTTAAATAA
    (14)
    TTGATTTATGTTTTAAAAGATTTATGTAATACTATTGCTGAAGTCTATGGCAAAAGTATT
    TTAAAAGGAGTTTTTATCATGAAACATACGCTTAAAGTTGATCAAGTACGTGACGGTTTA
    TGGCTAGATTCAGATATTACGTATACGCAAGTTCCTGGATGGCTTGGTAATACAACGCGA
    GATTTGAAGCTTTCAGTCATTCGACATTTTCAAACTAATGATGATACACGTTATCCAGTA
    ATTTTTTGGTTTGCTGGTGGCGGCTGGATGGATACTGACCACAATGTTCATCTGCCGAAT
    TTGGTTGATTTTGCTCGGCATGGTTACATTGTTGTCGGCGTCGAATATCGTGATAGCAAC
    AAAGTTCAGTTTCCTGGGCAATTAGAAGATGCTAAGGCTGCTATTCGTTATATGAGAGCT
    AATGCCAAGCGCTTCCAAGCTGATCCTAATCGGTTTATTGTGATGGGAGAATCGGCCGGT
    GGACATATGGCAAGTATGCTAGGTGTTACTAACGGCCTTAACCAATTTGACAAAGGTGCT
    AATTTAGATTACTCCAGTGATGTTCAAGTAGCAGTTCCTTTTTATGGTGTGGTTGATCCC
    TTAACCGCTAAAACAGGAAGTGCATCAAACGATTTTGATTTTGTTTACCGTAACTTGCTT
    GGTGCTGAGCCTGAAAACGCTCCTGAGCTTGATTCTGCCGCAAATCCCCTCACCTATGTA
    AATTCTAATTCTACGCCCTTTCTTATCTTTCATGGGACAGAAGATGTCGTTGTTCCAATT
    AAAGATAGTGAAAAGCTTTATGATGCATTAGTTGAAAACAACGTTCCTGCTGAATTATAC
    GAAATCGAAGGCGCAAGTCACATGGATGTGAAATTCCTTCAACCACAGGTATTTAAGATT
    GTGATGGACTTTTTAGATAAGTATTTAACTCGGTCATAG
    (15)
    ATGGAAATTAAAAGTGTTAACTTAGATCAACCATATTCGTCTCTAGATATTTATCATAGT
    AATACTGATAAAGCTTTGCCCGGTCTTGTTATTTTACCAGGAGGCAGTTATAACCAGATC
    ATGGAGCGAGATTCTGAACGGGTGGCATTAACGTTTGCAACCCATGCATGGCAAACATTT
    GTTGTACGATATCCGGTAGTTGAGCATAAGAATTATGAAGAAGCCAAAATAGCGGTTCAC
    CAAGCATTTGAATATATCGTCAACCATGCAGCTGAATTAGATGTTGACGCTGATCGGTTG
    GGGATTATTGGCTTTTCTGCAGGAGGCCAAATTGCCGCTGCATATAGTAATGAAAAACTA
    ACACACGCTAGATTCGCCGCATTAGGATATCCTGTTATTCAACCCTTGATTGATGAACGT
    ATGGGGGTTACAACAGAGAATGTAGCGAAATTAGTAAATCCGCAAACACCACCAACCTTT
    ATGTGGGGATCGGCAAAAGATGAACTGACTCCCTTTGTTGATCACCTTCAAGTATATGCA
    GATGCGTTAATTAAGAATGATATTCCATATGAATTACATGAGTTTGGCACTGGGGGACAT
    GGAATCGCGTTAGCTAACGAATATACTGGTATTGTTAATAATGATCGGGTAGATAATCAT
    ATGGGAAAGTGGTTCCCGCTATTTCTTGAGTGGTTAACTGAACTGAATTTAATTTAG
  • Examples of the “stringent condition” as referred to in the invention include a condition under which hybridization is carried out by preserving in a solution containing 6×SSC (composition of 1×SSC: 0.15 M of NaCl, 0.015 M of sodium citrate, pH 7.0), 0.5% SDS, 5×Denhardt and 100 μg/mL of thermally denatured herring sperm DNA together with a probe at a temperature of from 50 to 65° C. overnight.
  • Furthermore, the lactic acid bacterium of the invention having the transport gene (4) and the lactic acid bacteria of the invention having each of the genes (9) to (11) encoding a subunit of glycerol-degrading enzyme, the genes (32) to (38) encoding a glycerol-degrading enzyme, the genes (39) to (41) encoding an aldehyde dehydrogenase, the gene (42) encoding a glycerate kinase, the gene (43) encoding a glycerol kinase, the gene (44) encoding a glycerol-3-phosphate dehydrogenase, the genes (45) to (47) encoding triosephosphate isomerase and the gene (12) encoding an enteroadherent protein can also be obtained in the same manner as described above.
  • Representative examples of the lactic acid bacterium of the invention include Lactobacillus reuteri JCM1112T which is a standard strain of RIKEN, Japan.
  • The anti-obesity agent of the invention is prepared by processing the foregoing lactic acid bacterium of the invention into a live bacterial agent which can be orally administered and made to arrive at the intestinal tract in a live state as it is. The formulation is not particularly limited and may be, for example, a solid such as a powder, a granule, a tablet and a capsule, a semi-solid such as a jelly and a paste or a liquid such as a suspension and a syrup. These respective formulations can be produced by a known method in the pharmaceutical field.
  • The lactic acid bacterium of the invention which is blended in the foregoing anti-obesity agent can be cultured by applying a known culture method of lactic acid bacteria. With respect to this culture method, a culture obtained by liquid culturing the lactic acid bacterium of the invention by the ordinary method may be utilized as it is; bacterial cells collected from this culture by means of centrifugation or the like may be used; or a powder obtained by freeze-drying a culture may be used.
  • As a general production method of the anti-obesity agent of the invention which is a solid, there is exemplified a method in which the lactic acid bacterium of the invention is blended together with a carrier such as water, starch, microcrystalline cellulose, wheat flour and sugar and processed into a desired form. The foregoing carrier is also known and can be properly chosen and used in conformity with the use form. More specifically, powder may be prepared by freeze-drying a bacterial cell of the lactic acid bacterium of the invention as obtained by culturing by the ordinary method to form a powder and mixing it with sugar. Also, a tablet can be obtained by mixing a bacterial cell of the lactic acid bacterium of the invention together with an adequate carrier for tablet and subjecting to tablet making by the ordinary method. Furthermore, a wet bacterial cell of the lactic acid bacterium of the invention may be suspended in a syrup to form a syrup formulation. In preparing the anti-obesity agent of the invention, other components, for example, other microorganisms and active ingredients, sweeteners, flavors and coloring agents may be contained as the need arises.
  • The dose of the thus obtained anti-obesity agent can be properly determined while taking into consideration the physical state of a subject, for example, state of health, weight, age, medical history and other components to be used. In general, it is from about 108 to 109 CFU/day per an adult in terms of a bacterial number of the lactic acid bacterium of the invention.
  • Also, in order to prepare an anti-obesity food and drink by using the lactic acid bacterium of the invention, an orally ingestible fermented food may be prepared by utilizing a conventionally known culture method of lactic acid bacteria. Specifically, fermented milk such as yogurt, lactic acid bacteria beverage and fermented sausage can be prepared, and the production of such a food can be achieved by processing apart or the whole of used lactic acid bacteria into the lactic acid bacterium of the invention. Also, the lactic acid bacterium of the invention can be processed into a form containing a larger amount thereof to prepare a healthy food or functional good. In preparing this anti-obesity food, needless to say, other lactic acid bacteria may be contained instead of single use of the lactic acid bacterium of the invention, and food additives or seasonings or the like may be added.
  • The lactic acid bacterium of the invention shows a significant body weight gain-inhibiting effect (slimming effect) as described later in Examples, and the reasons for this are thought as follows.
  • That is, as illustrated in FIG. 1, the lactic acid bacterium of the invention degrades a fat in a digestive tract into glycerol and a fatty acid by the action of three lipases (lipases (1) to (3)). The degraded glycerol is then taken into a bacterial cell by a transporter (PduF; encoded by the nucleotide (4)) of the lactic acid bacterium and metabolized by a glycerol-degrading enzyme gene in the bacterial cell (PduCDE; composed of the subunits (5) to (7)) to produce reuterin, or converted into an energy source of the bacterium per se.
  • On the other hand, the fatty acid is utilized as a bacterial cell component of the present bacterium but not absorbed in a living body. Furthermore, the peptide (8) has such a function to fix the lactic acid bacterium of the invention to the intestinal tract of a human being or a mammal and enables the lactic acid bacterium of the invention to stably exist in the intestinal tract for a fixed period of time.
  • As has been described previously, since the lactic acid bacterium of the invention stably exists in the intestinal tract, positively degrades a fat and utilizes its metabolites or further metabolizes them, it inhibits the absorption of a lipid from the intestinal tract into the body and even when a normal meal is ingested, is able to prevent obesity from occurring and bring maintenance and improvement of a slimming effect.
  • Also, an embodiment of the invention includes the use of the lactic acid bacterium of the invention for the prevention or therapy of obesity and further includes the use of the lactic acid bacterium of the invention for the production of an anti-obesity agent.
  • Moreover, another embodiment of the invention includes a method for therapy of obesity, which is characterized by administering a patient suffering from obesity with the lactic acid bacterium of the invention and also a method for therapy of obesity, which is characterized by administering the anti-obesity agent of the invention.
    • EXAMPLES
  • The invention is hereunder described in more detail with reference to the following Examples, but it should be construed that the invention is not limited to these Examples at all.
    • Example 1 Anti-obesity Effect Test of L. reuteri JCM1112T
  • An anti-obesity effect of L. reuteri JCM1112T was examined by the following materials and method.
    • Materials and Test Method: (1) Experimental Animal:
  • Wistar rats of SPF grade (males of 8-week-old; Japan SLC, Inc.) having a body weight of from about 180 to 200 g were used, an acclimatization period of 7 days from the day for the sending in a laboratory was provided, and the experiment was then started. The breeding circumstance was set up at a temperature of 22±1° C., a humidity of 55±5% and a lighting time of 12 hours (from 8:00 to 20:00); and the rats were caged individually and provided with free access to sterile distilled water through a watering bottle and a radiation-sterilized solid diet* for rat (CE-2, CLEA Japan, Inc.) by a feeder, respectively. All of the breeding instruments to be used were ones sterilized by a high-pressure steam sterilizer.
  • *: Use for breeding and propagation (crude fat: 4.6%)
    • (2) Preparation of Test Bacterial Solution:
  • As test bacteria, L. reuteri JCM1112T (a standard strain of RIKEN, Japan, which was received from the same) and L. rhamnosus ATCC53103 (GG strain) were used. These test bacteria were inoculated in an MRS liquid medium (Oxid) and cultured at 37° C. overnight to prepare pre-culture solutions. An MRS liquid medium was newly added such that the concentration of this pre-culture solution was 1% and cultured at 37° C. for 18 hours to prepare a test bacterial solution.
    • (3) Administration of Test Bacterial Solution:
  • The foregoing experimental animals were divided into two test bacterial groups and a control group (five animals per group), and the foregoing test bacterial solutions were orally administered in the test bacterial groups respectively. The test bacterial solution was forcibly administered via probe. The bacterial solutions were prepared at the time of use, and the bacterial dose was set up at 109 CFU per rat. Also, the control group was administered with the same volume of PBS.
    • (4) Measurement of Body Weight and General Observation of Symptoms:
  • The body weight of the experimental animal was measured every day by a scale. General observation of symptoms was made every day. The symptoms were recorded for every individual, and symptom items at which a remarkable change was observed were expressed in terms of number of the animals.
    • (5) Results:
  • FIG. 2 shows the body weight gain with time in the rats of the groups administered either of the foregoing Lactobacillus bacteria and the control group. As is clear from this drawing, in the L. reuteri JCM1112T group, the body weight gain was significantly inhibited as compared with the control group, and the degree of the inhibition was larger than that in the L. rhamnosus ATCC53103 group.
  • As well as favorable progress of the body weight gain, a medical examination of the rats of each group confirmed that the state of health of the animals was good.
    • Example 2 Genome Analysis of L. reuteri JCM1112T
  • DNA was obtained from L. reuteri JCM1112T by using the following chemicals in the following method, thereby achieving genome analysis.
    • Method for Obtaining DNA: (Chemicals)
      • Nuclei Lysis solution (Wizard genome DNA purification kit; manufactured by Promega)
      • Physiological saline
      • 50 mM EDTA (pH: 7.0)
      • 50 mg/mL lysozyme solution (prepared on the day by dissolving a prescribed amount of lysozyme in a TE buffer solution, 0.25 M Tris-HCl (pH 8.0) or 10 mM Tris-HCl (pH 8.0)/10 mM EDTA/0.5% SDS)
      • 2 mg/mL EDTA
      • Phenol/chloroform/isoamyl alcohol (25:24:1) mixed solution (hereinafter abbreviated as “PCI”)
      • Chloroform/isoamyl alcohol (24:1) mixed solution (hereinafter abbreviated as “CIA”)
      • 99% Ethanol
      • 70% Ethanol
      • TE buffer solution
    (Method)
  • 1. L. reuteri JCM1112T is cultured at 37° C. for 24 hours under static conditions, and the obtained culture solution (50 mL) is centrifuged at 3,500 r.p.m. for 15 minutes and then suspended in physiological saline.
    2. The suspension as obtained in 1 is centrifuged at 3,500 r.p.m. for 15 minutes, a supernatant is removed, and the residue is suspended in 5 mL of 50 mM EDTA.
    3. To the suspension as obtained in 2, 200 μL of a 50 mg/mL lysozyme solution is added and incubated at 37° C. for 60 minutes. (On that occasion, in the case where an air incubator is used, the incubation time is set up at from 2 to 3 hours.)
    4. After the incubation, the resultant is centrifuged at 3,500 r.p.m. for 15 hours, and a supernatant is removed.
    5. To the precipitate as obtained in 4, 5 mL of a Nuclei Lysis solution is added and incubated at 80° C. for 10 minutes.
    6. 1 μL of 2 mg/mL RNase A is added and incubated at 37° C. for 45 minutes.
    7. 10 mL of PCI is added and mixed, and the mixture is centrifuged at 3,500 r.p.m. for 15 minutes.
    8. A supernatant is transferred into a new tube, and 10 mL of PCI is further added and mixed.
    9. The same operations are repeated 3 times in total. (The operations are carried out until no protein layer is identified.)
    10. 10 mL of CIA is added and gently mixed, and the mixture is then centrifuged at 3,500 r.p.m for 15 minutes (removal of phenol).
    11. A supernatant is transferred into a new tube, followed by precipitation with ethanol.
    12. A precipitate as obtained in 11 is dissolved in 1 mL of a TE buffer solution to obtain a lactic acid bacterium DNA.
    • Genome Analysis Method:
  • The DNA thus obtained was subjected to structural gene prediction and annotation. The structural gene prediction and the like were carried out by combining the results of GENOMEGAMBLER (Sakiyama, T., Takami, H., Ogasawara, N., Kuhara, S., Kozuki, T., Doga, K., Ohyama, A., Horikoshi, K., “An automated system for genome analysis to support microbial whole-genome shotgun sequencing”, Biosci. Biotechnol. Biochem., 64: 670 to 673 2000), GLIMMER 2.0 (Salzberg, SL., Delcher, A L., Kasif, S., and White, O., “Microbial gene identification using interpolated Markov models”, Nucleic. Acid. Res., 26: 544 to 548, 1998) and BLAST program blastp (Altschul, SF., Gish, W., Miller, W., Myers, EW., and Lipman, DJ., “Basic local alignment search tool”, J. Mol. Biol., 215: 403 to 410, 1990).
  • Also, INTERPRO (Mulder, N J., Apweiler, R., Attwood, T K., Bairoch, A., Barrell, D., Bateman, A., Binns, D., Biswas, M., Bradley, P., Bork, P., Bucher, P., Copley, R R., Courcelle, E., Das, U., Durbin, R., Falquet, L., Fleischmann, W., Griffiths-Jones, S., Haft, D., Harte, N., Hulo, N., Kahn, D., Kanapin, A., Krestyaninova, M., Lopez, R., Letunic, I., Lonsdale, D., Silventoinen, V., Orchard, S E., Pagni, M., Peyruc, D., Ponting, CP., Selengut, J D., Servant, F., Sigrist, CJ., Vaughan, R., and Zdobnov, E M., “The InterPro Database, 2003 brings increased coverage and new features”, Nucleic. Acids. Res., 31: 315 to 318, 2003; http://www.ebi.ac.uk/interpro) was used for the analysis of a domain structure; and CLUSTALW (Thompson, JD., Higgins, DG., and Gibson, TJ., “CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, Nucleic. Acids. Res., 22: 4673 to 4680, 1994; http://clustalw.genome.ad.jp/) was used for the preparation of a molecular phylogenetic tree.
