JP2015216863A - Novel lipase and gene thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel lipase, polynucleotide encoding the enzyme, a recombinant vector comprising the polynucleotide, a transformant having the vector introduced thereinto, a production method for the enzyme, and a use method for the enzyme.SOLUTION: A novel lipase is obtained from Chlorella algae such as Chlorella kessleri.

Description

  The present invention relates to a novel lipase, a polynucleotide encoding the enzyme, a recombinant vector containing the polynucleotide, a transformant introduced with the vector, a method for producing the enzyme, and a method for using the enzyme .

  “Lipase” is a general term for enzymes that hydrolyze glycerolipids.

  “Glycerolipid” refers to a lipid having a glycerol skeleton. Examples of the glycerolipid include glyceroglycolipid, glycerophospholipid, and neutral fat.

  “Glyceroglycolipid” refers to a compound in which a sugar chain is covalently bonded to a free hydroxyl group of diacylglycerol. Examples of the glyceroglycolipid include galactolipids such as monogalactosyl diacylglycerol and digalactosyl diacylglycerol. Glyceroglycolipids such as galactolipids exist as constituent lipids in cyanobacterial cell membranes and chloroplast thylakoid membranes (Non-patent Document 1).

An enzyme that exhibits lipase activity on glyceroglycolipid is also referred to as galactolipase. Galactolipase is known to exist in higher plants such as spinach having chloroplasts (Non-patent Document 2). As galactolipase, galactolipase derived from Aspergillus japonicus (patent document 1) and galactolipase derived from Chlamydomonas reinhardtii (non-patent document 3) are also known.

  Galactolipase is used, for example, as a quality improving agent for breadmaking. Moreover, galactolipase is utilized as a component of a detergent in combination with chlorophytase, for example.

  However, the known galactolipase has a problem that, for example, its production amount is low and it is difficult to apply it to industries. Moreover, since the galacto lipase described in Patent Document 1 has a lipase activity for lecithin, when used for processing a material containing lecithin, there may be a problem that a by-product is generated. Furthermore, since the galactolipase described in Patent Document 1 is derived from mold, it is not suitable for processing food materials because it requires removal of antibiotics and allergens in order to use it in the food field. Further, the galacto lipase described in Patent Document 1 has a drawback that there is no mass production method since an expression method by a gene recombination method has not been established.

JP2008-206515

Koichi Kobayashi Photosynthesis Research 2009 19: 52-58 Sakaki T, et al., Plant Physiol. 1990 Oct; 94 (2): 773-80. Li X, et al., Plant Cell 11, 4670 (2012)

  An object of the present invention is to provide a novel lipase.

  As a result of intensive studies to solve the above problems, the present inventor found galactolipase from Chlorella kessleri and completed the present invention. The homology of the amino acid sequence between this enzyme and the known galactolipase was very low, and this enzyme was a novel galactolipase that was completely different from the known galactolipase.

That is, the present invention is as follows.
[1]
Lipases having the following properties (1) to (3):
(1) having an activity of catalyzing a reaction of hydrolyzing a carboxyl ester bond of glycerolipid;
(2) the molecular weight is from 50,000 to 60,000;
(3) Derived from the genus Chlorella.
[2]
The lipase, wherein the glycerolipid is a glyceroglycolipid.
[3]
The lipase, wherein the Chlorella algae is Chlorella kessleri.
[4]
Lipase, which is a protein described in the following (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 8 or 11;
(B) the amino acid sequence shown in SEQ ID NO: 8 or 11, comprising an amino acid sequence containing substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and hydrolyzing the carboxyl ester bond of glyceroglycolipid A protein having an activity of catalyzing a decomposing reaction.
[5]
A polynucleotide encoding the lipase.
[6]
The polynucleotide according to (A) or (B) below:
(A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 7 or 10;
(B) encodes a protein comprising a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 7 or 10 and having an activity of catalyzing a reaction of hydrolyzing a carboxyl ester bond of glyceroglycolipid Polynucleotide.
[7]
A recombinant vector comprising the polynucleotide.
[8]
A transformant introduced with the recombinant vector.
[9]
A method for producing a lipase, comprising culturing the transformant in a medium to produce the lipase, and collecting the lipase from the culture.
[10]
A method for hydrolyzing glycerolipid, which comprises allowing the lipase to act on glycerolipid.
[11]
The method, wherein the glycerolipid is a food or drink containing glycerolipid or a raw material thereof. [12]
The method to modify food-drinks or its raw material including making the said lipase act on the food-drinks containing glycerolipid, or its raw material.
[13]
A method for producing glycoglycerol, comprising causing the lipase to act on glyceroglycolipid.

  According to the present invention, a novel lipase is provided. In one embodiment, the functionality of the food material can be improved using the enzyme.

The photograph which shows the analysis result by SDS-PAGE of galactolipase (it describes as chGL) derived from Chlorella kessleri 11h strain (UTEX 263). Lane M: marker, Lane 1: purified enzyme. The figure which shows the substrate specificity of ChGL. The figure which shows the temperature dependence of the activity of ChGL. The figure which shows the temperature stability of ChGL. The figure which shows the pH dependence of the activity of ChGL. The figure which shows the pH stability of ChGL. The figure which shows the Ca2 ⁺ ion concentration dependence of the activity of ChGL. The figure which shows the TritonX-100 density | concentration dependence of the activity of ChGL. The photograph of SDS-PAGE analysis which shows the result of expressing TF-chGL fusion protein in Escherichia coli. Photo of SDS-PAGE analysis showing the results of treating TF-chGL fusion protein with Factor-Xa. The figure which shows the result of the homology comparison with ChGL and galactolipase derived from Aspergillus japonicus. The figure which shows the result of the homology comparison with ChGL and galactolipase derived from Chlamydomonas reinhardtii. The figure which compared the temperature stability of ChGL and lipopan F. The figure which shows the physical-property change effect of flour by ChGL. The figure of the HPLC analysis which shows the result of having processed digalactosyl diacylglycerol (DGDG) with the TF-chGL fusion protein processed with FactorXa.

<1> Lipase of the present invention The present invention provides a novel lipase. The lipase provided in the present invention is also referred to as “the lipase of the present invention”.

  In the present invention, “lipase” refers to a protein having an activity of catalyzing a reaction of hydrolyzing an ester bond (carboxyl bond) of glycerolipid. More specifically, the “ester bond (carboxyl bond)” is an ester bond between a hydroxyl group of a glycerol skeleton constituting a glycerolipid and a fatty acid. Carboxylic acid (fatty acid) is liberated by hydrolysis of the ester bond. This activity is also referred to as “lipase activity”.

  “Glycerolipid” refers to a lipid having a glycerol skeleton. Examples of the glycerolipid include glyceroglycolipid, glycerophospholipid, and neutral lipid.

“Neutral lipid” is a general term for monoacylglycerol, diacylglycerol, and triacylglycerol. “Monoacylglycerol” refers to a compound in which a fatty acid is ester-bonded to one of the three hydroxyl groups of glycerol. “Diacylglycerol” refers to a compound in which a fatty acid is ester-bonded to two of the three hydroxyl groups of glycerol. “Triacylglycerol” refers to a compound in which fatty acids are ester-bonded to all three hydroxyl groups of glycerol. The position of the hydroxyl group to which the fatty acid is bonded is not particularly limited. In the case of monoacylglycerol, the hydroxyl group to which the fatty acid is bonded may be the 1-position, 2-position, or 3-position hydroxyl group of the glycerol skeleton. In the case of diacylglycerol, the hydroxyl group to which the fatty acid is bonded may be the 1st and 2nd, 1st and 3rd, or 2nd and 3rd hydroxyl groups of the glycerol skeleton.

  “Glyceroglycolipid” refers to a compound in which a sugar chain is covalently bonded to a free hydroxyl group of diacylglycerol. “Free hydroxyl group” refers to a hydroxyl group that is not ester-bonded to a fatty acid. For example, when diacylglycerol is 1,2-diacylglycerol, the hydroxyl group at the 3-position of the glycerol skeleton is a free hydroxyl group. The type and length of the sugar chain are not particularly limited. Examples of the glyceroglycolipid include galactolipid. “Galactolipid” refers to a compound in which a galactose chain (for example, one molecule or two molecules of galactose) is covalently bonded to a free hydroxyl group of diacylglycerol. Examples of the galactolipid include monogalactosyl diacylglycerol and digalactosyl diacylglycerol. Other glyceroglycolipids include sulfoquinovosyl diacylglycerol. Monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol are glyceroglycolipids when the sugar chain is one molecule of galactose, two molecules of galactose, and one molecule of sulfoquinobose, respectively. .

  “Glycerophospholipid” refers to a compound in which a phosphoryl base is covalently bonded to a free hydroxyl group of diacylglycerol. Examples of the base include choline, ethanolamine, inositol, serine, and glycerol. Examples of the glycerophospholipid include lecithin.

  The position and number of ester bonds to be hydrolyzed are not particularly limited. In the case of glyceroglycolipids, either one or both of the two ester bonds may be hydrolyzed. In the case of glycerophospholipids, either one or both of the two ester bonds may be hydrolyzed. In the case of a neutral lipid, 1 to 3 ester bonds among 1 to 3 ester bonds may be hydrolyzed.

  The lipase may exhibit lipase activity only for one glycerolipid, or may exhibit lipase activity for two or more glycerolipids. The lipase preferably exhibits lipase activity for at least glyceroglycolipid, and more preferably exhibits lipase activity for at least galactolipid. The lipase activity for glyceroglycolipid is also called “galactolipase activity”, and the lipase having the same activity is also called “galactolipase”.

  The lipase may have a molecular weight measured by SDS-PAGE of 50,000-60,000.