  • Furthermore, the used DNA and amino acid sequences were obtained from National Center of Biological Information (NCBI, http://www.ncbi.nlm.nih.gov) and KEGG database (Ogata, H., Goto, S., Sato, K., Fujibuchi, W., Bono, H., and Kanehisa, M., “KEGG: Kyoto Encyclopedia of Genes and Genomes”, Nucleic. Acids. Res., 27: 29 to 34, 1999; http://www.genome.ad.jp/kegg/kegg2.html); and a draft sequence of the Lactobacillus bacterium was obtained from DOE JOINT GENOME INSTITUTE (JGI; http://www.jgi.doe.gov/JGI_microbial/html/index.html).
  • Based on the foregoing information, the genes depicted in (13) to (15) were identified as encoding the lipases; the gene depicted in (4) as encoding a transporter gene; the genes depicted in (9), (10) and (11) as encoding a glycerol-degrading enzyme; and the gene depicted in (12) as an adhesive gene, respectively. Also, the genes depicted in (32) to (38) were identified as encoding a glycerol-degrading enzyme; the genes depicted in (39) to (41) as encoding an aldehyde dehydrogenase; the gene depicted in (42) as encoding a glycerate kinase; the gene depicted in (43) as encoding a glycerol kinase; the gene depicted in (44) as encoding a glycerol-3-phosphate dehydrogenase; and the genes depicted in (45) to (47) as encoding triosephosphate isomerase, respectively.
    • Example 3 Amplification of Glycerol-degrading Gene
  • PCR was carried out by using DNA as purified in Example 2 as a template and the following nucleotide sequences as primers and using the following reaction solutions. The PCR condition is also shown below.
  • (Primer)
    pduCDE(F):
    CACCATGAAACGTCAAAAACGATTT
    pduCDE(R):
    AAAAGCTTAGTTATCGCCCTTTAGC
  • (PCR reaction solution)
    Template DNA:  1 μL
    KOD-plus:  1 μL
    10 × KOD-plus buffer solution:  5 μL
    dNTP (2 mM each):  5 μL
    Primer (20 mm):  1 μL each
    MgSO4 (25 mm):  2 μL
    Deionized water (D.W.): 34 μL
    • (PCR Condition)
  • (1) To hold at 94° C. for 3 minutes.
    (2) To hold at 94° C. for 15 seconds.
    (3) To hold at 56° C. (Tm) for 30 seconds.
    (4) To hold at 68° C. for 3 minutes 30 seconds.
    (5) To perform (2) to (4) in 30 cycles.
    (6) To preserve at 4° C.
    • Example 4 Amplification of L. reuteri-derived Lipase Gene and Adhesive Gene
  • PCR was carried out in the same manner as in Example 3, except using the following sequences as primers, thereby amplifying the lipase gene and adhesive gene.
  • (Primer)
    Lipase (1)
    5′-ATGGTGAAATTGATGACAAT
    5′-TTATTTAAATTGATCGCCAA
    Lipase (2)
    5′-TTGATTTATGTTTTAAAAGA
    5′-CTATGACCGAGTTAAATACT
    Lipase (3)
    5′-ATGGAAATTAAAAGTGTTAA
    5′-CTAAATTAAATTCAGTTCAG
    Adhesive gene
    5′-ATGTTCGGTCACGATGGCCG
    5′-TCAAATTTCAGAAGGATCAT
    • INDUSTRIAL APPLICABILITY
  • As is clear from the results of the foregoing Examples using L. reuteri JCM1112T which is a representative of the lactic acid bacterium of the invention, the administration of this microorganism could inhibit the body weight gain without affecting the health of the experimental animals and without particularly limiting nutrition intake.
  • Accordingly, the anti-obesity agent or anti-obesity food and drink utilizing the lactic acid bacterium of the invention is able to prevent obesity and to bring a slimming effect without requiring particular therapy or treatment other than intake of the agent, or the food or drink per se.
  • Also, by incorporating genes encoding subunits of a glycerol-degrading enzyme or a gene encoding an enteroadherent protein, each of which has been found out from the lactic acid bacterium of the invention, into other lactic acid bacterium by a known measure, it becomes possible to obtain a lactic acid bacterium with high glycerol-degrading properties or a lactic acid bacterium with a long intestinal residence time. It is also possible to advantageously use it for the modification of other useful lactic acid bacteria.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIG. 1 is a drawing showing a metabolism map of L. reuteri JCM1112T.
  • FIG. 2 is a drawing showing the body weight gain with time in the rats administered with L. reuteri JCM1112T in comparison with the comparative group and the control group.

Claims (60)

1. An anti-obesity agent comprising, as an active ingredient, a microorganism belonging to the species Lactobacillus reuteri and capable of producing lipases depicted in the following amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3):
(1) MVKLMTIHELANNPTLSGQVRLIENIVYGA MDGEALHMSILAPWTQRFPKQYQTEPRPLI VFVQGSSWRTPKMGEEIPQLVQFVRAGYIV ATVQHRSSIDSHPFPAFLQDVKTAIRFLRA NAQKYAIDPQQVAIWGTSSGANAAMLVGLT GDDPRYKVDLYQDESDAVDAVVSCFAPMDV EKTFEYDANVPGNKLLQYCLLGPDVSKWPE IEKQMSPLYQVKDGQNYPPFLLFHGDADKV VPYEQMEKMYMRLKDNGNSVEAYRVKGANH ERDFWSPTIYNIVQKFLGDQFK (2) LIYVLKDLCNTIAEVYGKSILKGVFIMKHT LKVDQVRDGLWLDSDITYTQVPGWLGNTTR DLKLSVIRHFQTNDDTRYPVIFWFAGGGWM DTDHNVHLPNLVDFARHGYIVVGVEYRDSN KVQFPGQLEDAKAAIRYMRANAKRFQADPN RFIVMGESAGGHMASMLGVTNGLNQFDKGA NLDYSSDVQVAVPFYGVVDPLTAKTGSASN DFDFVYRNLLGAEPENAPELDSAANPLTYV NSNSTPFLIFHGTEDVVVPIKDSEKLYDAL VENNVPAELYEIEGASHMDVKFLQPQVFKI VMDFLDKYLTRS (3) MEIKSVNLDQPYSSLDIYHSNTDKALPGLV ILPGGSYNQIMERDSERVALTFATHAWQTF VVRYPVVEHKNYEEAKIAVHQAFEYIVNHA AELDVDADRLGIIGFSAGGQIAAAYSNEKL THARFAALGYPVIQPLIDERMGVTTENVAK LVNPQTPPTFMWGSAKDELTPFVDHLQVYA DALIKNDIPYELHEFGTGGHGIALANEYTG IVNNDRVDNHMGKWFPLFLEWLTELNLI
2. The anti-obesity agent according to claim 1, wherein the microorganism belonging to the species Lactobacillus reuteri has nucleotide sequences encoding the amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3), and a nucleotide sequence depicted in the following formula (4):
(4) ATGCATGGATTTATTGGCGAATTTTTTGGCACCATGGTTTTAATCCTATTAGGAGCAGGA TGTTGTGCTGGTAATAGTTTGAATAAAACATATGGGAAACAAAGTGGCTGGTGGTTTATC TGTATTTCATGGGGCTTAGCAGTTACAATGGGAGTTTATGTTGCAGGATTTCTGGGTTCA TTAGGGCACTTAAATCCCGCTGTAACAATTCCTTTTGCTATTTTTGGCTTATTCCCATGG AGTAACGTTATACGTTACTTACTTGGTCAATTTCTTGGTGCGTTTGTTGGTGCAGTATTA GTAATTATTCAATTCTATCCACAATTTAAAGCAACCCCAAATGAAGAAGAAGGAAATAAT GTTGGTATTTTTGCTACTCGTCCAGCGATAAATAGTCCAATTTTTAACTTTTTCTCAGAA GTGATTGCGACCTTTGCATTTATTTTCATCTTATTAAATCTTGGCAACTTTACACAGGGA TTGAAGCCATTTATCGTAGGAATGGTTATTGCAGTTGTTGGTACATGTCTCGGGACAACT ACTGGCTTTGCATTAAACCCAGCTCGTGATTGGTCACCACGTTTAGCATATACTATTTTG CCAATTCCTAATAAGGGTGTTTCAGAATGGTGGTATGCATGGGTTCCAATGTGTGGCCCA ATTGTTGGGGGCCTTCTTGCTTGTGCTTTACAAACGGCACTAGTTTAG
3. The anti-obesity agent according to claim 1 or 2, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-degrading enzyme composed of subunits depicted in the following amino acid sequences (5) to (7) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7):
(5) MKRQKRFEELEKRPIHQDTFVKEWPEEGFV AMMGPNDPKPSVKVENGKIVEMDGKKLEDF DLIDLYIAKYGINIDNVEKVMNMDSTKIAR MLVDPNVSRDEIIEITSALTPAKAEEIISK LDFGEMIMAVKKMRPRRKPDNQCHVTNTVD NPVQIAADAADAALRGFPEQETTTAVARYA PFNAISILIGAQTGRPGVLTQCSVEEATEL QLGMRGFTAYAETISVYGTDRVFTDGDDTP WSKGFLASCYASRGLKMRFTSGAGSEVLMG YPEGKSMLYLEARCILLTKASGVQGLQNGA VSCIEIPGAVPNGIREVLGENLLCMMCDIE CASGCDQAYSHSDMRRTERFIGQFIAGTDY INSGYSSTPNYDNTFAGSNTDAMDYDDMYV MERDLGQYYGIHPVKEETIIKARNKAAKAL QAVFEDLGLPKITDEEVEAATYANTHDDMP KRDMVADMKAAQDMMDRGITAIDIIKALYN HGFKDVAEAILNLQKQKVVGDYLQTSSIFD KDWNVTSAVNDGNDYQGPGTGYRLYEDKEE WDRIKDLPFALDPEHLEL (6) MADIDENLLRKIVKEVLSETNQIDTKIDFD KSNDSTATATQEVQQPNSKAVPEKKLDWFQ PVGEAKPGYSKDEVVIAVGPAFATVLDKTE TGIPHKEVLRQVIAGIEEEGLKARVVKVYR SSDVAFCAVQGDHLSGSGIAIGIQSKGTTV IHQKDQDPLGNLELFPQAPVLTPETYRAIG KNAAMYAKGESPEPVPAKNDQLARIHYQAI SAIMHIRETHQVVVGKPEEEIKVTFD (7) MSEVDDLVAKIMAQMGNSSSANSSTGTSTA STSKEMTADDYPLYQKHRDLVKTPKGHNLD DINLQKVVNNQVDPKELRITPEALKLQGEI AANAGRPAIQKNLQRAAELTRVPDERVLEM YDALRPFRSTKQELLNIAKELRDKYDANVC AAWFEEAADYYESRKKLKGDN
4. The anti-obesity agent according to any one of claims 1 to 3, wherein the microorganism belonging to the species Lactobacillus reuteri further produces an enteroadherent protein depicted in the following amino acid sequence (8) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8):
(8) MFGHDGRIVTKVYQWAGTYYYFDPNTYLRV DNDYRQSQWGDWYMFGPDGRIVTGLKEWYG SYYYFDPTTYLKVTNKWIDNKYFGPAGQQA ISRFERLDNKYYYFDANGAVLNIHDQFKNI DNHTYYFGADGACYTSQFLNKDGKQYYFDN DGIMLTDQEKIIDGKFYHFNVNGEAIQVND PSEI
5. The anti-obesity agent according to any one of claims 1 to 4, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-degrading enzyme depicted in any of the following amino acid sequences (16) to (20) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (16) to (20):
(16) MKAAVINDPVDGFVTVKDVQLRDLKPGEAL VDMEYCGLCHTDLHVAAGDFGKKPGRIIGH EGVGRVSKVAPGVTSLKVGDRVSIAWFFKG CGHCEYCLTGRETLCRNVLNAGYTADGAMA EQCIVPADYAVKVPEGLDPVEATSLTCAGV TMYKALKVADIKPGQWVSIVGAGGLGNLGI QLAHNVFGAHVIAVDGNPDKLEAAKKNGAE ILINRHDGDVDKQIQEKVGGVHAAVVTAVS ASAFDQAVDSLRPDGKLVAVALPQGDMKLN IAKTVLDGIIVAGSLVGTRQDLAECFQFGA EGKVHPIVKTRKLSEINDMIQELKDNKVVG RNVVDFVHNDND (17) MEKRENAIPKTMKAWAVTTPGPIDGKESPI EFTEKPVPTPKRGEVLVKVITCGVCHTDLH VTEGDLPVHHEHVTPGHEIVGKVVGFGPET QRFKFGERIGIPWFRHACGVCKFCRSGHEN LCPHSLYTGWDHDGGYAEYVTVPEGFAYRL PEKFDSLEAAPLLCAGIIGYRAFERANVPA GGRLGLYGFGGSAHITAQIALAQGIEVHVF TRGEDAKKFALELGCASVQGSYDPAPVPLD SSIIFAPVGDMVLPALASLVPGGTLALAGI HMTDIPTMNYQKEIFHEKTLTSVESNTRRD GEEFLTLADRLNIHPEVHEYPLAKADEALR YVKHGDIKGACVLRVSED (18) MQIKAALATKPNADLEIQTVELDEPKENEV LIKIASTGFCHTDIVGRSGATTPLPVVLGH EGAGVVQKVGANVTDVKPGDHVVLSFSYCG HCYNCTHNHQGLCENFNQLNFEGKTYDGTH RLHLDDGTPVSVFFGQSSFATYVTANVHNI VKVDQDVDLNLLGPLGCGMQTGAGTVLNYI KPAPEDAIAVFGAGAVGLAAIMAAKIAGVK HIIAINRNGNHLDLAKELGATETINNTAED PVKAIKEIVPRGVTYAIDTTGNTGVIKSAI DSLATAGECVLLGVGGDITLDLMNDILSES KKISGVVEGDSNPQEFIPQLVKYYKQSKFP LDKLVKYYDFADINQVIADSTNGKVIKPII KIDPELAKLPLTNDGSNVQKMVAEAGLADQ ITIDSAGTSNIAEGSPADSRTKAILDKYHI KDDGMIARQLQDRDYYDADYIIAMDQMNVR DAKDMAPAGLENKVHGIFEATPGKENCYIV DPWITH (19) MKKAIFEKAGQMKIVDVDRPTIEKPDDVII KVVRTCVCGSDLWNFRGINPVEKDSENSGH EAIGIVEEVGEDITTVKPGDFVIAPFTHGC GHCAACRAGFDGSCQSHNDNFSSGVQAQYV RFQHGQWALVKVPGKPSDYSEGMLKSLLTL ADVMATGYHAARVANVSDGDTVVVMGDGAV GLCAIIAAKMRGAKKIISTSRHADRQALAK EFGATDNVAERSDEAVQKIMELTNGAGADA VLECVGTEQSTDTAMKVGRPGTIVGRVGLP HTPKMDMTVLFYNNTIVGGGPASVTTYDKD VLLKAVLDGDINPGKVFTKSFDLDQIQEAY EAMDKREAIKSYIIMDGFERD (20) MGRLDNKVAIITGGSKGIGAAVAKKFIEEG AKVVLTARKMDEGQKVADQLGDNAIFIQQD VARKGDWDRVIRQTVQVFGKLNIVVNNAGI AEYADVEKTDAEIWDKTIAVNLTGTMWGTK LGIEAMKNNGEKNSIINMSSIEGLIGDPDL FAYNASKGGVRLLTKSAALDCARKGYDIRV NTIHPGYISTPLVDNLVKDDPKAEGHLESL HPLGRLGKPEEIANLALYLASDESSFSTGS EFVADGGYTAQ