Examples of the lipase include galactolipase of the genus Chlorella. Examples of Chlorella algae include Chlorella kessleri, Chlorella pyrenoidosa, and Chlorella vulgaris. As Chlorella kessleri, Chlorella kessleri
11h strain (UTEX 263). Chlorella kessleri 11h shares the University of Texas at Austin, The Culture Collection of
Algae (UTEX), 1 University Station A6700, Austin, TX 78712-0183, USA). That is, the lipase may be a protein derived from such an algae, for example.

The amino acid sequence of galactolipase of Chlorella kessleri 11h strain (UTEX 263) and the base sequence of the gene (cDNA) encoding it are shown in SEQ ID NOs: 8 and 7, respectively. That is, the lipase may be a protein having the amino acid sequence shown in SEQ ID NO: 8, for example. The lipase may be a protein encoded by a gene having the base sequence shown in SEQ ID NO: 7, for example. In addition, the expression “having an (amino acid or base) sequence” includes the case of “including the (amino acid or base) sequence” and the case of “consisting of the (amino acid or base) sequence”.

  The amino acid sequence shown in SEQ ID NO: 8 includes a pro region. The lipase may be, for example, a protein having a portion of the amino acid sequence shown in SEQ ID NO: 8 excluding the pro region (ie, the mature protein amino acid sequence). The lipase may be, for example, a protein encoded by a gene having a base sequence encoding the amino acid sequence of the mature protein in the base sequence shown in SEQ ID NO: 7. The amino acid sequence of the mature protein of galactolipase of Chlorella kessleri 11h strain (UTEX 263) and the base sequence of the gene (cDNA) encoding it are shown in SEQ ID NOs: 11 and 10, respectively.

  The lipase may be a variant of the lipase exemplified above (for example, a protein having the amino acid sequence shown in SEQ ID NO: 8 or 11) as long as the original function is maintained. Similarly, a lipase-encoding gene (also referred to as a “lipase gene”) can be used as long as the original function is maintained, that is, as long as it encodes a protein with the original function maintained (for example, It may be a variant of the gene having the base sequence shown in SEQ ID NO: 7 or 10. Such a variant in which the original function is maintained may be referred to as a “conservative variant”. Examples of conservative variants include the lipases exemplified above, homologues of genes encoding them, and artificially modified variants.

  “The original function is maintained” means that the variant of the protein has an activity (or property) corresponding to the activity (or property) of the original protein. That is, “the original function is maintained” means that, in lipase, a protein variant has lipase activity.

Examples of lipase homologs include proteins obtained from public databases by BLAST search or FASTA search using the amino acid sequence as a query sequence. The homologue of the lipase gene can be obtained, for example, by PCR using chromosomes of various microorganisms (prokaryotes) as templates and oligonucleotides prepared based on these known gene sequences as primers. On the other hand, in the case of a eukaryotic microorganism, a homolog of the above lipase gene can be obtained by PCR using a cDNA library as a template.

  As long as the original function of the lipase is maintained, one or several amino acids at one or several positions are substituted or deleted in the amino acid sequence (for example, the amino acid sequence shown in SEQ ID NO: 8 or 11). Or a protein having an inserted or added amino acid sequence. The above “one or several” varies depending on the position and type of the protein in the three-dimensional structure of the amino acid residue, and specifically, for example, 1 to 50, 1 to 40, 1 to 30, Preferably 1 to 20, more preferably 1 to 10, more preferably 1 to 5, particularly preferably 1 to 3.

One or several amino acid substitutions, deletions, insertions or additions described above are conservative mutations in which the function of the protein is maintained normally. A typical conservative mutation is a conservative substitution. Conservative substitution means that when the substitution site is an aromatic amino acid, Phe, Trp, Tyr,
When the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val, when it is a polar amino acid, it is between Gln and Asn, and when it is a basic amino acid, it is between Lys, Arg, and His. In the case of an acidic amino acid, it is a mutation that substitutes between Asp and Glu. In the case of an amino acid having a hydroxyl group, it is a mutation that substitutes between Ser and Thr. Specifically, substitutions considered as conservative substitutions include substitution from Ala to Ser or Thr, substitution from Arg to Gln, His or Lys, substitution from Asn to Glu, Gln, Lys, His or Asp, Asp to Asn, Glu or Gln, Cys to Ser or Ala, Gln to Asn, Glu, Lys, His, Asp or Arg, Glu to Gly, Asn, Gln, Lys or Asp Substitution, Gly to Pro substitution, His to Asn, Lys, Gln, Arg or Tyr substitution, Ile to Leu, Met, Val or Phe substitution, Leu to Ile, Met, Val or Phe substitution, Substitution from Lys to Asn, Glu, Gln, His or Arg, substitution from Met to Ile, Leu, Val or Phe, substitution from Phe to Trp, Tyr, Met, Ile or Leu, Ser to Thr or Ala Substitution, substitution from Thr to Ser or Ala, substitution from Trp to Phe or Tyr, substitution from Tyr to His, Phe or Trp, and substitution from Val to Met, Ile or Leu. In addition, amino acid substitutions, deletions, insertions, additions, or inversions as described above include naturally occurring mutations (mutants or variants) such as those based on individual differences or species differences in the organism from which the protein is derived. Also included by

  In addition, as long as the original function is maintained, the lipase is 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, particularly preferably relative to the entire amino acid sequence. It may be a protein having a homology of 99% or more. In the present specification, “homology” may refer to “identity”.

In addition, as long as the original function is maintained, the lipase is a probe that can be prepared from the above base sequence (for example, the base sequence shown in SEQ ID NO: 7 or 10), for example, a complementary sequence to the whole or a part of the base sequence, Or a protein encoded by DNA that hybridizes under stringent conditions. Such a probe can be prepared, for example, by PCR using an oligonucleotide prepared based on the base sequence as a primer and a DNA fragment containing the base sequence as a template. “Stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, highly homologous DNAs, for example, 80% or more, preferably 90% or more, more preferably 95% or more, further preferably 97% or more, particularly preferably 99% or more. Is hybridized and DNAs with lower homology do not hybridize with each other, or normal Southern hybridization washing conditions of 60 ° C, 1 × SSC, 0.1% SDS, preferably 60 ° C, 0.1 × SSC , 0.1% SDS, more preferably 68 ° C., 0.1 × SSC, a salt concentration and temperature corresponding to 0.1% SDS, and conditions for washing once, preferably 2 to 3 times. For example, when a DNA fragment having a length of about 300 bp is used as the probe, hybridization washing conditions include 50 ° C., 2 × SSC, and 0.1% SDS.

  The lipase may be one obtained by removing the lipase from which the amino acid sequence or the base sequence of the gene encoding the lipase is known at the time of filing of the present invention.

  The lipase gene may be any gene in which any codon is replaced with an equivalent codon as long as the original function is maintained. For example, the lipase gene may be modified to have an optimal codon according to the codon usage frequency of the host to be used.

In the present invention, the term “gene” is not limited to DNA as long as it encodes a target protein, and may include any polynucleotide. That is, the “lipase gene” may mean any polynucleotide encoding lipase. The lipase gene may be DNA, RNA, or a combination thereof. The lipase gene may be single-stranded or double-stranded. The lipase gene may be single-stranded DNA or single-stranded RNA. The lipase gene may be double-stranded DNA, double-stranded RNA, or a hybrid strand composed of a DNA strand and an RNA strand. The lipase gene may contain both DNA and RNA residues in a single polynucleotide chain. When the lipase gene contains RNA, the description regarding DNA such as the exemplified base sequence may be appropriately read according to RNA. The mode of the lipase gene can be appropriately selected according to various conditions such as the usage mode.

<2> Manufacture of lipase Lipase can be manufactured using the host which has the capability to produce lipase. That is, the present invention provides a method for producing lipase, comprising culturing a host capable of producing lipase in a medium to produce lipase, and collecting lipase from the culture. The lipase can also be produced by expressing the lipase gene in a cell-free protein synthesis system.

  The host having the ability to produce lipase may be one that inherently has the ability to produce lipase, or may have been modified to have the ability to produce lipase.

  Examples of the host having the ability to produce lipase include the chlorella algae as described above, for example, Chlorella kessleri 11h strain (UTEX 263).

  Examples of a host having the ability to produce lipase also include a host into which a lipase gene has been introduced.

The host into which the lipase gene is introduced is not particularly limited as long as it can express a functional lipase. Examples of the host include bacteria, actinomycetes, yeast, fungi, plant cells, insect cells, and animal cells. Preferred hosts include microorganisms such as bacteria and yeast. More preferred hosts include bacteria. Examples of bacteria include gram negative bacteria and gram positive bacteria. Examples of Gram-negative bacteria include bacteria belonging to the family Enterobacteriaceae such as Escherichia bacteria, Enterobacter bacteria, Pantoea bacteria and the like. Examples of Gram-positive bacteria include coryneform bacteria such as Bacillus bacteria and Corynebacterium bacteria. Examples of actinomycetes include bacteria belonging to the genus Streptmyces. In particular, Escherichia coli can be preferably used as the host.

The lipase gene can be obtained by cloning from an organism having the lipase gene. For cloning, nucleic acids such as genomic DNA and cDNA containing the same gene can be used. The lipase gene can also be obtained by chemical synthesis (Gene, 60 (1), 115-127 (1987)
).

Moreover, the variant can also be obtained by appropriately modifying the obtained lipase gene. The gene can be modified by a known method. For example, a target mutation can be introduced into a target site of DNA by site-specific mutagenesis. That is, for example, the coding region of a gene can be modified by site-directed mutagenesis so that an amino acid residue at a specific site of the encoded protein includes substitution, deletion, insertion or addition. As a site-specific mutation method, a method using PCR (Higuchi, R., 61, in PCR technology, Erlich, HA Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)) and methods using phage (Kramer, W. and Frits, HJ, Meth. In Enzymol., 154, 350 (1987); Kunkel, TA et al., Meth. In Enzymol., 154, 367 ( 1987)).