6. The anti-obesity agent according to any one of claims 1 to 5, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-degrading enzyme depicted in the following amino acid sequence (21) or (22) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (21) or (22):
(21) MTNVPTVKLNNGVEMPTLGFEVFQVPDLSQ AEQAVTDALEVGYRLIDTAAAYQNEEAVGK AIKNSSVNREDVFVTSKLWVSDFNYKRAKA GIDASLQKLGLDYMDLYLLHQPYGDTMGAW RALQEAQKEGKIRAIGVSNFYADQLKDLEL TMPVKPAVNQIEVNPWYQQDQEVKFAQSED IRVEAWAPFAEGKHDIFTNEIIAEIAAKYG KSNGQVILRWLLQRGITVIPKSVHKNRMEE NIDVFDFELSNDDMKKIASLNKKESQFFDH RDPVTIEQIFGSSLKMVQDDEK (22) MILDETITLNSGVKIPKFALGTWMIDDDQA AEAVRNAIKMGYRHIDTAQAYDNERGVGEG VRTAGIDRDKIFVTSKIAAEHKDYDVTKKS IDETLEKMGLDYIDMMLIHSPQPWKEVNQS DNRYLEGNLAAWRAMEDAVNEGKIRTIGVS NFKKADLENIIKNSDTVPAVDQVLAHIGHT PFNLLSFTHEHDIAVEAYSPVAHGAALDNP VIEKMAKKYNVSVPQLCIRYDWQIGMIVLP KTTNPEHMKENTEIDFEISEADMDLLRRVK PLDYGDFDIYPVYGGKM
7. The anti-obesity agent according to any one of claims 1 to 6, wherein the microorganism belonging to the species Lactobacillus reuteri further produces an aldehyde dehydrogenase depicted in any of the following amino acid sequences (23) to (25) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (23) to (25):
(23) MPANNKKQVEKKELTAEEKKQNAQKLVDDL MTKSQAAFEKLRYYSQEQVDKICQAMALAA EEHHMDLAVDAANETGRGVAEDKAIKNIYA SEYIWNNIRHDKTVGIIEDNDEDQTIKIAD PLGVIAGIVPVTNPTSTTIFKSIISAKTRN TIIFSFHRQAMKSSIKTAKILQEAAEKAGA PKNMIQWLPESTRENTTALLQHPNTATILA TGGPSLVKAAYSSGNPALGVGPGNGPAYIE KTANIERSVYDIVLSKTFDNGMICATENSV VVDEEIYDKVKEEFQKWNCYFLKPNEIDKF TDGFIDPDRHQVRGPIAGRSANAIADMCGI KVPDNTKVIIAEYEGVGDKYPLSAEKLSPV LTMYKATSHENAFDICAQLLHYGGEGHTAA IHTLDDDLATKYGLEMRASRIIVNSPSGIG GIGNIYNNMTPSLTLGTGSYGSNSISHNVT DWDLLNIKTIAKRRENRQWVKIPPKVYFQR NSLKELQDIPNINRAFIVTGPGMSKRGYVQ RVIDQLRQRQNNTAFLVFDDVEEDPSTNTV EKGVAMMNDFKPDTIIALGGGSPMDAAKAM WMFYEHPETSWYGVMQKYLDIRKRAYQIKK PTKSQLIGIPTTSGTGSEVTPFAVITDSKT HVKYPLADYALTPNIAIVDSQFVETVPAKT TAWTGLDVLCHATESYVSVMATDYTRGWSL QTIKGVMENLPKSVQGDKLARRKMHDFSTM AGMAFGQAFLGINHSLAHKMGGAFGLPHGL LIAIAMPQVIRFNAKRPQKLALWPHYETYH ATKDYADIARFIGLKGNTDEELAEAYAKKV IELAHECGVKLSLKDNGVTREEFDKAVDDL ARLAYEDQCTTTNPVEPLVSQLKELLERCY DGTGVEEK (24) MAYQSINPFTNQVEKTFENTTDEELEQTLT TAHQLYLDWRKYNDLEERKRQILKLGQILR ERRVEYATVMSKEMGKLISEAEGEVDLCAS FCDYYAAHADEFLQPKIIATTSGRAKVLKQ SLGILVAVEPWNFPFYQIARVFIPNFIAGN PMILKDASNCPASAQAFNDAVKEAGAPAGS LTNLFLSYDQVNKAIADKRVAGVCLTGSER GGATVAKEAGANLKKSTLELGGNDAFIILD DADWDLVEKVAPAARLYNAGQVCTSSKRFI VLEKDYDRFLKMMKDAFSKVKMGDPLDPLT TLAPLSSKKAKEKLQQQVATAVENGAKVYY GNKPVDMEGQFFMPTILTDITPDNPIFDTE MFGPVASVYKVSSEEEAIELANNSSYGLGN TIFSNDSEHAERVAAKIETGMSWINAGWAS LPELPFGGVKNSGYGRELSSYGIDEFTNKH LIYEARQ (25) MQINDIESAVRKILAEELDNASSSSANVAA TTDNGHRGIFTNVNDAIAAAKAAQEIYRDK PIAVRQQVIDAIKEGFRPYIEKMAKDIKEE TGMGTVEAKIAKLNNALYNTPGPEILEPVV ENGDGGMVMYERLPYGVIGAVGPSTNPSET VIANAIMMLAGGNTLYFGAHPGAKNVTRWT IEKMNDFIADATGLHNLVVSIETPTIESVQ QMMKHPDIAMLAVTGGPAVVHQAMTSGKKA VGAGPGNPPAMVDATADIDLAAHNIITSAS FDNDILCTAEKEVVAESSIKDELIRKMQDE GAFVVNREQADKLADMCIQENGAPDRKFVG KDATYILDQANIPYTGHPVEIICELPKEHP LVMTEMLMPILPVVSCPTFDDVLKTAVEVE KGNHHTATIHSNNLKHINNAAHRMQCSIFV VNGPSYVGTGVADNGAHSGASALTIATPTG EGTCTARTFTRRVRLNSPQGFSVRNWY
8. The anti-obesity agent according to any one of claims 1 to 7, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerate kinase depicted in the following amino acid sequence (26) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (26):
(26) MKFVIAPDSFKGGLTAKEAANVMAEGIKRV FPNAEYALVPMADGGEGTVQSLVDATNGQK MIAKVHNPLNKLVNAEYGILGDGETAVIEM AAASGLQFVNKETANPLITTTYGTGELIKD ALDHNIKKIIIGIGGSATVDGGAGMAQALG ARLLDADNHEIGLGGGELASLEQVDFGGLD PRLKNVDIQIASDVTNPLTGKNGAAPVFGP QKGADEEMVNILDKNLHHYARKIVAAGGPD VEQTAGAGAAGGLGAGLIAFTGATMKRGVE LVIEATQLQKKAVGADYVFTGEGGIDFQTK FGKTPYGVAKATKEVAPTAPVIVLAGNIGK GVNDLYSSTAIDAIFATPEGAKPLKTALAD APIDIAQTAENVARLIKVSHVSN
9. The anti-obesity agent according to any one of claims 1 to 8, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol kinase depicted in the following amino acid sequence (27) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (27):
(27) LSEQQYIMAIDQGTTSSRAIIFDHDGNKVA ISQQEFPQYFPQPGWVEHDPLEIWDSVQSV ISNVMIKSQIKPYKIAAIGITNQRETTVIW DRHTGKPIYNAIVWQSKQTSDIAEQLIKDG YKDMIHQKTGLVIDSYFAATKIKWILDHVP GAREKAAKGDLMFGTIDTWLLWNLSGRRVH ATDVTNASRTMLFNIHTLDWDQDILDLLDI PQSLLPVVKPSSAIYGYTGDYHFYGVQIPI AGIAGDQQAALFGQAAYDKGSIKNTYGTGA FIVMNTGLKPTLSDNGLLTTIAYGLDGQTH YALEGSIFVAGSAVQWLRDGLKMFDKASES EQMAVDAKTTGGVYVVPAFTGLGAPYWDQE VRGAMFGLTRGTERGHIIRATLEAIAYQTK DVVDTMVKDTQLPLTALTVNGGASRNNFMM QFQADILQTPIKRAAMEETTALGAAFLAGL AVDFWEDQDELRKLSRIGDQFDPQMDPQKA ADLYRGWQRAIAAAQFYGKD
10. The anti-obesity agent according to any one of claims 1 to 9, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-3-phosphate dehydrogenase depicted in the following amino acid sequence (28) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (28):
(28) MAEKIAVLGAGSWGSVLANMLTENGHDVTL WSRNEEQVKQLNTEHTNPRYMKDFVYSTNL TATTDMKKAVKGASVVLIVIPTKGLREVAK QLNAILTELHQKPLVIHATKGLEQNTYKRP SEMLSEDISPENRQAIVVLSGPSHAEDVAI KDMTAVTAACEDLASAKKAQKLFSNSYFRV YTNDDVIGAEFGAALKNIIAIGAGAIQGLG YHDNARAALITRGLAEIRRLGVAFGANPMT FIGLSGVGDLVVTATSKNSRNWRAGYQLGQ GKKLQDVIDNMGMVIEGVYTTKAAYELSRK RQVQMPITEALYRVLYEGEDIKTAISQLMD RDLTSENE
11. The anti-obesity agent according to any one of claims 1 to 10, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a triosephosphate isomerase depicted in any of the following amino acid sequences (29) to (31) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (29) to (31):
(29) MRKPFIAANWKMHKNVQESVEFVDAIKGKL PDPQEVEVGIAAQAFALPSMVQAADDSGLK IIAQNAAAEYSGAFTGEISLRGLADAGVSY VMLGHIERRHLFHEDNELVNRKVLAALQMG VTPIICTDETMVQKEVNGEIHYVFQQLMSV LRGVSLDQIKNVVVSYEPSWAVGYGQHANP VLAEEGCRQIRRTIADNYTYEIADKIRILY GGSVNPDNIGMIMNKPDVDGVLIGRASLDV DNFLRMVNYLKNDQEK (30) MRKPFIIANWKMNKNVHESVAFVKAIKEKL PADKEIGIAAQAVSLYNMKKVASSSNLQII AQNASAELEGPYTGEISMRSLADAGVTYVM LGHLERRRLFNESNDSINQKVLAALNAGII PIICTDEEMVQTEVNGQIHYVFRQLKSVLK GVPANKLSQIVISYEPSWAVGSTHQANPDI AEEGCQAIRQSLVEMYGNEIGEQVRILYGG SVNPENIGQIMSKPNVDGALIGRASLEIES FLQMINYIELASKQKLQVI (31) MRVPIIAGNWKMHKDVQEAVSFIEKVKNQL PPADQLETAIAAPTLCLVPMVKAAEESPLK IMAENCYYKNEGAYTGETSPYALYQAGIHH VILGHSERRTYFNETDELINKKVKAALVNG LCPIVCCDDTMRRRVAGKKVHWVVSRILAD LHGLTNDEICHVTVAYEPSWAIGTGESADP EQAAEGCYLIRQTISDMYGDEVANNVRILY GGSVTTSNINALMAKNDIDGVLVGAASLNP ETFLQLVHH
12. The anti-obesity agent according to any one of claims 1 to 11, wherein the microorganism belonging to the species Lactobacillus reuteri is Lactobacillus reuteri JCM1112T.
13. An anti-obesity food and drink comprising, as an active ingredient, a microorganism belonging to the species Lactobacillus reuteri and capable of producing lipases depicted in the following amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3):
(1) MVKLMTIHELANNPTLSGQVRLIENIVYGA MDGEALHMSILAPWTQRFPKQYQTEPRPLI VFVQGSSWRTPKMGEEIPQLVQFVRAGYIV ATVQHRSSIDSHPFPAFLQDVKTAIRFLRA NAQKYAIDPQQVAIWGTSSGANAAMLVGLT GDDPRYKVDLYQDESDAVDAVVSCFAPMDV EKTFEYDANVPGNKLLQYCLLGPDVSKWPE IEKQMSPLYQVKDGQNYPPFLLFHGDADKV VPYEQMEKMYMRLKDNGNSVEAYRVKGANH ERDFWSPTIYNIVQKFLGDQFK (2) LIYVLKDLCNTIAEVYGKSILKGVFIMKHT LKVDQVRDGLWLDSDITYTQVPGWLGNTTR DLKLSVIRHFQTNDDTRYPVIFWFAGGGWM DTDHNVHLPNLVDFARHGYIVVGVEYRDSN KVQFPGQLEDAKAAIRYMRANAKRFQADPN RFIVMGESAGGHMASMLGVTNGLNQFDKGA NLDYSSDVQVAVPFYGVVDPLTAKTGSASN DFDFVYRNLLGAEPENAPELDSAANPLTYV NSNSTPFLIFHGTEDVVVPIKDSEKLYDAL VENNVPAELYEIEGASHMDVKFLQPQVFKI VMDFLDKYLTRS (3) MEIKSVNLDQPYSSLDIYHSNTDKALPGLV ILPGGSYNQIMERDSERVALTFATHAWQTF VVRYPVVEHKNYEEAKIAVHQAFEYIVNHA AELDVDADRLGIIGFSAGGQIAAAYSNEKL THARFAALGYPVIQPLIDERMGVTTENVAK LVNPQTPPTFMWGSAKDELTPFVDHLQVYA DALIKNDIPYELHEFGTGGHGIALANEYTG IVNNDRVDNHMGKWFPLFLEWLTELNLI
14. The anti-obesity food and drink according to claim 13, wherein the microorganism belonging to the species Lactobacillus reuteri has nucleotide sequences encoding the amino acid sequences (1) to (3) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (1) to (3), and a nucleotide sequence depicted in the following formula (4):
(4) ATGCATGGATTTATTGGCGAATTTTTTGGCACCATGGTTTTAATCCTATTAGGAGCAGGA TGTTGTGCTGGTAATAGTTTGAATAAAACATATGGGAAACAAAGTGGCTGGTGGTTTATC TGTATTTCATGGGGCTTAGCAGTTACAATGGGAGTTTATGTTGCAGGATTTCTGGGTTCA TTAGGGCACTTAAATCCCGCTGTAACAATTCCTTTTGCTATTTTTGGCTTATTCCCATGG AGTAACGTTATACCTTACTTACTTGGTCAATTTCTTGGTGCGTTTGTTGGTGCAGTATTA GTAATTATTCAATTCTATCCACAATTTAAAGCAACCCCAAATGAAGAAGAAGGAAATAAT GTTGGTATTTTTGCTACTCGTCCAGCGATAAATAGTCCAATTTTTAACTTTTTCTCAGAA GTGATTGCGACCTTTGCATTTATTTTCATCTTATTAAATCTTGGCAACTTTACACAGGGA TTGAAGCCATTTATCGTAGGAATGGTTATTGCAGTTGTTGGTACATGTCTCGGGACAACT ACTGGCTTTGCATTAAACCCAGCTCGTGATTGGTCACCACGTTTAGCATATACTATTTTG CCAATTCCTAATAAGGGTGTTTCAGAATGGTGGTATGCATGGGTTCCAATGTGTGGCCCA ATTGTTGGGGGCCTTCTTGCTTGTGCTTTACAAACGGCACTAGTTTAG
15. The anti-obesity food and drink according to claim 13 or 14, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-degrading enzyme composed of subunits depicted in the following amino acid sequences (5) to (7) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7):