The method for introducing the lipase gene into the host is not particularly limited. In the host, the lipase gene may be retained so that it can be expressed under the control of a promoter that functions in the host. In the host, the lipase gene may be present on a vector that autonomously replicates outside the chromosome, such as a plasmid, or may be introduced on the chromosome. The host may have only one copy of the lipase gene or may have two or more copies. The host may have only one type of lipase gene, or may have two or more types of lipase genes.

The promoter for expressing the lipase gene is not particularly limited as long as it functions in the host. The “promoter that functions in the host” refers to a promoter having promoter activity in the host. The promoter may be a host-derived promoter or a heterologous promoter. The promoter may be a native promoter of the lipase gene or a promoter of another gene. Specific examples of the promoter include T7 promoter, trp promoter, trc promoter, lac promoter, tac promoter, tet promoter, araBAD promoter, rpoH promoter, cspA promoter, PR promoter, and PL promoter. Moreover, as a promoter, you may acquire and utilize the highly active type of a conventional promoter by using various reporter genes. For example, the activity of the promoter can be increased by bringing the −35 and −10 regions in the promoter region closer to the consensus sequence (International Publication No. 00/18935). Methods for evaluating promoter strength and examples of strong promoters are described in Goldstein et al. (Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995)).

A terminator for termination of transcription can be arranged downstream of the lipase gene. The terminator is not particularly limited as long as it functions in the host. The terminator may be a host-derived terminator or a heterologous terminator. The terminator may be a specific terminator of the lipase gene or may be a terminator of another gene. As the terminator, specifically, for example, T7 terminator, T4 terminator, fd phage terminator, tet terminator,
And trpA terminator.

The lipase gene can be introduced into the host using a vector containing the gene, for example. A vector containing a lipase gene is also referred to as a lipase gene expression vector or a recombinant vector. A lipase gene expression vector can be constructed by, for example, ligating a DNA fragment containing a lipase gene with a vector that functions in a host. By transforming the host with a lipase gene expression vector, a transformant into which the vector has been introduced can be obtained, that is, the gene can be introduced into the host. As the vector, a vector capable of autonomous replication in a host cell can be used. The vector is preferably a multicopy vector. Further, the vector preferably has a marker such as an antibiotic resistance gene in order to select a transformant. Moreover, the vector may be provided with a suitable promoter or terminator for expressing the inserted gene. The vector may be, for example, a vector derived from a bacterial plasmid, a vector derived from a yeast plasmid, a vector derived from a bacteriophage, a cosmid, or a phagemid. As vectors capable of autonomous replication in bacteria of the Enterobacteriaceae family such as Escherichia coli, specifically, for example, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all available from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene), pTrc99A
(Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen), pCold TF DNA (TaKaRa), pACYC, wide host range Vector RSF1010 is mentioned. Specific examples of vectors capable of autonomous replication in coryneform bacteria include, for example, pHM1519 (Agric, Biol. Chem., 48, 2901-2903 (1984)); pAM330 (Agric. Biol. Chem., 48, 2901- 2903 (1984)); plasmids having improved drug resistance genes; plasmid pCRY30 described in JP-A-3-210184; plasmid pCRY21 described in JP-A-2-72876 and US Pat. No. 5,185,262. PCRY2KE, pCRY2KX, pCRY31, pCRY3KE and pCRY3KX; plasmids pCRY2 and pCRY3 described in JP-A-1-91686; pAJ655, pAJ611 and pAJ1844 described in JP-A-58-192900; JP-A-57-134500 Examples include pCG1 described in the publication; pCG2 described in JP-A-58-35197; pCG4 and pCG11 described in JP-A-57-183799. As a vector capable of autonomous replication in actinomycetes, specifically, for example, pUC702 (Molnar et al., J. Ferment. Bioeng., Vol.72, 368-372 (Escherichia coli and actinomycetes shuttle vector) 1991)). When constructing an expression vector, for example, a lipase gene containing a unique promoter region may be incorporated into the vector as it is, or the lipase coding region may be incorporated downstream of the above promoter and then incorporated into the vector. A lipase coding region may be incorporated downstream of the promoter inherent in the vector.

  For vectors, promoters, and terminators that can be used in various microorganisms, see “Basic Course of Microbiology 8 Genetic Engineering, Kyoritsu Shuppan, 1987”. Et al., John Innes Foundation, 2000), etc., and are described in detail.

Moreover, a lipase gene can be introduce | transduced on the chromosome of a host, for example. Introduction of a gene onto a chromosome can be performed using, for example, homologous recombination. Examples of gene transfer methods using homologous recombination include Red-driven integration (WO2005 / 010175), transduction using phages such as P1 phage, methods using conjugative transfer vectors, and functions in the host. And a method using a suicide vector that does not have an origin of replication. Only one copy of the gene may be introduced, or two copies or more may be introduced. For example, multiple copies of a gene can be introduced into a chromosome by performing homologous recombination with a sequence having multiple copies in the chromosome as a target. Examples of sequences having multiple copies in the chromosome include repetitive DNA sequences (repetitive DNA sequences).
DNA) and inverted repeats present at both ends of the transposon. In addition, for example, a gene can be randomly introduced onto a chromosome by a method using transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985, US Pat. No. 5,882,888, EP805867B1). When introducing a gene into a chromosome, for example, a lipase gene containing a unique promoter region may be incorporated into the chromosome as it is, or the lipase coding region may be incorporated downstream into the promoter and then incorporated into the chromosome. Alternatively, the lipase coding region may be incorporated downstream of the promoter originally present on the chromosome.

  The introduction of a gene onto a chromosome can be attributed to, for example, Southern hybridization using a probe having a base sequence complementary to all or part of the gene, or a primer prepared based on the base sequence of the gene. Can be confirmed by PCR.

Lipases can also be expressed as fusion proteins with other amino acid sequences (proteins and peptides). The type of other amino acid sequence is not particularly limited. Other amino acid sequences can be appropriately selected according to various conditions such as the type of host and the intended use of the lipase. Specifically, other amino acid sequences include tag sequences such as trigger factor (TF) and His tag, pro sequences, and signal sequences. For example, lipase can be efficiently produced as an active enzyme by using a prosequence. Further, for example, by using a signal sequence, lipase can be efficiently secreted and produced outside the cell. Examples of the pro sequence include a pro sequence of galactolipase of Chlorella algae such as Chlorella kessleri 11h strain (UTEX 263). Examples of the signal sequence include a phospholipase signal sequence. In addition, by designing a protease recognition sequence between the lipase and another amino acid sequence, the lipase can be cut out and obtained from the fusion protein using a protease, if necessary. Examples of the protease include Factor Xa protease and proTEV protease. The recognition sequences for these proteases are known.

The transformation method is not particularly limited, and a conventionally known method can be used. As a transformation method, for example, a method of increasing the permeability of DNA by treating a recipient cell with calcium chloride as reported for Escherichia coli K-12 (Mandel, M. and Higa, A., J. Mol.
Biol. 1970, 53, 159-162), as described for Bacillus subtilis, a method of preparing competent cells from proliferating cells and introducing DNA (Duncan, CH, Wilson, GA and Young, FE., 1997. Gene 1: 153-167). In addition, as a transformation method, recombinant DNA is prepared by transforming DNA-receptive cells, such as those known for Bacillus subtilis, actinomycetes, and yeast, into a protoplast or spheroplast state that easily incorporates recombinant DNA. (Shang, S. and Choen, SN, 1979. Mol. Gen. Genet. 168: 111-115; Bibb, MJ, Ward, JM and Hopwood, OA 1978. Nature 274: 398- 400; Hinnen, A., Hicks, JB and Fink, GR 1978. Proc. Natl. Acad. Sci. USA 75: 1929-1933). As a transformation method, an electric pulse method (Japanese Patent Laid-Open No. 2-207791) as reported for coryneform bacteria can also be used. In particular, for actinomycetes, transformation can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Innes Foundation, 2000)”.

  The lipase can be expressed by culturing a host having the ability to produce the lipase as described above in a medium. At that time, gene expression may be induced as necessary. Host culture conditions and gene expression induction conditions may be appropriately selected according to various conditions such as marker type, promoter type, and host type. The medium used for the culture is not particularly limited as long as the host can proliferate and can express lipase. As the medium, for example, a normal medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions, and other organic components as required can be used.

  Examples of the carbon source include sugars such as glucose, fructose, sucrose, molasses, and starch hydrolysates, alcohols such as glycerol and ethanol, and organic acids such as fumaric acid, citric acid, and succinic acid.

  Examples of the nitrogen source include inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, organic nitrogen such as soybean hydrolysate, ammonia gas, and aqueous ammonia.

  Examples of the sulfur source include inorganic sulfur compounds such as sulfate, sulfite, sulfide, hyposulfite, and thiosulfate.

  Examples of inorganic ions include calcium ions, magnesium ions, manganese ions, potassium ions, iron ions, and phosphate ions.

  Other organic components include organic trace nutrient sources. Examples of organic micronutrients include required substances such as vitamin B1, yeast extract containing them, and the like.

The culture method may be liquid culture or solid culture, but liquid culture is preferred. The culture is preferably performed aerobically. Examples of the aerobic culture method include a shaking culture method and an aerobic deep culture method using a jar fermenter. The oxygen concentration at that time may be adjusted to, for example, 5 to 50%, preferably about 10% with respect to the saturation concentration. The culture temperature may be, for example, 10 to 50 ° C, preferably 20 to 45 ° C, more preferably 25 to 40 ° C. Medium pH
May be adjusted, for example, to 3-9, preferably 5-8. For the pH adjustment, inorganic or organic acidic or alkaline substances such as calcium carbonate, ammonia gas, aqueous ammonia and the like can be used. The culture period may be, for example, 12 hours to 20 days, preferably 1 day to 7 days.