(5) MKRQKRFEELEKRPIHQDTFVKEWPEEGFV AMMGPNDPKPSVKVENGKIVEMDGKKLEDF DLIDLYIAKYGINIDNVEKVMNMDSTKIAR MLVDPNVSRDEIIEITSALTPAKAEEIISK LDFGEMIMAVKKMRPRRKPDNQCHVTNTVD NPVQIAADAADAALRGFPEQETTTAVARYA PFNAISILIGAQTGRPGVLTQCSVEEATEL QLGMRGFTAYAETISVYGTDRVFTDGDDTP WSKGFLASCYASRGLKMRFTSGAGSEVLMG YPEGKSMLYLEARCILLTKASGVQGLQNGA VSCIEIPGAVPNGIREVLGENLLCMMCDIE CASGCDQAYSHSDMRRTERFIGQFIAGTDY INSGYSSTPNYDNTFAGSNTDAMDYDDMYV MERDLGQYYGIHPVKEETIIKARNKAAKAL QAVFEDLGLPKITDEEVEAATYANTHDDMP KRDMVADMKAAQDMMDRGITAIDIIKALYN HGFKDVAEAILNLQKQKVVGDYLQTSSIFD KDWNVTSAVNDGNDYQGPGTGYRLYEDKEE WDRIKDLPFALDPEHLEL (6) MADIDENLLRKIVKEVLSETNQIDTKIDFD KSNDSTATATQEVQQPNSKAVPEKKLDWFQ PVGEAKPGYSKDEVVIAVGPAFATVLDKTE TGIPHKEVLRQVIAGIEEEGLKARVVKVYR SSDVAFCAVQGDHLSGSGIAIGIQSKGTTV IHQKDQDPLGNLELFPQAPVLTPETYRAIG KNAAMYAKGESPEPVPAKNDQLARIHYQAI SAIMHIRETHQVVVGKPEEEIKVTFD (7) MSEVDDLVAKIMAQMGNSSSANSSTGTSTA STSKEMTADDYPLYQKHRDLVKTPKGHNLD DINLQKVVNNQVDPKELRITPEALKLQGEI AANAGRPAIQKNLQRAAELTRVPDERVLEM YDALRPFRSTKQELLNIAKELRDKYDANVC AAWFEEAADYYESRKKLKGDN
16. The anti-obesity food and drink according to any one of claims 13 to 15, wherein the microorganism belonging to the species Lactobacillus reuteri further produces an enteroadherent protein depicted in the following amino acid sequence (8) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8):
(8) MFGHDGRIVTKVYQWAGTYYYFDPNTYLRV DNDYRQSQWGDWYMFGPDGRIVTGLKEWYG SYYYFDPTTYLKVTNKWIDNKYFGPAGQQA ISRFERLDNKYYYFDANGAVLNIHDQFKNI DNHTYYFGADGACYTSQFLNKDGKQYYFDN DGIMLTDQEKIIDGKFYHFNVNGEAIQVND PSEI
17. The anti-obesity food and drink according to any one of claims 13 to 16, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-degrading enzyme depicted in any of the following amino acid sequences (16) to (20) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (16) to (20):
(16) MKAAVINDPVDGFVTVKDVQLRDLKPGEAL VDMEYCGLCHTDLHVAAGDFGKKPGRIIGH EGVGRVSKVAPGVTSLKVGDRVSIAWFFKG CGHCEYCLTGRETLCRNVLNAGYTADGAMA EQCIVPADYAVKVPEGLDPVEATSLTCAGV TMYKALKVADIKPGQWVSIVGAGGLGNLGI QLAHNVFGAHVIAVDGNPDKLEAAKKNGAE ILINRHDGDVDKQIQEKVGGVHAAVVTAVS ASAFDQAVDSLRPDGKLVAVALPQGDMKLN IAKTVLDGIIVAGSLVGTRQDLAECFQFGA EGKVHPIVKTRKLSEINDMIQELKDNKVVG RNVVDFVHNDND (17) MEKRENAIPKTMKAWAVTTPGPIDGKESPI EFTEKPVPTPKRGEVLVKVITCGVCHTDLH VTEGDLPVHHEHVTPGHEIVGKVVGFGPET QRFKFGERIGIPWFRHACGVCKFCRSGHEN LCPHSLYTGWDHDGGYAEYVTVPEGFAYRL PEKFDSLEAAPLLCAGIIGYRAFERANVPA GGRLGLYGFGGSAHITAQIALAQGIEVHVF TRGEDAKKFALELGCASVQGSYDPAPVPLD SSIIFAPVGDMVLPALASLVPGGTLALAGI HMTDIPTMNYQKEIFHEKTLTSVESNTRRD GEEFLTLADRLNIHPEVHEYPLAKADEALR YVKHGDIKGACVLRVSED (18) MQIKAALATKPNADLEIQTVELDEPKENEV LIKIASTGFCHTDIVGRSGATTPLPVVLGH EGAGVVQKVGANVTDVKPGDHVVLSFSYCG HCYNCTHNHQGLCENFNQLNFEGKTYDGTH RLHLDDGTPVSVFFGQSSFATYVTANVHNI VKVDQDVDLNLLGPLGCGMQTGAGTVLNYI KPAPEDAIAVFGAGAVGLAAIMAAKIAGVK HIIAINRNGNHLDLAKELGATETINNTAED PVKAIKEIVPRGVTYAIDTTGNTGVIKSAI DSLATAGECVLLGVGGDITLDLMNDILSES KKISGVVEGDSNPQEFIPQLVKYYKQSKFP LDKLVKYYDFADINQVIADSTNGKVIKPII KIDPELAKLPLTNDGSNVQKMVAEAGLADQ ITIDSAGTSNIAEGSPADSRTKAILDKYHI KDDGMIARQLQDRDYYDADYIIAMDQMNVR DAKDMAPAGLENKVHGIFEATPGKENCYIV DPWITH (19) MKKAIFEKAGQMKIVDVDRPTIEKPDDVII KVVRTCVCGSDLWNFRGINPVEKDSENSGH EAIGIVEEVGEDITTVKPGDFVIAPFTHGC GHCAACRAGFDGSCQSHNDNFSSGVQAQYV RFQHGQWALVKVPGKPSDYSEGMLKSLLTL ADVMATGYHAARVANVSDGDTVVVMGDGAV GLCAIIAAKMRGAKKIISTSRHADRQALAK EFGATDNVAERSDEAVQKIMELTNGAGADA VLECVGTEQSTDTAMKVGRPGTIVGRVGLP HTPKMDMTVLFYNNTIVGGGPASVTTYDKD VLLKAVLDGDINPGKVFTKSFDLDQIQEAY EAMDKREAIKSYIIMDGFERD (20) MGRLDNKVAIITGGSKGIGAAVAKKFIEEG AKVVLTARKMDEGQKVADQLGDNAIFIQQD VARKGDWDRVIRQTVQVFGKLNIVVNNAGI AEYADVEKTDAEIWDKTIAVNLTGTMWGTK LGIEAMKNNGEKNSIINMSSIEGLIGDPDL FAYNASKGGVRLLTKSAALDCARKGYDIRV NTIHPGYISTPLVDNLVKDDPKAEGHLESL HPLGRLGKPEEIANLALYLASDESSFSTGS EFVADGGYTAQ
18. The anti-obesity food and drink according to any one of claims 13 to 17, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-degrading enzyme depicted in the following amino acid sequence (21) or (22) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (21) or (22):
(21) MTNVPTVKLNNGVEMPTLGFEVFQVPDLSQ AEQAVTDALEVGYRLIDTAAAYQNEEAVGK AIKNSSVNREDVFVTSKLWVSDFNYKRAKA GIDASLQKLGLDYMDLYLLHQPYGDTMGAW RALQEAQKEGKIRAIGVSNFYADQLKDLEL TMPVKPAVNQIEVNPWYQQDQEVKFAQSED IRVEAWAPFAEGKHDIFTNEIIAEIAAKYG KSNGQVILRWLLQRGITVIPKSVHKNRMEE NIDVFDFELSNDDMKKIASLNKKESQFFDH RDPVTIEQIFGSSLKMVQDDEK (22) MILDETITLNSGVKIPKFALGTWMIDDDQA AEAVRNAIKMGYRHIDTAQAYDNERGVGEG VRTAGIDRDKIFVTSKIAAEHKDYDVTKKS IDETLEKMGLDYIDMMLIHSPQPWKEVNQS DNRYLEGNLAAWRAMEDAVNEGKIRTIGVS NFKKADLENIIKNSDTVPAVDQVLAHIGHT PFNLLSFTHEHDIAVEAYSPVAHGAALDNP VIEKMAKKYNVSVPQLCIRYDWQIGMIVLP KTTNPEHMKENTEIDFEISEADMDLLRRVK PLDYGDFDIYPVYGGKM
19. The anti-obesity food and drink according to any one of claims 13 to 18, wherein the microorganism belonging to the species Lactobacillus reuteri further produces an aldehyde dehydrogenase depicted in any of the following amino acid sequences (23) to (25) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (23) to (25):
(23) MPANNKKQVEKKELTAEEKKQNAQKLVDDL MTKSQAAFEKLRYYSQEQVDKICQAMALAA EEHHMDLAVDAANETGRGVAEDKAIKNIYA SEYIWNNIRHDKTVGIIEDNDEDQTIKIAD PLGVIAGIVPVTNPTSTTIFKSIISAKTRN TIIFSFHRQAMKSSIKTAKILQEAAEKAGA PKNMIQWLPESTRENTTALLQHPNTATILA TGGPSLVKAAYSSGNPALGVGPGNGPAYIE KTANIERSVYDIVLSKTFDNGMICATENSV VVDEEIYDKVKEEFQKWNCYFLKPNEIDKF TDGFIDPDRHQVRGPIAGRSANAIADMCGI KVPDNTKVIIAEYEGVGDKYPLSAEKLSPV LTMYKATSHENAFDICAQLLHYGGEGHTAA IHTLDDDLATKYGLEMRASRIIVNSPSGIG GIGNIYNNMTPSLTLGTGSYGSNSISHNVT DWDLLNIKTIAKRRENRQWVKIPPKVYFQR NSLKELQDIPNINRAFIVTGPGMSKRGYVQ RVIDQLRQRQNNTAFLVFDDVEEDPSTNTV EKGVAMMNDFKPDTIIALGGGSPMDAAKAM WMFYEHPETSWYGVMQKYLDIRKRAYQIKK PTKSQLIGIPTTSGTGSEVTPFAVITDSKT HVKYPLADYALTPNIAIVDSQFVETVPAKT TAWTGLDVLCHATESYVSVMATDYTRGWSL QTIKGVMENLPKSVQGDKLARRKMHDFSTM AGMAFGQAFLGINHSLAHKMGGAFGLPHGL LIAIAMPQVIRFNAKRPQKLALWPHYETYH ATKDYADIARFIGLKGNTDEELAEAYAKKV IELAHECGVKLSLKDNGVTREEFDKAVDDL ARLAYEDQCTTTNPVEPLVSQLKELLERCY DGTGVEEK (24) MAYQSINPFTNQVEKTFENTTDEELEQTLT TAHQLYLDWRKYNDLEERKRQILKLGQILR ERRVEYATVMSKEMGKLISEAEGEVDLCAS FCDYYAAHADEFLQPKIIATTSGRAKVLKQ SLGILVAVEPWNFPFYQIARVFIPNFIAGN PMILKDASNCPASAQAFNDAVKEAGAPAGS LTNLFLSYDQVNKAIADKRVAGVCLTGSER GGATVAKEAGANLKKSTLELGGNDAFIILD DADWDLVEKVAPAARLYNAGQVCTSSKRFI VLEKDYDRFLKMMKDAFSKVKMGDPLDPLT TLAPLSSKKAKEKLQQQVATAVENGAKVYY GNKPVDMEGQFFMPTILTDITPDNPIFDTE MFGPVASVYKVSSEEEAIELANNSSYGLGN TIFSNDSEHAERVAAKIETGMSWINAGWAS LPELPFGGVKNSGYGRELSSYGIDEFTNKH LIYEARQ (25) MQINDIESAVRKILAEELDNASSSSANVAA TTDNGHRGIFTNVNDAIAAAKAAQEIYRDK PIAVRQQVIDAIKEGFRPYIEKMAKDIKEE TGMGTVEAKIAKLNNALYNTPGPEILEPVV ENGDGGMVMYERLPYGVIGAVGPSTNPSET VIANAIMMLAGGNTLYFGAHPGAKNVTRWT IEKMNDFIADATGLHNLVVSIETPTIESVQ QMMKHPDIAMLAVTGGPAVVHQAMTSGKKA VGAGPGNPPAMVDATADIDLAAHNIITSAS FDNDILCTAEKEVVAESSIKDELIRKMQDE GAFVVNREQADKLADMCIQENGAPDRKFVG KDATYILDQANIPYTGHPVEIICELPKEHP LVMTEMLMPILPVVSCPTFDDVLKTAVEVE KGNHHTATIHSNNLKHINNAAHRMQCSIFV VNGPSYVGTGVADNGAHSGASALTIATPTG EGTCTARTFTRRVRLNSPQGFSVRNWY
20. The anti-obesity food and drink according to any one of claims 13 to 19, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerate kinase depicted in the following amino acid sequence (26) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (26):
(26) MKFVIAPDSFKGGLTAKEAANVMAEGIKRV FPNAEYALVPMADGGEGTVQSLVDATNGQK MIAKVHNPLNKLVNAEYGILGDGETAVIEM AAASGLQFVNKETANPLITTTYGTGELIKD ALDHNIKKIIIGIGGSATVDGGAGMAQALG ARLLDADNHEIGLGGGELASLEQVDFGGLD PRLKNVDIQIASDVTNPLTGKNGAAPVFGP QKGADEEMVNILDKNLHHYARKIVAAGGPD VEQTAGAGAAGGLGAGLIAFTGATMKRGVE LVIEATQLQKKAVGADYVFTGEGGIDFQTK FGKTPYGVAKATKEVAPTAPVIVLAGNIGK GVNDLYSSTAIDAIFATPEGAKPLKTALAD APIDIAQTAENVARLIKVSHVSN
21. The anti-obesity food and drink according to any one of claims 13 to 20, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol kinase depicted in the following amino acid sequence (27) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (27):
(27) LSEQQYIMAIDQGTTSSRAIIFDHDGNKVA ISQQEFPQYFPQPGWVEHDPLEIWDSVQSV ISNVMIKSQIKPYKIAAIGITNQRETTVIW DRHTGKPIYNAIVWQSKQTSDIAEQLIKDG YKDMIHQKTGLVIDSYFAATKIKWILDHVP GAREKAAKGDLMFGTIDTWLLWNLSGRRVH ATDVTNASRTMLFNIHTLDWDQDILDLLDI PQSLLPVVKPSSAIYGYTGDYHFYGVQIPI AGIAGDQQAALFGQAAYDKGSIKNTYGTGA FIVMNTGLKPTLSDNGLLTTIAYGLDGQTH YALEGSIFVAGSAVQWLRDGLKMFDKASES EQMAVDAKTTGGVYVVPAFTGLGAPYWDQE VRGAMFGLTRGTERGHIIRATLEAIAYQTK DVVDTMVKDTQLPLTALTVNGGASRNNFMM QFQADILQTPIKRAAMEETTALGAAFLAGL AVDFWEDQDELRKLSRIGDQFDPQMDPQKA ADLYRGWQRAIAAAQFYGKD
22. The anti-obesity food and drink according to any one of claims 13 to 21, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a glycerol-3-phosphate dehydrogenase depicted in the following amino acid sequence (28) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (28):
(28) MAEKIAVLGAGSWGSVLANMLTENGHDVTL WSRNEEQVKQLNTEHTNPRYMKDFVYSTNL TATTDMKKAVKGASVVLIVIPTKGLREVAK QLNAILTELHQKPLVIHATKGLEQNTYKRP SEMLSEDISPENRQAIVVLSGPSHAEDVAI KDMTAVTAACEDLASAKKAQKLFSNSYFRV YTNDDVIGAEFGAALKNIIAIGAGAIQGLG YHDNARAALITRGLAEIRRLGVAFGANPMT FIGLSGVGDLVVTATSKNSRNWRAGYQLGQ GKKLQDVIDNMGMVIEGVYTTKAAYELSRK RQVQMPITEALYRVLYEGEDIKTAISQLMD RDLTSENE
23. The anti-obesity food and drink according to any one of claims 13 to 22, wherein the microorganism belonging to the species Lactobacillus reuteri further produces a triosephosphate isomerase depicted in any of the following amino acid sequences (29) to (31) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in any of the amino acid sequences (29) to (31):
(29) MRKPFIAANWKMHKNVQESVEFVDAIKGKL PDPQEVEVGIAAQAFALPSMVQAADDSGLK IIAQNAAAEYSGAFTGEISLRGLADAGVSY VMLGHIERRHLFHEDNELVNRKVLAALQMG VTPIICTDETMVQKEVNGEIHYVFQQLMSV LRGVSLDQIKNVVVSYEPSWAVGYGQHANP VLAEEGCRQIRRTIADNYTYEIADKIRILY GGSVNPDNIGMIMNKPDVDGVLIGRASLDV DNFLRMVNYLKNDQEK (30) MRKPFIIANWKMNKNVHESVAFVKAIKEKL PADKEIGIAAQAVSLYNMKKVASSSNLQII AQNASAELEGPYTGEISMRSLADAGVTYVM LGHLERRRLFNESNDSINQKVLAALNAGII PIICTDEEMVQTEVNGQIHYVFRQLKSVLK GVPANKLSQIVISYEPSWAVGSTHQANPDI AEEGCQAIRQSLVEMYGNEIGEQVRILYGG SVNPENIGQIMSKPNVDGALIGRASLEIES FLQMINYIELASKQKLQVI (31) MRVPIIAGNWKMHKDVQEAVSFIEKVKNQL PPADQLETAIAAPTLCLVPMVKAAEESPLK IMAENCYYKNEGAYTGETSPYALYQAGIHH VILGHSERRTYFNETDELINKKVKAALVNG LCPIVCCDDTMRRRVAGKKVHWVVSRILAD LHGLTNDEICHVTVAYEPSWAIGTGESADP EQAAEGCYLIPQTISDMYGDEVANNVRILY GGSVTTSNINALMAKNDIDGVLVGAASLNP ETFLQLVHH
24. The anti-obesity food and drink according to any one of claims 13 to 23, wherein the microorganism belonging to the species Lactobacillus reuteri is Lactobacillus reuteri JCM1112T.