Moreover, it illustrates below about the cultivation method of microalgae, such as Chlorella algae. There is a lot of knowledge about the culture of microalgae. For example, algae such as Chlorella algae, Arthrospira algae (Spirulina), and Dunaliella salina are cultivated industrially on a large scale (Spolaore). , P. et al. 2006. J. Biosci. Bioeng. 101: 87-96). The culture of microalgae may be performed with reference to such knowledge, for example.

  Microalgae culture uses autotrophic culture that uses photosynthesis and does not use organic compounds, heterotrophic culture that uses organic compounds without using photosynthesis, or both photosynthesis and organic compounds. It can be performed by mixed nutrient culture (mixotrophic culture). Microalgae may be cultured usually by autotrophic culture.

  The culture of microalgae may be performed in an open system or a closed system. For example, culture can be performed in an open culture system called an open pond. For example, culture can be performed in a closed culture system called a closed photobioreactor.

The medium used for culturing microalgae is not particularly limited as long as microalgae can be grown and lipase can be expressed. The culture medium may contain, for example, a nitrogen source and various inorganic salts. Moreover, the culture medium may contain other components, such as a carbon source, as needed. A person skilled in the art can appropriately set the type and concentration of the medium components. As the medium, for example, a normal medium used for culturing microalgae can be used. As such a medium, specifically, for example, 0.3 × HSM medium (Oyama, Y. et al. 2006. Planta 224: 646-654), 0.2
X Gamborg medium (Izumo, A. et al. 2007. Plant Science 172: 1138-1147), modified NORO medium (Yamaberi, K. et al. 1998. J. Mar. Biotechnol. 6: 44-48; Takagi, M et al. 2000. Appl. Microbiol. Biotechnol. 54: 112-117), Bold's Basal Medium (Tornabene, TG et al. 1983. Enzyme and Microb. Technol. 5: 435-440; Archibald, P.
A. and Bold, HC 1970. Phytomorphology 20: 383-389), F / 2 medium (Lie, C.-P. and
Lin, L.-P. 2001. Bot. Bull. Acad. Sin. 42: 207-214), TAP medium.

Microalgae can be cultured using a liquid medium. The culture temperature may be, for example, 20 to 40 ° C, preferably 25 ° C to 35 ° C, more preferably around 30 ° C. The initial pH of the medium may be, for example, near neutrality. Near neutrality may be, for example, pH 7-9. During the culture, the pH may or may not be adjusted. A suitable inorganic or organic acidic or alkaline substance can be used for pH adjustment. The culture may be performed with aeration. The aeration amount may be, for example, 0.1 to 2 vvm (volume per volume per minute) as an aeration amount for 1 minute per culture medium unit volume. The culture medium may be further supplied to CO _2. The supply amount of CO ₂ may be, for example, 0.5 to 5% (v / v) with respect to the ventilation amount. CO ₂ and air may be supplied separately to the culture solution, or mixed and supplied to the culture solution. When using photosynthesis, light is supplied to the culture system. The light can be supplied using a suitable light source. Examples of the light source include a white fluorescent lamp, a white light emitting diode, a high pressure sodium lamp, and sunlight. These light sources may be used in appropriate combination. For example, the illuminance of light is 1,000
It can be ~ 10,000 lux. The culture solution may be appropriately stirred or circulated. Various operations such as light supply, air supply, CO ₂ supply, agitation, and circulation may be performed continuously or intermittently. Conditions for various operations may or may not be constant throughout the culture. The culture period may be, for example, 1 to 40 days.

  By culturing under the conditions as described above, a culture containing lipase can be obtained. The lipase accumulates in, for example, the host cell and / or medium. “Bacteria” may be appropriately read as “cells” or “algae” depending on the type of host.

  The lipase may be used as it is contained in the microbial cells or the like, and may be appropriately separated and purified from the microbial cells and used as a crude enzyme fraction or a purified enzyme.

  That is, for example, when lipase accumulates in the host cell, the cell can be appropriately disrupted, dissolved, or extracted, and the lipase can be recovered. The cells can be recovered from the culture by centrifugation or the like. Cell disruption, lysis, extraction or the like can be performed by a known method. Examples of such a method include ultrasonic crushing method, dynomill method, bead crushing, French press crushing, and lysozyme treatment. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. For example, when lipase accumulates in the medium, the culture supernatant can be obtained by centrifugation or the like, and the lipase can be recovered from the culture supernatant.

  The lipase can be purified by a known method used for enzyme purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric point precipitation. One of these methods may be used alone, or two or more thereof may be used in appropriate combination. The lipase can be purified to a desired degree.

  The purified lipase can be used as a “lipase” for hydrolysis of glycerolipids. The lipase may be used in a free state or may be used in the state of an immobilized enzyme immobilized on a solid phase such as a resin.

  Further, the lipase is not limited to purified lipase, and any fraction containing lipase may be used as “lipase” for hydrolysis of glycerolipid. The fraction containing lipase is not particularly limited as long as the lipase is contained so that lipase can act on glycerolipid. As such a fraction, for example, a culture of a host having the ability to produce lipase, a microbial cell recovered from the culture (cultured microbial cell), a crushed product of the microbial cell, a lysate of the microbial cell, Extracts of the same cells (cell-free extract), treated cells such as immobilized cells obtained by immobilizing the same cells with acrylamide, carrageenan, etc., culture supernatants recovered from the same culture, and partial purification thereof Product (crude product), and combinations thereof. Any of these fractions may be used alone or with a purified lipase.

  The collected lipase may be appropriately formulated. The dosage form is not particularly limited, and can be appropriately set according to various conditions such as the intended use of lipase. Examples of the dosage form include solutions, suspensions, powders, tablets, pills, and capsules. In formulation, for example, excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, flavoring agents, fragrances, diluents, surfactants and other pharmacologically acceptable additives. Can be used.

<3> Use of lipase In the present invention, lipase can be used to hydrolyze glycerolipids. That is, the present invention provides a method for hydrolyzing glycerolipid, which comprises allowing lipase to act on glycerolipid. This method is also referred to as “the method of the present invention”. One embodiment of the method is a method for producing a hydrolyzate of glycerolipid, which comprises allowing lipase to act on glycerolipid.

  By hydrolysis of glycerolipid, fatty acid and glycerolipid in which the fatty acid is liberated (that is, the ester bond is hydrolyzed to generate a hydroxyl group) are produced. These components may be obtained together as a hydrolyzate of glycerolipid and used. Of these components, a desired component may be separated and recovered and used. The components can be recovered by a known method used for separation and purification of compounds. Examples of such a method include an ion exchange resin method and a membrane treatment method. These methods can be used in appropriate combination.

  For example, when the glycerolipid is a glyceroglycolipid, glycoglycerol can be produced by hydrolysis of the glyceroglycolipid. That is, one embodiment of the method of the present invention may be a method for producing glycoglycerol, which comprises allowing lipase to act on glyceroglycolipid.

  “Sugar glycerol” refers to a compound in which a sugar chain is glycosidically bonded to any hydroxyl group of glycerol. Specific examples of the sugar glycerol include galactosylglycerol, digalactosylglycerol, and sulfoquinovosylglycerol. Galactosylglycerol is a sugar glycerol in which galactose is bonded to the hydroxyl group of any one carbon of glycerol. Digalactosylglycerol is a sugar glycerol in which digalactose is bound to the hydroxyl group of any one carbon of glycerol. Sulfoquinovosyl glycerol is a sugar glycerol in which sulfo quinose is bound to the hydroxyl group of any one carbon of glycerol. Unless otherwise specified, galactosylglycerol, digalactosylglycerol, and sulfoquinovosylglycerol all have a sugar chain bonded to the hydroxyl group of the 1st carbon of glycerol, and the sugar chain to the hydroxyl group of the 2nd carbon of glycerol. It may be a bond, a sugar chain bonded to the hydroxyl group of the 3-position carbon of glycerol, or a mixture thereof.

  The type of sugar glycerol produced corresponds to the type of glyceroglycolipid to be hydrolyzed. That is, for example, hydrolysis of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, and sulfoquinovosyl diacylglycerol can produce galactosylglycerol, digalactosylglycerol, and sulfoquinovosylglycerol, respectively. In addition, the position of the sugar chain in the produced sugar glycerol corresponds to the position of the sugar chain in the glyceroglycolipid to be hydrolyzed. That is, for example, by hydrolysis of a glyceroglycolipid having a sugar chain bonded to the 3rd position of the glycerol skeleton, a sugar glycerol having a sugar chain bonded to the 3rd position of the glycerol skeleton can be generated. In the present invention, only one sugar glycerol may be produced, or two or more sugar glycerols may be produced.

  The glycerolipid may be glycerolipid itself or a material containing glycerolipid. In other words, the glycerolipid may be subjected to hydrolysis by lipase alone (that is, in an isolated state), or may be subjected to hydrolysis by lipase in a state of being contained in an arbitrary material. That is, “to make lipase act on glycerolipid” is not limited to the case where lipase is allowed to act on glycerolipid itself unless otherwise specified, and includes the case where lipase is allowed to act on a material containing glycerolipid. Similarly, for example, “to make lipase act on glyceroglycolipid” is not limited to the case where lipase acts on glyceroglycolipid itself unless otherwise specified, and when lipase acts on a material containing glyceroglycolipid. Is included. The glycerolipid may be a natural product or an artificial product. That is, examples of the glycerolipid include agricultural, aquatic and livestock products containing glycerolipid, processed products thereof, glycerolipid separated from them, and artificially synthesized glycerolipid. Examples of the material containing glycerolipid include wheat flour. The material containing glycerolipid may contain one type of glycerolipid, or may contain two or more types of glycerolipid. As the glycerolipid, one glycerolipid may be used, or two or more glycerolipids may be used.