25. A glycerol-degrading enzyme composed of subunits depicted in the following amino acid sequences (5) to (7) or amino acid sequences having deletion, substitution or addition of one or more amino acids in the amino acid sequences (5) to (7):
(5) MKRQKRFEELEKRPIHQDTFVKEWPEEGFV AMMGPNDPKPSVKVENGKIVEMDGKKLEDF DLIDLYIAKYGINIDNVEKVMNMDSTKIAR MLVDPNVSRDEIIEITSALTPAKAEEISK LDFGEMIMAVKKMRPRRKPDNQCHVTNTVD NPVQIAADAADAALRGFPEQETTTAVARYA PFNAISILIGAQTGRPGVLTQCSVEEATEL QLGMRGFTAYAETISVYGTDRVFTDGDDTP WSKGFLASCYASRGLKMRFTSGAGSEVLMG YPEGKSMLYLEARCILLTKASGVQGLQNGA VSCIEIPGAVPNGIREVLGENLLCMMCDIE CASGCDQAYSHSDMRRTERFIGQFIAGTDY INSGYSSTPNYDNTFAGSNTDAMDYDDMYV MERDLGQYYGIHPVKEETIIKARNKAAKAL QAVFEDLGLPKITDEEVEAATYANTHDDMP KRDMVADMKAAQDMMDRGITAIDIIKALYN HGFKDVAEAILNLQKQKVVGDYLQTSSIFD KDWNVTSAVNDGNDYQGPGTGYRLYEDKEE WDRIKDLPFALDPEHLEL (6) MADIDENLLRKIVKEVLSETNQIDTKIDFD KSNDSTATATQEVQQPNSKAVPEKKLDWFQ PVGEAKPGYSKDEVVIAVGPAFATVLDKTE TGIPHKEVLRQVIAGIEEEGLKARVVKVYR SSDVAFCAVQGDHLSGSGIAIGIQSKGTTV IHQKDQDPLGNLELFPQAPVLTPETYRAIG KNAAMYAKGESPEPVPAKNDQLARIHYQAI SAIMHIRETHQVVVGKPEEEIKVTFD (7) MSEVDDLVAKIMAQMGNSSSANSSTGTSTA STSKEMTADDYPLYQKHRDLVKTPKGHNLD DINLQKVVNNQVDPKELRITPEALKLQGEI AANAGRPAIQKNLQRAAELTRVPDERVLEM YDALRPFRSTKQELLNIAKELRDKYDANVC AAWFEEAADYYESRKKLKGDN
26. Nucleotide sequences encoding the glycerol-degrading enzyme subunits according to claim 25, which are depicted in the following (9) to (11):
(10) ATGGCTGATATTGATGAAAACTTATTACGTAAAATCGTTAAAGAAGTTTTAAGCGAAACT AATCAAATCGATACTAAGATTGACTTTGATAAAAGTAATGATAGTACTGCAACAGCAACT CAAGAGGTGCAACAACCAAATAGTAAAGCTGTTCCAGAAAAGAAACTTGACTGGTTCCAA CCAGTTGGAGAAGCAAAACCTGGATATTCTAAGGATGAAGTTGTAATTGCAGTCGGTCCT GCATTCGCAACTGTTCTTGATAAGACAGAAACTGGTATTCCTCATAAAGAAGTGCTTCGT CAAGTTATTGCTGGTATTGAAGAAGAAGGGCTTAAGGCGCGGGTAGTTAAAGTTTACCGG AGTTCAGATGTAGCATTCTGTGCTGTCCAAGGTGATCACCTTTCTGGTTCAGGAATTGCT ATTGGTATCCAATCAAAAGGGACGACAGTTATTCACCAAAAGGATCAAGACCCTCTTGGT AACCTTGAGTTATTCCCACAAGCGCCAGTACTTACTCCCGAAACTTATCGTGCAATTGGT AAGAATGCCGCTATGTATGCTAAGGGTGAATCTCCAGAACCAGTTCCAGCTAAAAACGAT CAACTTGCTCGTATTCACTATCAAGCTATTTCAGCAATTATGCATATTCGTGAAACTCAC CAAGTTGTTGTTGGTAAGCCTGAAGAAGAAATTAAGGTTACGTTTGATTAA (11) ATGAGTGAAGTTGATGATTTAGTAGCAAAGATCATGGCTCAGATGGGAAACAGTTCATCT GCTAATAGCTCTACAGGTACTTCAACTGCAAGTACTAGTAAGGAAATGACAGCAGATGAT TACCCACTTTATCAAAAGCACCGTGATTTAGTAAAAACACCAAAAGGACACAATCTTGAT GACATCAATTTACAAAAAGTAGTAAATAATCAAGTTGATCCTAAGGAATTACGGATTACA CCAGAAGCATTGAAACTTCAAGGTGAAATTGCAGCTAATGCTGGCCGTCCAGCTATTCAA AAGAATCTTCAACGAGCTGCAGAATTAACACGAGTACCTGACGAACGGGTTCTTGAAATG TATGATGCATTGCGTCCTTTCCGTTCAACTAAGCAAGAATTATTGAACATTGCAAAGGAA TTACGGGACAAGTATGACGCTAATGTTTGCGCAGCATGGTTTGAAGAAGCTGCTGATTAT TATGAAAGTCGTAAGAAGCTAAAGGGCGATAACTAA
27. An enteroadherent protein depicted in the following amino acid sequence (8) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (8):
(8) MFGHDGRIVTKVYQWAGTYYYFDPNTYLRV DNDYRQSQWGDWYMFGPDGRIVTGLKEWYG SYYYFDPTTYLKVTNKWIDNKYFGPAGQQA ISRFERLDNKYYYFDANGAVLNIHDQFKNI DNHTYYFGADGACYTSQFLNKDGKQYYFDN DGIMLTDQEKIIDGKFYHFNVNGEAIQVND PSEI
28. A nucleotide sequence encoding the enteroadherent protein according to claim 27, which is depicted in the
(9) ATGAAACGTCAAAAACGATTTGAAGAACTAGAAAAACGGCCAATTCATCAAGATACATTT GTTAAAGAATGGCCAGAAGAAGGTTTCGTTGCAATGATGGGGCCTAATGACCCTAAGCCT AGTGTAAAAGTTGAAAATGGCAAGATCGTAGAGATGGATGGTAAAAAGCTCGAAGATTTT GATTTGATTGACTTGTACATTGCTAAGTATGGAATCAATATTGACAACGTTGAAAAAGTT ATGAATATGGATTCTACCAAGATTGCACGGATGCTTGTTGATCCTAATGTTTCTCGTGAT GAAATTATTGAAATTACATCAGCTTTGACTCCTGCTAAGGCTGAAGAGATCATCAGTAAG CTTGATTTTGGTGAAATGATTATGGCTGTCAAGAAGATGCGCCCACGTCGTAAGCCTGAC AACCAGTGTCACGTTACCAATACTGTTGATAACCCAGTTCAAATTGCTGCTGATGCTGCT GATGCCGCTCTTCGTGGATTTCCAGAACAAGAAACCACGACAGCTGTGGCACGTTATGCA CCATTCAATGCTATTTCAATTTTAATTGGTGCACAAACAGGTCGCCCTGGTGTATTGACA CAATGTTCTGTTGAAGAAGCTACTGAATTGCAATTAGGTATGCGTGGTTTTACCGCATAT GCTGAAACCATTTCAGTTTACGGTACTGATCGTGTATTTACCGATGGTGATGATACTCCA TGGTCTAAAGGCTTCTTGGCATCTTGTTATGCATCACGTGGTTTGAAGATGCGATTTACT TCAGGTGCCGGTTCAGAAGTTTTGATGGGTTATCCAGAAGGTAAGTCAATGCTTTACCTT GAAGCGCGTTGTATTTTACTTACTAAGGCTTCAGGTGTTCAAGGACTTCAAAATGGTGCC GTAAGTTGTATTGAAATTCCTGGTGCTGTTCCTAATGGTATTCGTGAAGTTCTCGGTGAA AACTTGTTATGTATGATGTGTGACATCGAATGTGCTTCTGGTTGTGACCAAGCATACTCA CACTCCGATATGCGGCGGACTGAACGGTTTATTGGTCAATTTATTGCCGGTACTGATTAT ATTAACTCTGGTTACTCATCAACTCCTAACTACGATAATACCTTCGCTGGTTCAAACACT GATGCTATGGACTACGATGATATGTATGTTATGGAACGTGACTTGGGTCAATATTATGGT ATTCACCCTGTTAAGGAAGAAACCATTATTAAGGCACGTAATAAGGCCGCTAAAGCCCTT CAAGCAGTATTTGAAGATCTTGGATTACCAAAGATTACTGATGAAGAGGTCGAAGCAGCA ACGTATGCTAACACCCATGATGACATGCCAAAGCGGGATATGGTTGCAGATATGAAGGCT GCTCAAGATATGATGGATCGTGGAATTACTGCTATTGATATTATCAAGGCATTGTACAAC CACGGATTTAAAGATGTCGCTGAAGCAATTTTGAACCTTCAAAAACAAAAAGTTGTTGGT GATTACCTTCAAACATCTTCTATTTTTGATAAAGATTGGAACGTCACTTCTGCTGTTAAC GACGGAAATGATTATCAAGGACCAGGTACTGGATACCGTCTATATGAAGACAAGGAAGAA TGGGATCGGATTAAAGACTTACCATTCGCCCTTGATCCAGAACATTTGGAACTGTAG
following (12):
(12) ATGTTCGGTCACGATGGCCGCATTGTTACTAAAGTTTACCAATGGGCTGGCACGTATTAC TACTTTGATCCGAATACTTATTTGCGAGTAGATAATGATTACCGTCAATCTCAGTGGGGC GATTGGTATATGTTTGGCCCAGATGGTCGTATCGTTACAGGGTTAAAGGAATGGTACGGT AGTTATTATTACTTTGATCCGACGACTTACTTAAAAGTAACTAATAAGTGGATAGATAAT AAGTACTTTGGTCCAGCTGGTCAGCAAGCTATTTCACGCTTTGAGAGACTTGATAATAAG TATTACTATTTCGATGCTAATGGGGCAGTTCTTAATATCCATGATCAATTTAAGAATATT GATAACCACACTTATTACTTTGGAGCTGATGGTGCTTGTTATACCAGTCAATTCTTAAAT AAGGATGGTAAACAGTATTATTTCGATAATGATGGAATTATGCTCACTGATCAAGAGAAG ATCATTGACGGTAAATTCTATCATTTCAATGTTAATGGTGAAGCAATCCAAGTAAATGAT CCTTCTGAAATTTGA
29. A glycerol-degrading enzyme depicted in the following amino acid sequence (16) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (16):
(16) MKAAVINDPVDGFVTVKDVQLRDLKPGEAL VDMEYCGLCHTDLHVAAGDFGKKPGRIIGH EGVGRVSKVAPGVTSLKVGDRVSIAWFFKG CGHCEYCLTGRETLCRNVLNAGYTADGAMA EQCIVPADYAVKVPEGLDPVEATSLTCAGV TMYKALKVADIKPGQWVSIVGAGGLGNLGI QLAHNVFGAHVIAVDGNPDKLEAAKKNGAE ILINRHDGDVDKQIQEKVGGVHAAVVTAVS ASAFDQAVDSLRPDGKLVAVALPQGDMKLN IAKTVLDGIIVAGSLVGTRQDLAECFQFGA EGKVHPIVKTRKLSEINDMIQELKDNKVVG RNVVDFVHNDND
30. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 29, which is depicted in the following (32):
(32) ATGAAAGCTGCTGTTATTAATGATCCAGTAGACGGTTTTGTTACTGTTAAAGATGTTCAA CTTCGGGATTTGAAGCCCGGTGAAGCTTTAGTTGACATGGAATATTGTGGTCTTTGTCAC ACTGATCTACACGTTGCTGCTGGGGACTTTGGTAAGAAGCCCGGTCGTATTATCGGTCAC GAAGGGGTTGGTCGTGTATCTAAGGTTGCCCCTGGCGTTACTTCCTTGAAAGTTGGCGAC CGTGTATCAATTGCATGGTTCTTCAAGGGCTGTGGACACTGTGAATATTGTTTAACTGGT CGTGAAACTCTTTGTCGGAACGTTCTTAATGCGGGTTACACTGCTGACGGTGCAATGGCT GAACAATGTATCGTACCAGCTGACTACGCTGTTAAGGTTCCAGAAGGTCTTGATCCTGTT GAAGCTACTTCATTAACTTGTGCTGGTGTTACGATGTACAAGGCATTAAAGGTTGCTGAC ATCAAGCCAGGTCAATGGGTATCAATCGTTGGTGCTGGTGGTTTAGGTAACTTGGGTATT CAACTTGCTCACAACGTATTTGGTGCTCATGTTATCGCTGTTGATGGTAATCCTGATAAG CTTGAAGCCGCTAAGAAGAATGGTGCTGAAATTTTAATTAACCGTCATGACGGTGATGTT GATAAGCAAATTCAAGAAAAGGTTGGCGGTGTTCACGCTGCTGTAGTAACAGCTGTTTCT GCCTCTGCATTCGACCAAGCAGTTGATTCACTTCGCCCAGATGGTAAGCTTGTTGCCGTT GCGCTTCCACAAGGTGACATGAAGCTTAACATTGCTAAGACTGTTCTTGATGGTATCATT GTTGCTGGTTCATTAGTTGGTACCCGTCAAGACTTAGCTGAATGTTTCCAATTTGGTGCA GAAGGTAAGGTTCACCCAATTGTTAAGACTCGTAAGTTAAGCGAAATTAATGATATGATC CAAGAACTTAAGGATAACAAGGTTGTTGGTCGGAATGTTGTTGATTTTGTTCACAACGAT AACGACTAA
31. A glycerol-degrading enzyme depicted in the following amino acid sequence (17) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (17):
(17) MEKRENAIPKTMKAWAVTTPGPIDGKESPI EFTEKPVPTPKRGEVLVKVITCGVCHTDLH VTEGDLPVHHEHVTPGHEIVGKVVGFGPET QRFKFGERIGIPWFRHACGVCKFCRSGHEN LCPHSLYTGWDHDGGYAEYVTVPEGFAYRL PEKFDSLEAAPLLCAGIIGYRAFERANVPA GGRLGLYGFGGSAHITAQIALAQGIEVHVF TRGEDAKKFALELGCASVQGSYDPAPVPLD SSIIFAPVGDMVLPALASLVPGGTLALAGI HMTDIPTMNYQKEIFHEKTLTSVESNTRRD GEEFLTLADRLNIHPEVHEYPLAKADEALR YVKHGDIKGACVLRVSED
32. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 31, which is depicted in the following (33):
(33) ATGGAAAAACGCGAAAATGCTATTCCGAAAACAATGAAGGCTTGGGCAGTCACAACTCCT GGGCCGATTGATGGTAAGGAATCACCAATCGAATTTACCGAAAAGCCTGTGCCGACTCCT AAACGGGGAGAAGTCCTTGTTAAGGTAATAACGTGTGGAGTATGTCATACGGACTTGCAC GTGACTGAAGGAGACTTGCCGGTTCACCACGAACACGTTACTCCTGGTCATGAAATTGTT GGTAAAGTTGTCGGCTTTGGACCAGAGACACAACGATTTAAGTTTGGTGAGCGAATTGGG ATTCCATGGTTTCGGCATGCTTGTGGTGTATGCAAGTTTTGCCGATCAGGTCATGAGAAT CTCTGTCCTCATTCACTTTATACCGGTTGGGATCATGATGGCGGTTATGCAGAATATGTC ACAGTTCCAGAAGGATTTGCATATCGGCTTCCAGAAAAGTTTGATTCCCTAGAGGCAGCT CCGTTATTATGTGCAGGGATTATTGGTTATCGGGCCTTTGAACGTGCCAATGTTCCGGCT GGCGGTCGCCTAGGATTATATGGCTTCGGTGGTTCAGCTCATATTACAGCTCAAATTGCA CTTGCTCAGGGAATTGAAGTGCATGTCTTTACGCGTGGTGAGGATGCCAAGAAATTCGCC CTAGAATTAGGTTGTGCTTCTGTTCAGGGCTCCTATGACCCAGCACCAGTTCCTTTGGAT TCATCAATCATTTTTGCGCCGGTTGGTGATATGGTCTTGCCGGCTTTAGCTAGTTTAGTT CCAGGGGGGACATTAGCATTAGCCGGTATTCATATGACTGATATTCCAACAATGAATTAC CAAAAAGAAATATTCCACGAAAAGACATTAACGAGTGTTGAGAGTAATACTCGTCGTGAT GGGGAAGAATTCTTAACATTAGCTGATCGTCTTAATATCCATCCTGAAGTCCACGAATAT CCCCTAGCAAAGGCTGACGAAGCATTACGCTATGTTAAGCACGGTGATATTAAGGGAGCT TGTGTATTACGTGTTAGTGAGGACTAA
33. A glycerol-degrading enzyme depicted in the following amino acid sequence (18) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (18):
(18) MQIKAALATKPNADLEIQTVELDEPKENEV LIKIASTGFCHTDIVGRSGATTPLPVVLGH EGAGVVQKVGANVTDVKPGDHVVLSFSYCG HCYNCTHNHQGLCENFNQLNFEGKTYDGTH RLHLDDGTPVSVFFGQSSFATYVTANVHNI VKVDQDVDLNLLGPLGCGMQTGAGTVLNYI KPAPEDAIAVFGAGAVGLAAIMAAKIAGVK HIIAINRNGNHLDLAKELGATETINNTAED PVKAIKEIVPRGVTYAIDTTGNTGVIKSAI DSLATAGECVLLGVGGDITLDLMNDILSES KKISGVVEGDSNPQEFIPQLVKYYKQSKFP LDKLVKYYDFADINQVIADSTNGKVIKPII KIDPELAKLPLTNDGSNVQKMVAEAGLADQ ITIDSAGTSNIAEGSPADSRTKAILDKYHI KDDGMIARQLQDRDYYDADYIIAMDQMNVR DAKDMAPAGLENKVHGIFEATPGKENCYIV DPWITH
34. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 33, which is depicted in the following (34):
(34) ATGCAAATTAAAGCTGCTCTTGCAACCAAACCTAACGCTGATTTAGAGATTCAAACCGTC GAATTGGATGAACCAAAAGAAAATGAAGTATTAATAAAAATTGCTTCAACAGGTTTTTGT CATACAGATATTGTTGGTCGAAGCGGTGCCACTACCCCTCTCCCCGTTGTCCTCGGGCAT GAAGGTGCGGGCGTCGTCCAAAAAGTAGGAGCTAACGTTACGGACGTTAAACCCGGCGAC CATGTTGTTCTATCATTTAGCTACTGTGGCCATTGCTATAACTGTACTCATAATCATCAA GGCTTATGCGAAAACTTCAATCAGCTAAACTTTGAAGGAAAAACCTATGATGGTACTCAC CGCCTGCACTTAGATGATGGCACGCCAGTCAGTGTCTTTTTTGGTCAGTCTTCCTTTGCG ACCTATGTAACAGCCAATGTCCATAATATTGTTAAAGTTGATCAAGATGTTGATCTTAAC TTATTAGGGCCACTCGGTTGTGGAATGCAAACAGGTGCTGGAACCGTTCTAAATTATATT AAACCTGCTCCTGAAGATGCAATTGCCGTTTTCGGTGCTGGTGCTGTTGGCTTAGCCGCA ATTATGGCTGCTAAAATTGCTGGAGTTAAACATATTATTGCGATTAATCGTAACGGTAAC CACCTTGACCTGGCGAAGGAATTGGGCGCTACTGAAACGATTAATAATACGGCTGAAGAT CCCGTCAAAGCAATTAAAGAAATCGTTCCGCGTGGTGTAACTTATGCAATCGATACTACC GGAAACACCGGTGTAATTAAATCAGCAATTGATAGTCTTGCCACCGCTGGAGAATGTGTC CTCTTAGGAGTTGGCGGCGATATTACCTTAGACTTAATGAATGATATCTTATCAGAATCT AAGAAAATCTCTGGGGTTGTCGAAGGAGATAGCAATCCCCAAGAGTTTATTCCTCAACTA GTTAAGTACTACAAGCAAAGCAAGTTCCCCCTTGATAAGCTTGTTAAGTACTACGATTTT GCTGATATTAACCAAGTTATCGCTGACTCAACAAACGGAAAGGTTATTAAGCCAATCATC AAAATTGATCCTGAATTAGCTAAATAATTGCCGCTCACCAATGACGGAAGCAATGTTCAA AAAATGGTTGCAGAAGCTGGCCTTGCTGATCAAATTACTATTGATTCAGCCGGAACAAGT AACATTGCAGAAGGTTCACCTGCTGATAGTCGAACAAAAGCCATTCTCGATAAATATCAC ATTAAAGACGACGGAATGATTGCCCGTCAATTGCAGGACAGGGATTATTATGATGCCGAT TATATTATCGCAATGGATCAGATGAATGTCCGGGACGCAAAAGATATGGCACCAGCTGGG TTAGAAAATAAGGTTCATGGAATCTTTGAAGCTACCCCAGGAAAAGAAAATTGCTATATC GTTGACCCCTGGATCACTCACTGA
35. A glycerol-degrading enzyme depicted in the following amino acid sequence (19) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (19):
(19) MKKAIFEKAGQMKIVDVDRPTIEKPDDVII KVVRTCVCGSDLWNFRGINPVEKDSENSGH EAIGIVEEVGEDITTVKPGDFVIAPFTHGC GHCAACRAGFDGSCQSHNDNFSSGVQAQYV RFQHGQWALVKVPGKPSDYSEGMLKSLLTL ADVMATGYHAARVANVSDGDTVVVMGDGAV GLCAIIAAKMRGAKKIISTSRHADRQALAK EFGATDNVAERSDEAVQKIMELTNGAGADA VLECVGTEQSTDTAMKVGRPGTIVGRVGLP HTPKMDMTVLFYNNTIVGGGPASVTTYDKD VLLKAVLDGDINPGKVFTKSFDLDQIQEAY EAMDKREAIKSYIIMDGFERD
36. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 35, which is depicted in the following (35):
(35) ATGAAAAAAGCTATTTTTGAAAAGGCGGGTCAAATGAAGATTGTTGATGTTGACCGTCCA ACAATTGAAAAGCCTGATGACGTAATTATTAAGGTAGTGCGGACCTGTGTTTGTGGTTCT GACCTATGGAACTTCCGAGGAATTAATCCGGTTGAAAAAGATTCTGAAAACTCTGGCCAT GAAGCAATTGGAATTGTTGAAGAAGTTGGTGAAGATATCACTACTGTCAAACCTGGGGAC TTTGTGATTGCTCCATTTACTCATGGATGTGGGCACTGTGCTGCTTGTCGCGCGGGCTTC GATGGTTCTTGCCAAAGTCACAACGATAACTTTAGCTCTGGTGTGCAAGCTCAATACGTT CGGTTCCAACACGGTCAATGGGCGCTTGTTAAAGTTCCGGGCAAGCCAAGTGACTACAGT GAAGGAATGCTTAAGTCCCTCTTAACCCTTGCTGATGTTATGGCTACTGGTTACCACGCT GCACGAGTTGCTAACGTTAGTGATGGTGATACAGTTGTTGTAATGGGTGACGGTGCTGTT GGCCTTTGTGCGATTATTGCTGCTAAGATGCGGGGCGCTAAGAAGATCATTTCTACTAGT CGCCATGCTGACCGTCAAGCCCTTGCTAAGGAATTTGGTGCTACTGACAATGTTGCTGAA CGTAGTGACGAAGCGGTTCAAAAGATCATGGAACTCACTAACGGTGCCGGTGCTGATGCT GTCCTTGAATGCGTTGGTACTGAACAATCAACTGATACTGCCATGAAAGTTGGCCGTCCA GGTACCATCGTTGGTCGGGTTGGCTTACCTCATACCCCAAAGATGGACATGACGGTGCTA TTCTACAACAACACTATTGTCGGCGGTGGTCCAGCATCAGTAACCACTTACGACAAGGAC GTATTGTTGAAGGCTGTTCTTGATGGTGACATTAACCCTGGTAAGGTCTTTACTAAGAGC TTCGACCTTGACCAAATTCAAGAAGCTTATGAAGCAATGGATAAGCGTGAAGCAATCAAG TCTTACATTATTATGGATGGCTTTGAACGCGATTAA
37. A glycerol-degrading enzyme depicted in the following amino acid sequence (20) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (20):
(20) MGRLDNKVAIITGGSKGIGAAVAKKFIEEG AKVVLTARKMDEGQKVADQLGDNAIFIQQD VARKGDWDRVIRQTVQVFGKLNIVVNNAGI AEYADVEKTDAEIWDKTIAVNLTGTMWGTK LGIEAMKNNGEKNSIINMSSIEGLIGDPDL FAYNASKGGVRLLTKSAALDCARKGYDIRV NTIHPGYISTPLVDNLVKDDPKAEGHLESL HPLGRLGKPEEIANLALYLASDESSFSTGS EFVADGGYTAQ
38. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 37, which is depicted in the following (36):
(36) ATGGGTCGTTTAGATAATAAAGTTGCAATTATTACTGGTGGTTCTAAAGGAATTGGAGCT GCTGTCGCAAAAAAGTTTATCGAAGAAGGCGCAAAGGTTGTTTTAACCGCTCGGAAGATG GATGAGGGACAAAAAGTCGCTGACCAACTAGGTGACAATGCGATCTTTATCCAACAAGAC GTTGCTCGGAAAGGAGACTGGGACCGGGTAATCCGCCAAACTGTCCAAGTCTTTGGGAAG CTCAATATTGTGGTTAACAATGCGGGAATTGCCGAATACGCCGATGTTGAGAAGACGGAC GCTGAAATTTGGGATAAAACAATTGCCGTTAACCTTACCGGTACGATGTGGGGAACTAAG CTCGGTATTGAAGCAATGAAGAACAACGGGGAAAAGAATTCAATCATCAATATGTCATCC ATTGAAGGACTAATTGGTGATCCTGATCTCTTTGCATACAATGCTTCTAAGGGTGGTGTC CGCCTCTTAACTAAGTCCGCTGCGCTTGATTGTGCCCGGAAAGGCTATGACATCCGTGTA AATACAATTCATCCTGGTTATATCTCAACTCCACTAGTTGATAATTTGGTCAAGGATGAT CCAAAAGCAGAAGGACACCTAGAAAGCCTTCATCCCCTTGGCCGTCTTGGAAAGCCAGAA GAGATTGCTAACCTCGCTTTATACCTTGCTTCAGATGAATCAAGCTTTAGTACTGGTTCG GAATTTGTCGCTGATGGTGGCTATACGGCTCAATAA
39. A glycerol-degrading enzyme depicted in the following amino acid sequence (21) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (21):
(21) MTNVPTVKLNNGVEMPTLGFEVFQVPDLSQ AEQAVTDALEVGYRLIDTAAAYQNEEAVGK AIKNSSVNREDVFVTSKLWVSDFNYKRAKA GIDASLQKLGLDYMDLYLLHQPYGDTMGAW RALQEAQKEGKIRAIGVSNFYADQLKDLEL TMPVKPAVNQIEVNPWYQQDQEVKFAQSED IRVEAWAPFAEGKHDIFTNEIIAEIAAKYG KSNGQVILRWLLQRGITVIPKSVHKNRMEE NIDVFDFELSNDDMKKIASLNKKESQFFDH RDPVTIEQIFGSSLKMVQDDEK
40. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 39, which is depicted in the following (37):
(37) ATGACAAATGTACCAACAGTAAAATTAAATAACGGAGTAGAAATGCCAACCCTTGGATTT GAAGTATTCCAAGTTCCAGACTTAAGCCAAGCTGAACAAGCAGTTACCGATGCTCTTGAA GTCGGCTATCGTTTAATCGATACTGCTGCTGCTTACCAAAATGAAGAAGCAGTTGGAAAG GCAATTAAGAATAGTAGTGTAAACCGTGAAGATGTCTTTGTAACTTCTAAGTTATGGGTG TCTGATTTTAACTATAAGCGGGCTAAAGCAGGGATTGACGCTTCACTGCAAAAACTTGGC CTTGATTACATGGATCTTTACCTTCTCCATCAACCATATGGCGATACAATGGGGGCTTGG CGAGCATTACAAGAAGCACAGAAAGAAGGTAAGATTCGCGCAATCGGTGTATCGAACTTC TACGCTGATCAACTAAAGGATCTTGAATTAACAATGCCTGTTAAGCCAGCGGTCAACCAA ATTGAAGTTAACCCTTGGTACCAGCAAGATCAAGAGGTTAAGTTTGCGCAAAGTGAAGAT ATTCGTGTTGAAGCATGGGCACCATTTGCGGAAGGTAAGCATGATATTTTTACCAACGAA ATAATTGCGGAAATTGCTGCCAAGTATGGCAAGAGCAATGGTCAAGTAATTCTTCGCTGG CTTTTACAACGGGGTATTACTGTCATTCCAAAGTCAGTCCACAAGAACCGGATGGAAGAA AATATCGATGTCTTTGATTTTGAACTTTCCAATGATGATATGAAAAAGATAGCTAGTCTT AACAAGAAGGAAAGCCAATTCTTTGACCACCGTGATCCGGTTACGATTGAACAAATCTTT GGCTCCAGCTTAAAGATGGTTCAAGATGACGAAAAATAA
41. A glycerol-degrading enzyme depicted in the following amino acid sequence (22) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (22):
(22) MILDETITLNSGVKIPKFALGTWMIDDDQA AEAVRNAIKMGYRHIDTAQAYDNERGVGEG VRTAGIDRDKIFVTSKIAAEHKDYDVTKKS IDETLEKMGLDYIDMMLIHSPQPWKEVNQS DNRYLEGNLAAWRAMEDAVNEGKIRTIGVS NFKKADLENIIKNSDTVPAVDQVLAHIGHT PFNLLSFTHEHDIAVEAYSPVAHGAALDNP VIEKMAKKYNVSVPQLCIRYDWQIGMIVLP KTTNPEHMKENTEIDFEISEADMDLLRRVK PLDYGDFDIYPVYGGKM
42. A nucleotide sequence encoding the glycerol-degrading enzyme according to claim 41, which is depicted in the following (38):
(38) ATGATTTTAGATGAGACAATTACTCTTAATAGTGGTGTGAAAATTCCAAAGTTTGCATTA GGAACCTGGATGATTGATGATGACCAAGCAGCCGAAGCAGTTCGGAATGCGATTAAGATG GGATATCGGCACATCGATACAGCTCAGGCTTATGATAATGAGCGGGGAGTCGGTGAAGGT GTACGAACAGCCGGTATTGATCGGGATAAAATCTTTGTTACTTCAAAGATCGCTGCTGAA CACAAAGATTATGATGTAACTAAAAAGTCGATTGACGAGACTCTTGAAAAGATGGGTCTT GATTATATCGACATGATGCTTATTCATAGTCCTCAACCATGGAAAGAAGTAAATCAATCT GATAATCGTTACCTTGAAGGAAATCTCGCTGCTTGGCGAGCCATGGAAGATGCCGTTAAC GAAGGTAAGATTCGAACAATTGGCGTTTCTAATTTCAAAAAAGCCGATCTTGAAAATATT ATTAAGAATAGCGATACCGTTCCCGCTGTTGATCAAGTTTTAGCTCATATTGGTCATACT CCATTCAATCTTTTATCATTTACTCATGAACATGACATTGCGGTTGAAGCATATTCACCA GTTGCTCACGGCGCTGCTTTAGACAACCCCGTAATTGAAAAGATGGCTAAAAAGTACAAC GTTTCAGTCCCACAATTGTGCATTCGGTATGATTGGCAAATAGGAATGATCGTCTTACCA AAGACTACTAATCCAGAACACATGAAGGAAAACACTGAAATTGATTTTGAAATTTCTGAA GCTGATATGGACCTATTGCGGCGAGTAAAGCCATTAGACTATGGCGATTTTGATATCTAC CCTGTTTACGGTGGAAAAATGTAA
43. An aldehyde dehydrogenase depicted in the following amino acid sequence (23) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (23):
(23) MPANNKKQVEKKELTAEEKKQNAQKLVDDL MTKSQAAFEKLRYYSQEQVDKICQAMALAA EEHHMDLAVDAANETGRGVAEDKAIKNIYA SEYIWNNIRHDKTVGIIEDNDEDQTIKIAD PLGVIAGIVPVTNPTSTTIFKSIISAKTRN TIIFSFHRQAMKSSIKTAKILQEAAEKAGA PKNMIQWLPESTRENTTALLQHPNTATILA TGGPSLVKAAYSSGNPALGVGPGNGPAYIE KTANIERSVYDIVLSKTFDNGMICATENSV VVDEEIYDKVKEEFQKWNCYFLKPNEIDKF TDGFIDPDRHQVRGPIAGRSANAIADMCGI KVPDNTKVIIAEYEGVGDKYPLSAEKLSPV LTMYKATSHENAFDICAQLLHYGGEGHTAA IHTLDDDLATKYGLEMRASRIIVNSPSGIG GIGNIYNNMTPSLTLGTGSYGSNSISHNVT DWDLLNIKTIAKRRENRQWVKIPPKVYFQR NSLKELQDIPNINRAFIVTGPGMSKRGYVQ RVIDQLRQRQNNTAFLVFDDVEEDPSTNTV EKGVAMMNDFKPDTIIALGGGSPMDAAKAM WMFYEHPETSWYGVMQKYLDIRKRAYQIKK PTKSQLIGIPTTSGTGSEVTPFAVITDSKT HVKYPLADYALTPNIAIVDSQFVETVPAKT TAWTGLDVLCHATESYVSVMATDYTRGWSL QTIKGVMENLPKSVQGDKLARRKMHDFSTM AGMAFGQAFLGINHSLAHKMGGAFGLPHGL LIAIAMPQVIRFNAKRPQKLALWPHYETYH ATKDYADIARFIGLKGNTDEELAEAYAKKV IELAHECGVKLSLKDNGVTREEFDKAVDDL ARLAYEDQCTTTNPVEPLVSQLKELLERCY DGTGVEEK