  The glycerolipid may be, for example, a food or drink containing glycerolipid, or a raw material for food or drink containing glycerolipid. In other words, the glycerolipid may be subjected to hydrolysis by lipase in a state of being contained in a food or drink or a raw material thereof, for example. The type and form of the food or drink or its raw material are not particularly limited as long as the food or drink or its raw material contains glycerolipid. For example, any of the above-described glycerolipids and materials containing the same may be used alone as a food or drink or a raw material thereof, and two or more of them may be used in combination as appropriate as a food or drink or a raw material thereof. Alternatively, one or more of them and other components may be appropriately combined and used as a food or drink or a raw material thereof. By hydrolyzing the food / beverage products containing glycerolipid or its raw material with a lipase, the food / beverage products containing the hydrolyzate of glycerolipid or its raw material are obtained. Furthermore, the food / beverage products containing the glycerolipid hydrolyzate can be manufactured using the raw material of the food / beverage products containing the glycerolipid hydrolyzate. Seasonings are also included in food and drink. Examples of foods and drinks containing glycerolipids include processed wheat foods (noodle bands such as udon, bread, cookies, cakes, donuts, etc.) and processed soybean foods (soy milk, tofu, okara, miso, boiled soybeans, etc.) Etc.

The glycerolipid can be subjected to lipase hydrolysis in the form of, for example, a solution, a suspension, a slurry, or a paste. As the solvent or dispersion medium, for example, an aqueous medium such as water or an aqueous buffer can be used. Further, the glycerolipid may be appropriately subjected to a pretreatment such as emulsification and then subjected to hydrolysis with lipase. The concentration of glycerolipid in the reaction system is not particularly limited as long as hydrolysis is achieved to a desired degree. The concentration of glycerolipid in the reaction system can be appropriately set according to various conditions such as the type and form of glycerolipid and the desired hydrolysis rate. The reaction system (reaction solution or the like) may be composed of glycerolipid and a solvent, and may contain other components. Examples of other components include components used in foods and drinks and pharmaceuticals. Examples of other components include salts, saccharides, proteins, fragrances, humectants, colorants, and surfactants. Examples of the salts include Ca ²⁺ ions. Examples of the surfactant include Triton X-100.

The reaction conditions (enzyme amount, reaction time, reaction temperature, reaction pH, etc.) are not particularly limited as long as hydrolysis is achieved to a desired degree. The reaction conditions can be appropriately set according to various conditions such as the type and purity of lipase, the type and purity of glycerolipid, and the desired hydrolysis rate. The amount of the enzyme may be, for example, 0.01 to 100 U, preferably 0.1 to 10 U, with respect to 1 g of glycerolipid. The reaction temperature may be, for example, 20 to 60 ° C, preferably 30 to 40 ° C, more preferably 35 to 40 ° C. The pH of the reaction solution may be, for example, 4 to 8, preferably 5 to 7.5, more preferably 6 to 7. The reaction time may be, for example, 10 seconds to 48 hours, preferably 10 minutes to 24 hours.

  Hydrolysis can be performed to the desired extent. The hydrolysis rate (number of ester bonds hydrolyzed in glycerolipid / number of ester bonds before hydrolysis in glycerolipid) is, for example, 0.1% or more, 1% or more, 5% or more, 10% or more, Alternatively, it may be 20% or more, and may be 100% or less, 70% or less, or 50% or less.

The physical properties and functions of glycerolipids can be modified by hydrolysis. Therefore, for example, by hydrolyzing a food or drink containing glycerolipid or a raw material thereof with lipase, the physical properties or functions of the food or drink or the raw material can be modified. These modifications of physical properties and functions are collectively referred to as “reformation”. That is, in other words, the method of the present invention may be a method for modifying glycerolipid, which comprises allowing lipase to act on glycerolipid. Moreover, one aspect | mode of the method may be a method for modifying a food or drink or a raw material thereof, for example, by causing lipase to act on a food or drink or a raw material containing glycerolipid. Therefore, the lipase may be provided as a glycerolipid modifier (physical property modifier or functional modifier), for example, as a food or drink or a raw material modifier (physical property modifier or functional modifier). Also good. Examples of the modification include improvement of texture. Specifically, for example, the lipase may be provided as a quality improver for processed wheat foods or processed soybean foods.

  The lipase may also be provided as a test and research reagent such as a glycerolipid hydrolysis reagent.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to these.

Example 1: Culture of Chlorella quercerii strain 11h (UTEX 263) The University of Texas at Austin, The
The Chlorella kesleri 11h strain (UTEX 263) was obtained from Culture Collection of Algae (UTEX), 1 University Station A6700, Austin, TX 78712-0183, USA. Chlorella quercerii strain 11h was pre-cultured in 0.2 × Gamborg's B5 medium. The 0.2 × Gamborg's B5 medium was prepared by diluting Gamborg's B5 medium (the composition is described below) 5 times and then autoclaving (120 ° C., 15 min). 1 L of 16 ml of the obtained preculture solution and 800 ml of 0.2 × Gamborg's B5 medium
It is added to the medium bottle and mixed with air and CO ₂ using a portable gas mixing device PMG-1 (Coflock), and the gas adjusted to 3% CO ₂ concentration is blown at 500 ml / min at 30 ° C for 14 days. And stirred culture. All the cultures were carried out in a plant incubator CL-301 (Tomy Seiko Co., Ltd.), and the light irradiation condition was continuous irradiation at an illuminance of about 5,000 lux using a white fluorescent light as a light source.

<Composition of Gamborg's B5 medium (Nippon Pharmaceutical)>
KNO ₃ 2500 mg
MgSO ₄・ 7H ₂ O 250 mg
NaH ₂ PO ₄・ H ₂ O 150 mg
CaCl ₂・ 2H ₂ O 150 mg
(NH ₄ ) ₂ SO ₄ 134 mg
Na ₂・ EDTA 37.3 mg
FeSO ₄・ 7H ₂ O 27.8 mg
MnSO ₄・ H ₂ O 10 mg
H ₃ BO ₃ 3 mg
ZnSO ₄・ 7H ₂ O 2 mg
KI 0.75 mg
Na ₂ MoO ₄・ 2H ₂ O 0.25 mg
CuSO ₄・ 5H ₂ O 0.025 mg
CoCl ₂・ 6H ₂ O 0.025 mg
Prepare to 1 L with deionized water.

Example 2: Purification of galactolipase derived from Chlorella quercerii strain 11h (UTEX 263) The culture solution (2 L) obtained in Example 1 was centrifuged, and the supernatant was removed to remove the algae. Obtained. The obtained algal cells are suspended in 200 ml of 20 mM Tris-HCl (pH 8.0) buffer, and then a cycle of "1 minute ON-1 minute OFF" using a bead crusher (manufactured by Hamilton Beach) under cooling conditions. Was repeated 10 times to prepare a disruption liquid of algal cells. After that, crush the liquid (2,400xg, 4 ℃
And the supernatant was recovered as a cell-free extract. Subsequently, the Triton X-100 solution was added to the cell-free extract at a final concentration of 1%, and the mixture was stirred at 4 ° C. for 2 hours to solubilize the protein in the cell-free extract. Next, the solubilized solution was ultracentrifuged (210,000 × g, 1 hour at 4 ° C.), then 3 times its volume of acetone was added to the supernatant, and then left at -20 ° C. overnight. As a result, the protein was precipitated with acetone. Subsequently, the acetone precipitation solution was centrifuged (12,000 × g, 4 ° C. for 5 minutes), and the buffer fraction (20 mM Tris-HCl (pH 8.0) containing 1% TritonX-100) 150 was added to the precipitate fraction. After solubilization of protein by adding ml, DTT (dithiothreitol, final concentration 1 mM)
) And KCl (final concentration 2 M) were added to obtain a crude enzyme solution.

  The crude enzyme solution was applied to a hydrophobic interaction column Toyopearl Butyl-650M (Tosoh) equilibrated with buffer A containing 2M KCl to adsorb the enzyme. Thereafter, the column was washed with the same buffer. Next, the enzyme was eluted with a linear gradient from 2 M to 0 M KCl using buffer A as an eluent, and a fraction having galactolipase activity was collected. Galactolipase activity was measured according to the standard method for measuring galactolipase activity described in Example 4 using DGDG (digalactosyl diacylglycerol) as a substrate (the same applies hereinafter unless otherwise specified).

Subsequently, the active fraction was desalted and replaced with buffer A by Vivaspin 20-10K (GE Healthcare). The enzyme solution after desalting and buffer exchange was applied to an anion exchange column (Hitrap DEAE FF) equilibrated with buffer A to adsorb the enzyme. Thereafter, the column was washed with the same buffer. Next, 0 M using buffer A containing 1 M NaCl as the eluent.
Then, the enzyme was eluted with a linear gradient of 1 M NaCl, and a fraction having galactolipase activity was recovered. Next, potassium chloride was added to the obtained active fraction to a final concentration of 2 M, and the enzyme solution was equilibrated with the hydrophobic interaction column Hitrap Butyl HP (buffer A containing 2 M KCl). The enzyme was adsorbed. Thereafter, the column was washed with the same buffer. Next, using Buffer A as an eluent, the target enzyme was eluted with a linear gradient from 2 M to 0 M KCl, and a fraction having galactolipase activity was collected and used as a purified enzyme.

  The obtained purified enzyme was subjected to SDS-PAGE (Laemmli, UK, Nature, 227, 680 (1970)) using 12% polyacrylamide gel, and the molecular weight was measured. The purified enzyme had a single molecular weight of about 55,000. A band was shown (FIG. 1).

Example 3: Determination of amino acid sequence of galactolipase derived from Chlorella quercerii strain 11h (UTEX 263) The N-terminal amino acid sequence of the purified enzyme was determined as follows. That is, after the purified enzyme was subjected to SDS-PAGE by the method shown in Example 2, the migrated protein was transferred to a PVDF membrane. Next, the transcribed protein was analyzed with an amino acid sequence analyzer Procise 494 HT (Applied Biosystem), and its N-terminal amino acid sequence was determined. As a result, the N-terminal amino acid sequence shown in SEQ ID NO: 1 was obtained.

  Next, the internal amino acid sequence of the purified enzyme was determined as follows. That is, after the purified enzyme was subjected to SDS-PAGE by the method shown in Example 2, the band corresponding to the purified enzyme was excised and digested in gel using trypsin. Next, the obtained digest (peptide) was analyzed with a mass spectrometer (nanoLC-MS / MS), and the internal amino acid sequence of the purified enzyme was determined. As a result, the internal amino acid sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 were obtained.

Example 4: Characteristic analysis of galactolipase derived from Chlorella querseri 11h strain (UTEX 263) First, substrate specificity of galacto lipase derived from Chlorella kesleri 11h strain (UTEX 263) was analyzed. In the present invention, a galactolipase derived from the Chlorella quercerii strain 11h (UTEX 263) is also referred to as “chGL”.

The standard measurement method of galactolipase activity is as follows. That is, 1% (w / v) Triton
1% (w / v) substrate containing X-100 (DGDG; digalactosyl diacylglycerol, Larodan Fine
Chemicals, # 59-1210) 2.5 μL, 1% (w / v) 200 mM MES-NaOH (pH 6.5) 15 μL with Triton X-100, 20 mM CaCl ₂ 2.5 μL, 1% (w / v) Triton X-100 2.5 Mix 1 μL, 1% (w / v) Triton X-1
An enzyme solution containing 00 (here, purified enzyme) (2.5 μL) was added to prepare a reaction solution with a total volume of 25 μL. This reaction solution was incubated at 37 ° C. for 5 minutes for enzyme reaction, and then heated at 100 ° C. for 5 minutes to stop the reaction. The free fatty acid contained in 5 μL of the reaction solution was measured using a free fatty acid measurement kit “NEFA C-Test Wako” (Wako Pure Chemical Industries, Ltd.) as described in the instructions attached to the kit. Under these conditions, the enzyme activity that produces 1 μmol of free fatty acid per minute was defined as 1 unit (U) of galactolipase activity.

As substrates other than DGDG, MGDG (monogalactosyl diacylglycerol), SQDG (sulfoquinovosyl diacylglycerol), POPG (1-palmitoyl-2-oleyl-sn-glycero-3-phospho-rac- (1-glycerol) )), POPC (1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine), POPS (1-palmitoyl-2) -Oleyl-sn-glycero-3-phosphoserine), DMPA (1,2-dimyristoylphosphatidic acid), tristearate, dipalmitate, and olive oil were used. The lipase activity for each substrate other than DGDG was measured according to the standard measurement method for galactolipase activity, except that the same amount ((w / v)%) of each substrate was used instead of DGDG. Under these conditions, the enzyme activity that produces 1 μmol of free fatty acid per minute was defined as 1 unit (U) of lipase activity for each substrate. The lipase activity against olive oil was measured according to the method described in Japanese Patent No. 5179208. Specifically, olive oil (produced by Nacalai Tesque) 200 mg and gum arabic (produced by Wako Pure Chemical Industries, Ltd.) 100 mg are added with 10 ml of water, and the ultrasonic generator (Fine, Ultra sonic cleaner, 100W) Sonication for 1 minute and vortexing for 1 minute were emulsified under a total of 2 conditions, and this was used as a substrate solution.

  Note that each of DGDG, MGDG, and SQDG used in the examples is a glyceroglycolipid in which a sugar chain is bonded to the hydroxyl group at the 3-position of 1,2-diacylglycerol.

  FIG. 2 is a graph showing the activity for each substrate as a relative activity based on the activity for DGDG (100%). From this result, it was revealed that the present enzyme (Chlorella querseri-derived galactolipase) is a galactolipase that mainly acts on DGDG.

  The purified enzyme had a galactolipase activity of 27.6 U / mL and an activity per 2.5 μL of 69 mU. The specific activity of the purified enzyme was 531 U / mg.

Next, the temperature dependence of the activity of this enzyme was assayed as follows. Final concentration 0.1 (w / v)% substrate (DGDG), 0.3 (w / v)% Triton X-100, ₂ mM CaCl ₂ , 120 mM MES-NaOH (pH 6.5, at each set temperature), purified enzyme 69 mU Were mixed to prepare a reaction solution having a total volume of 25 μL. This reaction solution was kept warm for 5 minutes under each set temperature condition to carry out an enzyme reaction. Thereafter, galactolipase activity was measured according to a standard method for measuring galactolipase activity. FIG. 3 is a graph showing the galactolipase activity at each reaction temperature as a relative activity based on the galactolipase activity when the reaction temperature is 37 ° C. (100%). As shown in FIG. 3, this enzyme showed galactolipase activity at 25 ° C. to 55 ° C., and the optimum temperature for the reaction was around 37 ° C. (for example, 35 to 40 ° C.).

Next, the temperature stability of this enzyme was assayed as follows. The purified enzyme was diluted 5-fold with 0.2 M MES-NaOH (pH 6.5, at each set temperature) and then allowed to stand at each temperature for 30 minutes for treatment. Then, the enzyme reaction was performed according to the standard measuring method of galactolipase activity, and galactolipase activity was measured. FIG. 4 is a graph showing the residual galactolipase activity after treatment at each temperature as relative activity based on the galactolipase activity when the enzyme reaction was carried out without treatment (100%). As shown in FIG. 4, the present enzyme was confirmed to have residual galactolipase activity after treatment at temperatures from 4 ° C. to 60 ° C. Especially after treatment at temperatures below 40 ° C, 80% before treatment
The above galactolipase activity was maintained.

Next, the pH dependency of the activity of the enzyme was assayed as follows. Acetic acid-Na acetate (black circle), MES-NaOH (black triangle), HEPES-NaOH (white circle), Bis containing 0.1 (w / v)% substrate (DGDG) and 0.3 (w / v)% Triton X-100 -69 mU of purified enzyme is added to a buffer solution (140 mM, each pH, temperature 37 ° C) selected from -Tris (black square), Tris-HCl (white triangle), and Glycine-NaOH (white square), for a total volume of 25 μL A reaction solution was prepared. This reaction solution was subjected to an enzyme reaction for 5 minutes under each pH condition. Thereafter, galactolipase activity was measured according to a standard method for measuring galactolipase activity. FIG. 5 is a graph showing the galactolipase activity at each reaction pH as a relative activity based on the galactolipase activity when the reaction pH is 6.5 (MES-NaOH) (100%). As shown in FIG. 5, this enzyme showed activity in a wide range from pH 4 to pH 8, and the optimum pH of the reaction was around 6.5 (for example, 6 to 7).

Next, the pH stability of this enzyme was assayed as follows. Acetic acid-Na acetate (white circle), MES-NaOH (
Black circle), HEPES-NaOH (black triangle), Bis-Tris (white triangle), Tris-HCl (white square), and Glycine-NaOH (black square) buffer (50 mM, each pH, temperature 37 ° C) The purified enzyme was diluted 5-fold and then treated at 4 ° C. for 3 hours at each pH. Then, the enzyme reaction was performed according to the standard measuring method of galactolipase activity, and galactolipase activity was measured. FIG. 6 is a graph showing the residual galactolipase activity after treatment at each pH as relative activity based on the galactolipase activity when the enzyme reaction was carried out without treatment (100%). As shown in FIG. 6, the present enzyme was confirmed to have residual galactolipase activity after treatment at a pH of at least pH 4 to 10.

Next, the metal ions necessary for the activity of this enzyme were assayed as follows. Final concentration 0.1 (w / v)% substrate (DGDG), 0.3 (w / v)% Triton X-100, ₂ mM CaCl ₂ , 120 mM MES-NaOH (pH 6.5, at each set temperature), and various metals A reagent containing ions was added to a final concentration of 10 mM, or an inhibitor or the like was added to a final concentration of 2 mM, and purified enzyme 69 mU was added to prepare a reaction solution with a total volume of 25 μL. Separately, 0.1% (w / v) substrate (DGDG) and 0.3% (w / v) as final concentrations
Purified enzyme 69 mU was added to 120 mM MES-NaOH (pH 6.5) containing Triton X-100 to prepare a reaction solution (Ca ²⁺ free) with a total volume of 25 μL. This reaction solution was subjected to an enzymatic reaction for 5 minutes under conditions of pH 6.5 and 37 ° C. according to a standard measurement method for galacto lipase activity, and then galacto lipase activity was measured. Table 1 shows the galactolipase activity under the condition where various additives are added as the relative activity based on the galactolipase activity under the condition where 10 mM Ca ²⁺ is added (100%). The activity of this enzyme decreased slightly in the presence of Mn ²⁺ or an inhibitor. In addition, the activity of this enzyme was greatly reduced in the presence of Mg ²⁺ , Zn ²⁺ , Fe ²⁺ , Fe ³⁺ , or EDTA and in the condition where nothing was added (Ca ²⁺ free). Therefore, it was found that this enzyme is Ca ²⁺ auxotrophic. Then, next, the relationship between Ca ²⁺ ion concentration and galactolipase activity was examined. Final concentration of 0.1% (w / v) substrate (DGDG), 0.3% (w / v) Triton X-100, 120 mM MES-NaOH (pH 6.5) so that Ca ²⁺ ions have a predetermined final concentration Then, 69 mU of purified enzyme was added to prepare a reaction solution with a total volume of 25 μL. This reaction solution was subjected to an enzyme reaction at pH 6.5 and 37 ° C. for 5 minutes in accordance with a standard method for measuring galactolipase activity, and then galactolipase activity was measured. FIG. 7 is a graph showing the galactolipase activity under the condition where Ca ²⁺ of each concentration is added as the relative activity based on the galactolipase activity under the condition where 2 mM Ca ²⁺ is added (100%). . As shown in FIG. 7, the present enzyme was found to be optimal under the reaction conditions in which about 2 mM of Ca ²⁺ ions exist. In addition, since the activity was reduced by IAA (iodoacetamide), 2-ME (2-mercaptoethanol), and DTT (dithiothreitol), this enzyme has a cysteine residue or cystine structure that maintains the three-dimensional structure of the enzyme and exhibits its activity. Is implicated in In addition, the activity was reduced by PMSF (phenylmethylsulfonyl fluoride), suggesting that the serine residue of this enzyme is involved in the activity expression.