44. A nucleotide sequence encoding the aldehyde dehydrogenase according to claim 43, which is depicted in the following (39):
(39) ATGCCTGCTAACAACAAGAAACAAGTTGAAAAGAAAGAATTAACTGCTGAAGAAAAAAAG CAAAACGCCCAAAAGCTAGTTGACGATTTAATGACTAAGAGTCAAGCTGCTTTTGAAAAG TTACGTTACTATTCACAAGAACAAGTTGACAAGATTTGTCAGGCAATGGCTCTCGCTGCC GAAGAACACCACATGGACTTAGCTGTTGATGCTGCTAACGAAACTGGTCGTGGGGTTGCT GAAGATAAGGCTATCAAGAACATCTACGCAAGTGAATACATTTGGAACAACATCCGTCAC GATAAGACTGTTGGTATTATCGAAGACAATGATGAAGACCAAACTATCAAAATTGCTGAT CCACTTGGTGTCATTGCCGGAATTGTTCCAGTTACTAACCCTACTTCAACAACGATCTTC AAATCAATCATTAGTGCTAAGACACGGAATACAATCATCTTTTCTTTCCACCGTCAAGCA ATGAAGTCATCTATCAAGACTGCAAAGATTCTCCAAGAAGCTGCTGAAAAAGCCGGTGCG CCAAAGAACATGATTCAATGGCTCCCTGAAAGTACCCGCGAAAACACTACCGCATTACTC CAACACCCTAATACTGCTACTATTTTAGCAACCGGTGGTCCTTCATTAGTTAAGGCTGCC TACAGTTCTGGTAACCCTGCTCTTGGTGTTGGTCCTGGTAACGGTCCTGCTTACATCGAA AAAACTGCCAACATCGAACGTTCTGTTTACGACATCGTTCTTTCTAAGACATTCGATAAC GGTATGATTTGTGCCACTGAAAACTCAGTTGTTGTTGATGAAGAAATCTACGACAAGGTT AAAGAAGAATTCCAAAAGTGGAACTGTTACTTCTTGAAGCCAAACGAAATTGATAAATTT ACTGATGGCTTTATTGACCCAGATCGTCATCAAGTTCGTGGTCCAATCGCTGGTCGTTCA GCTAATGCTATTGCTGACATGTGTGGTATTAAAGTACCTGACAACACTAAGGTTATCATT GCTGAATACGAAGGTGTTGGTGACAAGTACCCACTTTCAGCTGAAAAGCTTTCACCAGTA TTAACAATGTACAAGGCAACCTCTCACGAAAATGCCTTTGATATCTGTGCTCAATTATTA CACTACGGTGGTGAAGGTCACACTGCTGCTATTCACACCCTTGATGATGATTTAGCTACT AAGTACGGTCTTGAAATGCGTGCTTCACGGATCATTGTTAACTCCCCATCTGGTATTGGT GGTATTGGTAACATCTACAACAACATGACTCCATCCCTTACTTTAGGTACTGGTTCATAC GGTAGTAACTCAATTTCTCACAACGTTACTGATTGGGACCTCTTAAACATCAAAACAATT GCAAAGCGGCGTGAAAACCGTCAATGGGTTAAGATTCCCCCAAAAGTATACTTTCAACGC AACTCACTAAAAGAATTGCAAGATATTCCAAACATTAACCGGGCATTCATCGTTACTGGT CCTGGAATGAGCAAGCGTGGTTACGTTCAACGTGTTATCGATCAATTGCGTCAACGCCAA AACAACACTGCTTTCTTAGTATTTGATGACGTTGAAGAAGATCCATCAACAAACACTGTT GAAAAAGGTGTTGCCATGATGAATGACTTCAAACCTGATACAATTATTGCTCTTGGTGGT GGTTCACCAATGGATGCTGCTAAGGCTATGTGGATGTTCTATGAGCACCCAGAAACTTCA TGGTATGGGGTTATGCAAAAGTACCTTGATATTCGGAAGCGTGCTTACCAAATCAAGAAG CCTACTAAGTCTCAACTTATTGGTATCCCTACTACATCAGGTACTGGTTCAGAAGTTACT CCATTTGCGGTTATTACCGATTCAAAAACTCATGTTAAGTACCCACTTGCTGACTACGCC TTAACACCAAACATTGCAATCGTTGACTCACAATTCGTTGAAACTGTTCCAGCAAAAACT ACTGCTTGGACTGGACTAGATGTTTTATGTCACGCTACTGAATCATATGTTTCTGTTATG GCAACTGACTACACTCGTGGTTGGTCACTACAAACCATCAAGGGTGTTATGGAAAACCTT CCTAAGTCAGTTCAAGGTGATAAGTTAGCTCGTCGTAAGATGCACGACTTCTCAACAATG GCCGGGATGGCATTTGGTCAAGCCTTCTTAGGAATTAACCACTCCCTTGCCCACAAGATG GGTGGAGCATTCGGTCTTCCTCACGGTTTGCTTATCGCTATTGCAATGCCACAAGTAATT CGCTTTAACGCAAAACGTCCACAAAAGCTTGCTCTCTGGCCTCACTATGAGACTTACCAT GCAACTAAGGACTACGCTGACATTGCACGGTTCATTGGTTTGAAAGGCAACACTGATGAA GAATTAGCTGAAGCATATGCTAAGAAAGTTATCGAACTTGCTCACGAATGTGGTGTTAAG CTTAGTCTTAAGGACAATGGTGTTACACGTGAAGAATTTGATAAGGCGGTTGACGATCTT GCTCGCTTAGCTTACGAAGATCAATGTACTACTACTAACCCAGTTGAACCACTTGTTAGC CAACTCAAGGAATTACTTGAACGTTGCTACGATGGTACTGGCGTTGAAGAAAAATAA
45. An aldehyde dehydrogenase depicted in the following amino acid sequence (24) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (24):
(24) MAYQSINPFTNQVEKTFENTTDEELEQTLT TAHQLYLDWRKYNDLEERKRQILKLGQILR ERRVEYATVMSKEMGKLISEAEGEVDLCAS FCDYYAAHADEFLQPKIIATTSGRAKVLKQ SLGILVAVEPWNFPFYQIARVFIPNFIAGN PMILKDASNCPASAQAFNDAVKEAGAPAGS LTNLFLSYDQVNKAIADKRVAGVCLTGSER GGATVAKEAGANLKKSTLELGGNDAFIILD DADWDLVEKVAPAARLYNAGQVCTSSKRFI VLEKDYDRFLKMMKDAFSKVKMGDPLDPLT TLAPLSSKKAKEKLQQQVATAVENGAKVYY GNKPVDMEGQFFMPTILTDITPDNPIFDTE MFGPVASVYKVSSEEEAIELANNSSYGLGN TIFSNDSEHAERVAAKIETGMSWINAGWAS LPELPFGGVKNSGYGRELSSYGIDEFTNKH LIYEARQ
46. A nucleotide sequence encoding the aldehyde dehydrogenase according to claim 45, which is depicted in the following (40):
(40) ATGGCATATCAAAGTATCAATCCATTTACGAACCAAGTAGAAAAAACGTTTGAAAATACA ACTGATGAAGAATTAGAACAAACATTAACTACGGCGCATCAATTATATTTAGATTGGCGG AAGTATAATGACCTTGAAGAACGGAAACGGCAAATTTTAAAGTTAGGTCAAATATTACGT GAACGGCGTGTTGAATATGCGACAGTTATGAGTAAGGAAATGGGAAAATTAATTAGCGAA GCAGAAGGCGAGGTTGACCTTTGTGCTTCTTTCTGTGATTATTATGCAGCCCATGCAGAT GAATTTCTGCAACCAAAAATTATTGCGACAACGAGTGGACGCGCCAAAGTTTTGAAGCAA TCATTAGGAATTTTAGTTGCAGTTGAACCTTGGAATTTCCCATTCTATCAAATTGCCCGG GTATTTATTCCCAACTTTATTGCAGGAAACCCCATGATCTTGAAGGATGCGTCGAATTGT CCAGCATCCGCCCAAGCATTTAACGATGCCGTTAAGGAAGCTGGTGCGCCAGCCGGCAGT TTAACTAATTTATTCCTTTCATATGACCAAGTAAATAAGGCAATTGCTGATAAGCGGGTA GCCGGCGTTTGTCTTACTGGTTCTGAACGTGGTGGTGCAACCGTTGCTAAAGAGGCTGGT GCTAATTTGAAGAAGAGCACTTTGGAACTTGGTGGTAATGATGCCTTTATTATCTTAGAC GATGCAGATTGGGATCTTGTCGAAAAAGTTGCCCCGGCAGCCCGTCTGTATAATGCTGGA CAAGTATGTACATCATCAAAACGTTTTATTGTCCTTGAAAAGGATTATGATCGTTTCTTA AAGATGATGAAAGATGCGTTCTCGAAAGTTAAAATGGGTGATCCCCTTGATCCATTAACA ACTCTGGCACCATTATCATCTAAGAAAGCAAAAGAAAAGCTCCAACAGCAAGTCGCAACA GCAGTAGAAAATGGGGCCAAAGTTTACTATGGTAATAAGCCGGTTGACATGGAAGGTCAA TTCTTTATGCCAACGATCTTAACTGATATCACTCCAGATAACCCAATATTTGATACGGAA ATGTTTGGGCCAGTGGCTTCGGTTTATAAGGTTAGTTCCGAAGAGGAAGCAATCGAACTG GCTAATAATTCAAGCTATGGGTTAGGAAACACTATCTTTAGCAATGATTCCGAACATGCG GAACGAGTAGCAGCGAAGATCGAAACTGGAATGAGTTGGATTAATGCCGGCTGGGCTTCA TTACCAGAATTACCATTTGGTGGTGTTAAGAATTCAGGTTACGGTCGTGAACTCAGCAGT TACGGAATTGATGAATTTACTAACAAACATCTAATTTACGAAGCACGACAATAA
47. An aldehyde dehydrogenase depicted in the following amino acid sequence (25) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (25):
(25) MQINDIESAVRKILAEELDNASSSSANVAA TTDNGHRGIFTNVNDAIAAAKAAQEIYRDK PIAVRQQVIDAIKEGFRPYIEKMAKDIKEE TGMGTVEAKIAKLNNALYNTPGPEILEPVV ENGDGGMVMYERLPYGVIGAVGPSTNPSET VIANAIMMLAGGNTLYFGAHPGAKNVTRWT IEKMNDFIADATGLHNLVVSIETPTIESVQ QMMKHPDIAMLAVTGGPAVVHQAMTSGKKA VGAGPGNPPAMVDATADIDLAAHNIITSAS FDNDILCTAEKEVVAESSIKDELIRKMQDE GAFVVNREQADKLADMCIQENGAPDRKFVG KDATYILDQANIPYTGHPVEIICELPKEHP LVMTEMLMPILPVVSCPTFDDVLKTAVEVE KGNHHTATIHSNNLKHINNAAHRMQCSIFV VNGPSYVGTGVADNGAHSGASALTIATPTG EGTCTARTFTRRVRLNSPQGFSVRNWY
48. A nucleotide sequence encoding the aldehyde dehydrogenase according to claim 47, which is depicted in the following (41):
(41) ATGCAGATTAATGATATTGAAAGTGCTGTACGCAAAATTCTTGCCGAAGAACTAGATAAT GCCAGCTCTTCAAGTGCAAACGTTGCAGCTACTACTGATAATGGTCATCGCGGAATTTTC ACTAATGTCAATGATGCAATTGCTGCTGCAAAAGCTGCTCAAGAAATATATCGGGATAAG CCAATTGCTGTTCGCCAACAAGTGATTGATGCCATTAAGGAAGGATTCCGCCCATATATT GAAAAAATGGCTAAAGATATCAAAGAAGAAACAGGAATGGGAACAGTAGAGGCCAAAATT GCTAAGTTAAACAATGCCTTGTACAACACTCCTGGTCCCGAGATTCTTGAACCAGTTGTA GAAAACGGTGACGGTGGGATGGTTATGTATGAACGGTTACCATATGGTGTTATTGGTGCG GTTGGCCCAAGTACAAACCCTTCAGAAACTGTAATTGCTAATGCGATCATGATGCTTGCC GGTGGTAATACTCTTTACTTTGGTGCTCACCCTGGCGCAAAGAATGTTACTCGCTGGACA ATTGAAAAGATGAACGATTTTATTGCAGATGCAACAGGCCTTCATAATTTAGTTGTAAGT ATTGAAACACCAACAATTGAATCAGTTCAACAAATGATGAAGCACCCCGACATTGCAATG TTAGCAGTAACTGGTGGCCCAGCTGTTGTTCACCAAGCAATGACCAGTGGTAAGAAAGCG GTTGGTGCTGGTCCTGGTAATCCTCCTGCAATGGTTGATGCTACTGCTGATATTGATTTA GCTGCTCATAATATCATTACATCTGCTTCATTTGATAATGATATTTTATGTACTGCTGAA AAGGAAGTAGTTGCAGAAAGTAGCATTAAAGATGAATTAATTCGTAAGATGCAAGATGAA GGTGCCTTTGTAGTTAACCGTGAACAAGCCGATAAATTAGCTGATATGTGTATCCAAGAA AATGGTGCTCCTGATCGTAAATTTGTTGGTAAGGATGCAACTTATATCTTAGACCAAGCT AATATTCCTTACACAGGCCACCCAGTTGAAATTATTTGTGAACTTCCTAAGGAACATCCA TTAGTAATGACTGAAATGTTAATGCCAATTTTACCAGTTGTTTCTTGTCCAACATTTGAT GATGTTTTGAAGACTGCTGTTGAAGTTGAAAAAGGTAACCATCACACAGCTACTATTCAT TCCAATAACCTTAAGCATATTAATAATGCTGCTCACCGGATGCAATGTTCAATCTTTGTT GTTAATGGCCCATCCTATGTTGGTACAGGTGTTGCAGATAATGGAGCTCACTCAGGTGCT TCAGCATTAACAATTGCTACGCCAACTGGTGAAGGAACATGTACTGCACGAACATTTACT CGTCGGGTTCGTTTGAACTCACCACAAGGATTCTCAGTACGTAACTGGTATTAA
49. A glycerate kinase depicted in the following amino acid sequence (26) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (26):
(26) MKFVIAPDSFKGGLTAKEAANVMAEGIKRV FPNAEYALVPMADGGEGTVQSLVDATNGQK MIAKVHNPLNKLVNAEYGILGDGETAVIEM AAASGLQFVNKETANPLITTTYGTGELIKD ALDHNIKKIIIGIGGSATVDGGAGMAQALG ARLLDADNHEIGLGGGELASLEQVDFGGLD PRLKNVDIQIASDVTNPLTGKNGAAPVFGP QKGADEEMVNILDKNLHHYARKIVAAGGPD VEQTAGAGAAGGLGAGLIAFTGATMKRGVE LVIEATQLQKKAVGADYVFTGEGGIDFQTK FGKTPYGVAKATKEVAPTAPVIVLAGNIGK GVNDLYSSTAIDAIFATPEGAKPLKTALAD APIDIAQTAENVARLIKVSHVSN
50. A nucleotide sequence encoding the glycerate kinase according to claim 49, which is depicted in the following (42):