Subsequently, the dependency of the enzyme activity on the surfactant Triton X-100 concentration was assayed as follows. Triton X-100 is added to a final concentration of 120 mM MES-NaOH (pH 6.5) containing 0.1% (w / v) substrate (DGDG) and 2 mM CaCl _2, and 69 mU of purified enzyme is added. Then, a reaction solution having a total volume of 25 μL was prepared. This reaction solution was adjusted to pH 6.5 according to the standard measurement method for galactolipase activity.
After the enzyme reaction at 37 ° C. for 5 minutes, the galactolipase activity was measured. FIG. 8 shows galactolipase activity under the condition of adding Triton X-100 at each concentration as the standard (100%) of galactolipase activity under the condition of adding 0.3% (w / v) Triton X-100. It is the graph shown as relative activity. Thus, the present enzyme was found to be optimal under conditions where about 0.3% (w / v) of Triton X-100 is present.

Example 5: Genomic DNA sequence analysis of Chlorella quercerii strain 11h (UTEX 263) Chlorella quercerii strain 11h (UTEX 263) was cultured for 7 days in the same manner as in Example 1, and the wet alga body weight was about 20 mg. Of alga bodies were collected. The algal bodies were placed in a mortar (autoclaved) cooled to −80 ° C. with liquid nitrogen, and the algal bodies were crushed with a pestle and powdered while adding liquid nitrogen. DNA was extracted and purified from a completely powdered algal body using NucleoSpinPlantII (manufactured by MACHEREY-NAGEL) to obtain about 10 μg of DNA.

Next, the obtained DNA was added to Nextera DNA Sample Preparation Kit (made by illumina), Nextera
A DNA sample was prepared by using Index Kit (manufactured by illumina), Agencourt AMPure XP (manufactured by BECKMAN COULTER), and DNA Clean & Concentration Kit-5 (manufactured by ZYMO RESEARCH) according to the manufacturer's recommended protocol. The DNA sample is then transferred to the Miseq Reagent Kit v2 500-cycles
It was loaded on (made by illumina), and base sequence information of genomic DNA was obtained by a MiSeq (made by illumina) DNA sequencer. When a contiguous sequence of genomic DNA prepared from the obtained base sequence was searched for a DNA sequence corresponding to SEQ ID NO: 1, which is the N-terminal amino acid sequence, the base sequence of SEQ ID NO: 4 was obtained.

Example 6: cDNA encoding galactolipase of Chlorella quercerii strain 11h (UTEX 263)
The total RNA was extracted and purified from the algal cells of Chlorella querseri 11h strain (UTEX 263) obtained in Example 5 using RNeasy mini (QIAGEN) to obtain about 8 μg of total RNA.

  Using the obtained total RNA as a template, “1st strand cDNA for 3′RACE and 5′RACE” was prepared using SMARTer RACE cDNA Amplification Kit (Clontech).

Subsequently, the gene fragment was amplified by the 3′RACE method and the 5′RACE method using “the first strand cDNA for 3′RACE and 5′RACE” as a template. As a primer for 3′RACE, a primer of SEQ ID NO: 5 designed based on SEQ ID NO: 4 and using genomic DNA sequence information was used. On the other hand, the primer for SEQ ID NO: 6 designed using genomic DNA information based on SEQ ID NO: 2 was used as the 5'RACE primer. KOD FX Neo (TOYOBO) was used as the polymerase. PCR was carried out 40 cycles of “98 ° C.-10 seconds, 68 ° C.-2 minutes” after heat denaturation at 94 ° C.-2 minutes. As a result, a DNA fragment of about 2 kbp was obtained by the 3′RACE method, and a DNA fragment of about 800 bp was obtained by the 5′RACE method.

The DNA fragments obtained by the 3′RACE method and the 5′RACE method were each subjected to sequence analysis, and the full-length cDNA sequence was obtained by overlapping the sequences. An open reading frame (ORF) consisting of 1530 bases was found in the cDNA. The ORF was capable of encoding 510 amino acid residues. SEQ ID NO: 7 shows the base sequence from the start codon to the end codon of the cDNA, including the base sequence of the ORF. An amino acid sequence that can be encoded by the ORF (that is, an amino acid sequence that can be encoded by the cDNA) is shown in SEQ ID NO: 8. In SEQ ID NO: 7, the nucleotide sequence of SEQ ID NO: 9 corresponding to SEQ ID NO: 3, which is the internal amino acid sequence of chGL, was confirmed, confirming that the cDNA can encode chGL. The amino acid sequence (SEQ ID NO: 8) that can be encoded by the cDNA is the amino acid sequence of chGL including the pro region. The amino acid sequence of mature chGL and the nucleotide sequence of the portion capable of encoding mature chGL in the cDNA are shown in SEQ ID NOs: 10 and 11, respectively.

Next, in order to confirm that the base sequence of the cDNA that can encode chGL is SEQ ID NO: 7, using the primers of SEQ ID NO: 12 and SEQ ID NO: 13, "1st strand for 3'RACE and 5'RACE" Perform PCR with 35 cycles of 98 ° C for 10 seconds, 68 ° C for 3 minutes after heat denaturation at 94 ° C for 2 minutes using KOD FX Neo as a template with cDNA as a template The DNA fragment was amplified. Then, the amplified DNA fragment was ligated to the attached pUC118 vector using Mighty Cloning Reagent Set (Blunt End) (TaKaRa), and the nucleotide sequence of the DNA fragment was determined. It was confirmed that the DNA fragment had

Example 7: Expression of chGL by Escherichia coli It was examined to express mature chGL in Escherichia coli. PCold TF DNA (TaKaRa) was selected as the expression plasmid. pCold TF DNA is a cold shock expression vector designed to express a target gene in a form in which the target protein is fused to a target protein using a trigger factor (TF), a chaperone, as a solubilizing tag. It is.

A cDNA capable of encoding mature chGL (SEQ ID NO: 10) was cloned into an expression plasmid using In-Fusion HD Cloning Kit (Clontech). pCold TF DNA fragment amplification is performed using pCold TF
PCR was performed using DNA as a template and primers of SEQ ID NO: 14 and SEQ ID NO: 15. Amplification of a cDNA fragment capable of encoding mature chGL was carried out by PCR using “1 ′ strand cDNA for 3′RACE and 5′RACE” as a template and primers of SEQ ID NO: 16 and SEQ ID NO: 17. KOD FX Neo (TOYOBO) was used as the polymerase. PCR was performed with 35 cycles of “98 ° C.-10 seconds, 55 ° C.-5 seconds, 68 ° C.-6 minutes” after heat denaturation at 94 ° C.-2 minutes. The amplified fragments were ligated by In-Fusion reaction, and E. coli JM109 strain was transformed with the reaction product. From the obtained transformant, it was possible to obtain a strain having an expression plasmid (named pCTF-chGL1) in which a cDNA capable of correctly encoding mature chGL was linked downstream of the DNA encoding TF.

Next, pCTF-chGL1 was extracted from the obtained strain using a plasmid extraction kit QIAprep Spin Miniprep Kit (QIAGEN). E. coli Rosetta2 strain (Novagen) was transformed with pCTF-chGL1 to produce a mature chGL expression strain E. coli pCTF-chGL1 / Rosetta2. This expression strain
LB medium with ampicillin added to a final concentration of 100 μg / ml (Trypton (Difco) 10 g, Yeast Extract (Difco) 5 g, deionized water added to 10 g NaCl, adjusted to 1 L, then adjusted to pH 7.0) And precultured at 37 ° C. overnight with shaking in an autoclave (121 ° C., 15 minutes)). Add 1/100 volume of preculture to a new LB medium supplemented with ampicillin to a final concentration of 100 μg / ml, and after shaking culture at 37 ° C. for about 3 hours, the final concentration becomes 1 mM. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added, and the culture temperature was lowered to 15 ° C. to induce transcription of pCTF-chGL1 from the cspA promoter (cold induction). After culturing at 15 ° C. for 24 hours, the cells were collected by centrifugation. As a control, the cells just before the low temperature induction were collected in the same manner.

Next, after suspending the bacterial cells for 1 ml of the culture in 350 μl of buffer S (20 mM Tris-HCl (pH 8.0)), an ultrasonic crusher UCD-250 (manufactured by Cosmo Bio) is used. Crushing was performed by repeating the cycle of “30 seconds ON−30 seconds OFF” 10 times under cooling conditions. The obtained bacterial cell disruption solution was centrifuged (21,600 × g, 4 ° C. for 10 minutes) to remove the insoluble fraction, and the supernatant fraction was used as a crude extract.

Next, 4 µl of crude extract, 2.5 µl of NuPAGE LDS sample buffer (Novex), NuPAGE
1 μl of sample reducing reagent (Novex) and 2.5 μl of deionized water were added, and after heat treatment at 70 ° C. for 10 minutes, electrophoresis was performed using NuPAGE 4-12% Bis-Tris gel (Novex). As a result, as shown in FIG. 9, a prominent protein band was observed in the vicinity of the target molecular weight of 105 × 10 ³ in the sample after the low temperature induction as compared with that before the low temperature induction with the expression plasmid. Since the theoretical molecular weight of TF is 52,100 and the theoretical molecular weight of chGL is 52,800, the molecular weight of the TF-chGL fusion protein, which is the target protein in this expression experiment, is estimated to be about 105 × 10 ³ .