(42) ATGAAATTTGTAATTGCTCCAGATTCATTTAAAGGCGGATTAACAGCAAAAGAAGCAGCA AATGTGATGGCAGAAGGAATCAAAAGAGTGTTTCCGAATGCCGAGTATGCTTTAGTTCCA ATGGCTGATGGAGGAGAGGGGACTGTTCAATCCTTAGTTGATGCGACTAACGGTCAAAAA ATGATTGCTAAAGTCCACAACCCATTAAATAAATTAGTTAATGCTGAGTACGGAATATTA GGTGATGGGGAAACGGCAGTGATTGAGATGGCGGCGGCAAGTGGCCTTCAATTTGTTAAT AAGGAGACTGCGAACCCGCTTATTACAACTACATATGGTACCGGCGAGTTAATTAAGGAT GCTCTTGACCATAACATTAAAAAAATAATTATTGGAATTGGTGGAAGTGCAACCGTTGAT GGCGGAGCGGGGATGGCCCAAGCACTTGGAGCACGTTTATTGGATGCTGATAATCATGAA ATTGGTTTAGGCGGTGGTGAGTTAGCAAGTTTAGAGCAAGTAGATTTTGGAGGATTAGAT CCTCGCTTAAAAAATGTAGATATTCAGATTGCATCAGACGTAACCAACCCATTAACAGGA AAAAATGGGGCAGCCCCAGTATTTGGCCCGCAAAAAGGAGCTGATGAAGAAATGGTGAAC ATCTTGGACAAAAATCTTCATCATTATGCCCGAAAAATAGTTGCAGCTGGTGGGCCAGAC GTTGAACAAACGGCAGGTGCAGGGGCAGCCGGTGGTTTAGGAGCCGGGTTGATAGCATTT ACCGGTGCGACAATGAAGCGAGGAGTAGAATTAGTGATTGAAGCAACTCAACTACAAAAA AAGGCAGTTGGCGCTGATTATGTTTTTACTGGTGAAGGAGGAATTGATTTCCAGACTAAA TTTGGTAAAACGCCATATGGAGTCGCTAAGGCAACTAAAGAGGTGGCTCCAACTGCTCCG GTAATTGTGTTGGCTGGAAATATTGGTAAAGGCGTAAATGATCTATATTCATCCACGGCC ATTGATGCAATTTTTGCAACTCCTGAAGGGGCTAAACCATTAAAAACAGCATTAGCAGAT GCACCTATTGATATTGCTCAAACAGCGGAAAACGTTGCACGTTTAATTAAAGTGAGTCAT GTTAGTAATTAA
51. A glycerol kinase depicted in the following amino acid sequence (27) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (27):
(27) LSEQQYIMAIDQGTTSSRAIIFDHDGNKVA ISQQEFPQYFPQPGWVEHDPLEIWDSVQSV ISNVMIKSQIKPYKIAAIGITNQRETTVIW DRHTGKPIYNAIVWQSKQTSDIAEQLIKDG YKDMIHQKTGLVIDSYFAATKIKWILDHVP GAREKAAKGDLMFGTIDTWLLWNLSGRRVH ATDVTNASRTMLFNIHTLDWDQDILDLLDI PQSLLPVVKPSSAIYGYTGDYHFYGVQIPI AGIAGDQQAALFGQAAYDKGSIKNTYGTGA FIVMNTGLKPTLSDNGLLTTIAYGLDGQTH YALEGSIFVAGSAVQWLRDGLKMFDKASES EQMAVDAKTTGGVYVVPAFTGLGAPYWDQE VRGAMFGLTRGTERGHIIRATLEAIAYQTK DVVDTMVKDTQLPLTALTVNGGASRNNFMM QFQADILQTPIKRAAMEETTALGAAFLAGL AVDFWEDQDELRKLSRIGDQFDPQMDPQKA ADLYRGWQRAIAAAQFYGKD
52. A nucleotide sequence encoding the glycerol kinase according to claim 51, which is depicted in the following (43):
(43) TTGAGTGAACAACAATATATCATGGCGATTGACCAGGGAACGACGAGCTCACGGGCGATT ATCTTTGACCATGACGGAAATAAGGTTGCGATCAGTCAGCAGGAATTTCCCCAATACTTC CCGCAGCCGGGGTGGGTTGAACATGATCCTCTAGAGATTTGGGATAGCGTTCAATCAGTG ATTTCAAATGTAATGATTAAGTCCCAGATCAAGCCCTATAAGATTGCGGCAATTGGGATT ACTAACCAACGGGAGACGACGGTTATTTGGGATCGCCATACCGGTAAGCCGATTTATAAC GCAATTGTCTGGCAATCGAAGCAAACGAGCGACATCGCCGAACAATTGATTAAAGATGGT TATAAGGATATGATCCACCAGAAGACTGGCTTGGTGATTGATTCGTATTTCGCGGCCACT AAGATCAAGTGGATCCTTGACCATGTTCCTGGTGCCCGGGAAAAAGCAGCAAAGGGAGAC TTGATGTTTGGGACTATCGATACTTGGTTACTATGGAATTTATCGGGACGGCGGGTCCAC GCAACGGATGTGACCAATGCCAGCCGGACGATGCTTTTTAATATCCATACCCTCGACTGG GATCAAGATATCCTTGACCTGCTTGATATTCCCCAGTCGCTTTTGCCAGTAGTAAAGCCA AGTTCAGCCATTTACGGTTATACTGGCGACTACCACTTCTATGGGGTGCAGATTCCAATT GCCGGGATTGCAGGTGACCAACAAGCAGCCCTCTTTGGTCAAGCAGCCTATGATAAAGGT TCAATCAAGAACACCTATGGGACTGGAGCCTTCATCGTCATGAATACGGGACTAAAACCC ACGCTTTCGGATAACGGCTTGTTGACGACGATTGCGTATGGCCTGGACGGGCAAACTCAT TACGCGCTTGAAGGAAGTATCTTTGTGGCCGGTTCTGCCGTTCAATGGTTGCGGGATGGT CTCAAGATGTTTGATAAGGCAAGCGAGTCCGAACAAATGGCTGTCGATGCCAAGACAACT GGCGGCGTTTATGTCGTCCCCGCCTTTACAGGATTAGGCGCACCGTACTGGGATCAAGAA GTGCGGGGCGCAATGTTTGGCCTTACCCGTGGAACTGAACGGGGACATATCATCCGTGCA ACTTTGGAAGCCATTGCCTACCAGACCAAAGATGTTGTCGATACGATGGTCAAGGACACC CAATTACCACTAACAGCACTAACGGTTAACGGGGGCGCTTCACGGAACAACTTCATGATG CAGTTCCAGGCCGATATCTTACAAACGCCAATCAAGCGGGCAGCAATGGAAGAGACAACC GCGCTGGGAGCAGCCTTTCTCGCTGGATTGGCCGTTGATTTCTGGGAAGACCAGGATGAG TTACGGAAGCTATCACGGATTGGCGACCAGTTTGATCCACAAATGGATCCGCAAAAGGCA GCTGACTTGTATCGGGGATGGCAACGGGCCATTGCAGCTGCGCAGTTTTATGGCAAAGAT TAA
53. A glycerol-3-phosphate dehydrogenase depicted in the following amino acid sequence (28) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (28):
(28) MAEKIAVLGAGSWGSVLANMLTENGHDVTL WSRNEEQVKQLNTEHTNPRYMKDFVYSTNL TATTDMKKAVKGASVVLIVIPTKGLREVAK QLNAILTELHQKPLVIHATKGLEQNTYKRP SEMLSEDISPENRQAIVVLSGPSHAEDVAI KDMTAVTAACEDLASAKKAQKLFSNSYFRV YTNDDVIGAEFGAALKNIIAIGAGAIQGLG YHDNARAALITRGLAEIRRLGVAFGANPMT FIGLSGVGDLVVTATSKNSRNWRAGYQLGQ GKKLQDVIDNMGMVIEGVYTTKAAYELSRK RQVQMPITEALYRVLYEGEDIKTAISQLMD RDLTSENE
54. A nucleotide sequence encoding the glycerol-3-phosphate dehydrogenase according to claim 53, which is depicted in the following (44):
(44) ATGGCAGAAAAAATTGCTGTTTTAGGTGCTGGTTCGTGGGGCAGTGTTTTAGCAAACATG CTTACAGAAAATGGCCACGATGTAACATTATGGTCTCGTAATGAGGAACAAGTTAAGCAA TTAAATACTGAACATACAAATCCTCGCTATATGAAAGATTTTGTTTATTCTACTAACTTA ACAGCAACAACGGACATGAAAAAAGCTGTTAAGGGTGCCAGTGTGGTCCTGATTGTAATT CCAACAAAGGGTCTTCGTGAAGTTGCTAAGCAATTAAATGCAATTTTGACTGAATTACAT CAAAAACCGCTAGTTATTCACGCAACGAAAGGCTTAGAACAAAATACTTATAAGCGGCCA TCGGAAATGCTTAGCGAAGATATTTCTCCTGAAAACCGTCAGGCAATTGTTGTTTTATCA GGTCCGAGTCATGCTGAAGATGTGGCGATTAAAGATATGACAGCTGTAACCGCAGCTTGT GAGGACCTGGCCAGTGCTAAAAAGGCGCAGAAGTTATTTAGTAATTCTTATTTCCGTGTG TACACTAATGACGATGTAATTGGTGCCGAATTTGGCGCAGCCTTAAAGAACATTATTGCA ATTGGTGCTGGAGCTATTCAGGGACTTGGTTATCATGATAATGCTCGGGCAGCGTTAATT ACTCGTGGACTTGCAGAAATTCGCCGATTGGGAGTTGCTTTTGGTGCCAACCCGATGACT TTTATTGGTCTTTCTGGGGTTGGTGACCTTGTTGTTACTGCTACCAGTAAAAATTCTCGA AATTGGCGTGCTGGCTATCAATTGGGGCAAGGAAAAAAGCTTCAAGATGTAATTGATAAT ATGGGAATGGTTATCGAAGGTGTCTATACTACCAAAGCCGCTTATGAATTAAGTCGTAAA CGACAAGTACAGATGCCAATTACCGAAGCTCTTTACCGTGTTTTGTATGAAGGCGAAGAT ATTAAAACTGCAATTTCTCAATTAATGGACCGAGATCTTACTTCAGAAAACGAATAA
55. A triosephosphate isomerase depicted in the following amino acid sequence (29) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (29):
(29) MRKPFIAANWKMHKNVQESVEFVDAIKGKL PDPQEVEVGIAAQAFALPSMVQAADDSGLK IIAQNAAAEYSGAFTGEISLRGLADAGVSY VMLGHIERRHLFHEDNELVNRKVLAALQMG VTPIICTDETMVQKEVNGEIHYVFQQLMSV LRGVSLDQIKNVVVSYEPSWAVGYGQHANP VLAEEGCRQIRRTIADNYTYEIADKIRILY GGSVNPDNIGMIMNKPDVDGVLIGRASLDV DNFLRMVNYLKNDQEK
56. A nucleotide sequence encoding the triosephosphate isomerase according to claim 55, which is depicted in the following (45):
(45) ATGCGCAAACCCTTTATTGCTGCTAATTGGAAGATGCATAAGAATGTCCAAGAATCGGTT GAATTTGTGGATGCAATTAAAGGAAAGCTACCAGATCCGCAAGAAGTTGAAGTCGGAATT GCAGCCCAAGCTTTTGCATTACCCAGTATGGTTCAAGCCGCTGATGATTCAGGATTAAAG ATAATCGCGCAAAACGCGGCGGCTGAATATTCGGGAGCTTTCACTGGTGAAATTAGCTTA CGAGGTTTAGCTGACGCCGGTGTTTCATATGTAATGTTAGGACATATTGAACGGCGCCAT TTATTCCACGAGGATAATGAGTTGGTTAATCGGAAAGTGTTGGCAGCCCTTCAAATGGGA GTTACCCCGATAATTTGTACGGATGAAACGATGGTCCAGAAAGAAGTTAATGGTGAAATT CACTACGTTTTCCAGCAATTGATGAGCGTATTGAGGGGCGTTTCTCTTGATCAAATTAAA AATGTAGTTGTTTCCTATGAACCAAGTTGGGCAGTTGGATATGGTCAGCATGCTAATCCA GTTCTTGCTGAAGAAGGATGCCGTCAAATTCGGCGAACGATTGCTGATAACTACACTTAT GAGATTGCTGATAAGATCAGGATTCTTTATGGGGGCAGTGTCAATCCAGATAATATCGGA ATGATTATGAACAAGCCAGATGTAGATGGGGTATTAATCGGTCGGGCAAGTTTAGATGTT GATAATTTTTTGCGAATGGTCAATTATTTAAAAAATGATCAAGAAAAATAA
57. A triosephosphate isomerase depicted in the following amino acid sequence (30) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (30):
(30) MRKPFIIANWKMNKNVHESVAFVKAIKEKL PADKEIGIAAQAVSLYNMKKVASSSNLQII AQNASAELEGPYTGEISMRSLADAGVTYVM LGHLERRRLFNESNDSINQKVLAALNAGII PIICTDEEMVQTEVNGQIHYVFRQLKSVLK GVPANKLSQIVISYEPSWAVGSTHQANPDI AEEGCQAIRQSLVEMYGNEIGEQVRILYGG SVNPENIGQIMSKPNVDGALIGRASLEIES FLQMINYIELASKQKLQVI
58. A nucleotide sequence encoding the triosephosphate isomerase according to claim 57, which is depicted in the following (46):
(46) ATGCGCAAACCGTTTATTATTGCGAACTGGAAAATGAATAAAAACGTTCATGAATCTGTT GCGTTTGTTAAAGCAATTAAAGAAAAGCTCCCGGCAGATAAAGAAATTGGGATCGCCGCG CAAGCAGTTTCGCTATATAACATGAAAAAAGTGGCGAGCTCTTCCAACTTACAAATTATT GCTCAAAATGCATCTGCTGAGTTAGAGGGACCATATACTGGAGAAATTAGCATGCGAAGT TTAGCAGATGCGGGCGTGACATACGTGATGCTAGGCCATTTAGAGCGCCGACGCCTTTTT AACGAGAGTAATGATTCAATTAATCAAAAAGTTTTAGCAGCCCTCAATGCTGGTATTATT CCAATCATTTGTACGGATGAAGAGATGGTCCAAACAGAAGTTAACGGACAAATTCATTAT GTATTTCGCCAACTAAAAAGCGTCCTTAAAGGGGTACCAGCTAATAAACTATCACAGATT GTTATTTCGTATGAACCAAGTTGGGCCGTTGGGAGCACGCATCAAGCAAATCCAGACATT GCGGAAGAGGGATGTCAGGCAATTCGTCAAAGCCTGGTTGAAATGTATGGTAATGAGATT GGCGAGCAAGTCCGAATACTCTATGGTGGCAGCGTTAATCCCGAGAACATTGGTCAAATT ATGAGTAAACCAAATGTTGATGGGGCGCTAATCGGTCGCGCAAGTCTCGAGATTGAAAGT TTCTTACAAATGATTAATTATATCGAATTAGCGAGCAAGCAGAAGTTACAGGTAATTTAG
59. A triosephosphate isomerase depicted in the following amino acid sequence (31) or an amino acid sequence having deletion, substitution or addition of one or more amino acids in the amino acid sequence (31):
(31) MRVPIIAGNWKMHKDVQEAVSFIEKVKNQL PPADQLETAIAAPTLCLVPMVKAAEESPLK IMAENCYYKNEGAYTGETSPYALYQAGIHH VILGHSERRTYFNETDELINKKVKAALVNG LCPIVCCDDTMRRRVAGKKVHWVVSRILAD LHGLTNDEICHVTVAYEPSWAIGTGESADP EQAAEGCYLIRQTISDMYGDEVANNVRILY GGSVTTSNINALMAKNDIDGVLVGAASLNP ETFLQLVHH
60. A nucleotide sequence encoding the triosephosphate isomerase according to claim 59, which is depicted in the following (47):
(47) ATGAGAGTACCGATTATTGCTGGTAATTGGAAAATGCATAAGGATGTACAAGAAGCTGTC TCTTTTATCGAAAAAGTAAAAAATCAGCTTCCGCCTGCCGACCAACTTGAAACAGCAATT GCTGCTCCTACTCTTTGTTTAGTACCAATGGTTAAAGCAGCTGAAGAATCCCCGTTAAAA ATAATGGCAGAAAACTGCTACTATAAGAATGAGGGAGCTTATACTGGTGAAACAAGTCCA TATGCTTTATACCAAGCAGGAATCCATCATGTGATTTTAGGCCATTCTGAACGCCGAACT TACTTTAATGAAACTGATGAATTAATTAATAAAAAAGTGAAGGCAGCATTAGTAAATGGG TTATGTCCGATTGTTTGTTGTGATGATACTATGCGTCGACGAGTTGCTGGAAAGAAAGTT CATTGGGTGGTGAGCCGAATTCTCGCTGACCTTCATGGATTGACCAATGACGAAATTTGT CATGTTACGGTTGCTTATGAACCAAGTTGGGCGATTGGAACAGGCGAGAGTGCTGATCCA GAACAAGCGGCGGAAGGTTGTTACCTTATTCGGCAAACGATTAGTGATATGTATGGCGAT GAAGTTGCAAATAACGTTCGAATTCTCTATGGCGGAAGTGTGACAACTTCTAATATCAAT GCACTAATGGCAAAAAATGATATTGATGGTGTTTTAGTCGGAGCGGCGAGCTTAAATCCA GAAACATTTTTACAATTAGTTCACCATTAG
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