It was examined whether galactolipase activity could be confirmed in the crude extract after low temperature induction of pCTF-chGL1 / Roseeta2. 2.5 μl of the crude extract obtained as described above was added to 17.5 μl of enzyme reaction solution (200 mM Tris-HCl solution (pH 7.1), “1% DGDG, 1% TritonX-100 mixed solution” 2.5 μl, 100
After adding to 22.5 μl of mM CaCl ₂ solution (2.5 μl), the mixture was incubated at 37 ° C. for 5 minutes. Thereafter, the enzyme was heat-inactivated by treatment at 100 ° C. for 10 minutes. Subsequently, the supernatant was collected by centrifugation (21,600 × g, 4 ° C. for 5 minutes), and the fatty acid therein was quantified. In addition, as a control sample, a crude extract previously treated at 100 ° C. for 10 minutes and heat-inactivated was prepared, and fatty acids were quantified after enzymatic reaction in the same manner. LabAssay NEFA (Wako Pure Chemical Industries) was used for quantification of fatty acids, and the amount of fatty acids produced was calculated in terms of oleic acid. A value obtained by subtracting the amount of fatty acid in the control sample heat-treated in advance from the amount of fatty acid quantified in the measurement sample was regarded as the amount of produced fatty acid, and the enzyme activity that produced 1 μmol of fatty acid per minute was defined as 1 unit of galactolipase activity. Absorbance plate reader SPECTRAMAX190 (manufactured by Molecular Devices) was used for absorbance measurement. As a result of enzyme activity measurement, the specific activity of galactolipase in the crude extract was 1.2 unit / mg protein. From the above results, it was shown that the cDNA obtained in Example 6 can encode chGL.

This fusion protein (TF-chGL fusion protein) has a His tag on the N-terminal side. Therefore, the fusion protein was purified by Ni-NTA resin of Amicon Pro Affinity Concentration Kit Ni-NTA (Millipore). Furthermore, since this fusion protein has the cleavage recognition site (Ile-Glu-Gly-Arg) of protease Factor Xa at the junction of TF and chGL, this fusion protein can be cleaved with Factor Xa (Novagen). So, only the chGL part can be acquired. Therefore, next, only the chGL part was obtained, and the galactolipase activity was confirmed only in the chGL part.

The purified TF-chGL fusion protein was treated with FactorXa for 1 hour. The treated sample was subjected to SDS-PAGE using NuPAGE 4-12% Bis-Tris gel (Novex). Compared with the sample before FactorXa treatment, when treated with FactorXa, the molecular weight band of TF-chGL disappeared and about 53,000 protein bands corresponding to TF and chGL were generated instead (FIG. 10). Also,
When the galactolipase activity of this treated sample was confirmed, the specific activity of galactolipase was about 8 units / mg protein. From the above results, it was found that the cDNA obtained in Example 6 was a cDNA encoding chGL.

Example 8: Expression of chGL by actinomycetes Expression of chGL by Streptomyces lividans was carried out as follows.

Using the “3′RACE and 5′RACE 1st strand cDNA” described in Example 6 as a template, the upstream and downstream fragments of DNA encoding the full length of chGL including the prosequence (SEQ ID NO: 8), Amplification was performed by PCR using the primer pair of SEQ ID NO: 20 and 21 and the primer pair of SEQ ID NO: 22 and 23, respectively. Next, both amplified DNA fragments were mixed to form a template, and PCR was performed using the primers of SEQ ID NOs: 21 and 22. As a result, a DNA fragment encoding the full length of chGL containing the pro sequence (SEQ ID NO: 8) was obtained by crushing the SphI site in the chGL gene.
In this DNA fragment, a SphI site is added to the 5 ′ end of the portion encoding the chGL prosequence, and a BglII site is added to the 3 ′ end of the portion encoding the mature chGL. This DNA fragment was digested with SphI and BglII and introduced into the known Escherichia coli and actinomycetes shuttle vector pUC702 (Molnar et al., J. Ferment. Bioeng., Vol.72, 368-372 (1991)). The inserted PLD promoter and PLD terminator (Ogino et al, Appl. Microbiol. Biotechnol., Vol. 64, 823-828 (2004)) were constructed to construct the chGL expression plasmid pUC702 / chGL.

  pUC702 / chGL was introduced into Streptomyces lividans strain 1326 (NBRC15675) by a known method. The transformant thus obtained was added to a PG medium containing 50 μg / ml thiostrepton (composition: 2% glucose, 0.5% yeast extract, 0.5% peptone, 0.8% K2HPO4, 0.05% MgSO4 · 7 water). Inoculated into ISP2 medium (composition: 10 g malt extract, 4 g yeast extract, and 4 g glucose per liter, pH 7.2) containing 50 μg / ml thiostrepton and at 23 ° C Cultured.

Next, after suspending the cells of 1 mL of the culture in 1 mL of buffer solution (20 mM Tris-HCl (pH 8.0) containing 1% Triton X-100), cooling conditions using an ultrasonic crusher The cells were crushed below. The obtained bacterial cell disruption solution was centrifuged (21,600 × g, 4 ° C. for 10 minutes) to remove the insoluble fraction, and the supernatant fraction was used as a crude extract. As a result of measuring enzyme activity using the crude extract, 1 mL of the culture solution was obtained on the 6th day of culture.
About 1.5 U of enzyme was obtained per unit. Thus, chGL was able to be expressed in the microbial cells in a form that can exhibit enzyme activity with actinomycetes as a host.

Example 9: Comparison of homology of amino acid sequence between chGL and known galactolipase
The homology of the amino acid sequence between chGL and known galactolipase was analyzed. For the analysis of homology, sequence multiple alignment software ClustalW was used. FIG. 11 shows a comparison result between chGL and galactolipase derived from Aspergillus japonicus (SEQ ID NO: 18; JP-A-2008-206515), and galactolipase derived from chGL and Chlamydomonas reinhardtii (SEQ ID NO: 19). ; Comparison results with Li X, et al., Plant Cell 11, 4670 (2012)) are shown in FIG. The homology at the amino acid level was found to be extremely low, 12% between chGL and galactolipase derived from Aspergillus japonicus, and 20% between chGL and galactolipase derived from Chlamydomonas reinhardtii. Therefore, it was shown that chGL acquired in this Example is a novel galactolipase. Note that. The galactolipase derived from Aspergillus japonics consists of 294 amino acid residues, and the galactolipase derived from Chlamydomonas reinhardi consists of 1117 amino acid residues, and the molecular weight of the protein is greatly different from chGL.

Example 10: Comparison of enzyme properties of lipopan F and chGL Temperature stability was compared between lipopan F (Novozyme) commercially available as lipase for breadmaking and chGL.

Lipopan F is added to the reaction preparation (20 mM Tris-HCl (pH 7.1), 1% Triton X-100) at 1 mg / ml (
In this enzyme activity measurement system, it was added so as to be equivalent to about 150 U / mg-protein) and used as an enzyme solution. On the other hand, chGL used the purified enzyme (enzyme concentration 66U / mg-protein) prepared in Example 2 as an enzyme solution. First, the enzyme solution was kept at 30 ° C., 35 ° C., 40 ° C., 45 ° C., 50 ° C., 60 ° C., and 70 ° C. for 30 minutes, and then 2.5 μl of the enzyme solution was added to the reaction solution (200 mM Tris- HCl (pH 7.1) 17.5 μl, “1% DGDG, 1% TritonX-100” mixed solution 2.5 μl, 100 mM CaCl ₂ solution 2.5 μl) 22.5
After mixing with μl, the mixture was incubated at 37 ° C. for 10 minutes. Thereafter, the mixture was heat-inactivated at 100 ° C. for 10 minutes, and then centrifuged (21,600 × g, 4 ° C. for 5 minutes) to obtain a supernatant. Subsequently, the fatty acid in the supernatant was quantified, and the galactolipase activity was calculated. All enzyme reactions were performed with n = 3 specimens. FIG. 13 is a graph showing the residual galactolipase activity after treatment at each temperature as relative activity based on the residual galactolipase activity after treatment at 40 ° C. (100%). As shown in FIG. 13, the temperature stability at 50 ° C. was shown to be superior to that of Lipopan F, and it was found that chGL has higher storage stability.

Example 11: Application of chGL to food ingredients
The effect of chGL on the modification of physical properties of food materials was tested using wheat flour. Add 25 ml of chGL solution for 10 units prepared to a final concentration of 20 mM Tris-HCl (pH 7.1), 0.2% Triton X-100, 30 mM NaCl to 3 g of weak flour (Nisshin Flour Mills). The fracture properties of the resulting product were evaluated using a texture analyzer (TA XTplus, manufactured by StableMicroSystems). Specifically, after the flour and chGL were reacted with stirring at 25 ° C. for 30 minutes, the temperature was raised to 95 ° C. over 7 minutes, and then gelatinization was performed by stirring at 95 ° C. for 110 seconds. The gelatinized sample was packed in a 1.5 ml Eppendorf tube and subjected to aging treatment (“freeze-thaw” was repeated three times) to prepare a simulated dough sample. The obtained dough sample was subjected to a break test. The results are shown in FIG. In FIG. 14, the change in the test force has two peaks because the fabric once broken due to the compression of the jig is compressed again. As a result of the break test, it was found that the hardness of the fabric sample treated with chGL tends to decrease (Table 2), that is, the effect of maintaining the fabric softness with respect to the old gelatinized sample. The possibility was suggested. On the other hand, with regard to “tackiness”, the results were almost the same between the dough sample treated with chGL and the control without addition of chGL (Table 2). Here, “hardness” is defined as the maximum test force (the magnitude of the force applied to the analyzer jig) received when the dough sample is compressed, and “tackiness” is defined as after the dough sample is compressed. It was defined as the value multiplied by the force and distance required to pull the jig apart.

Example 12: Production of sugar glycerol using chGL A sample prepared by treating the purified TF-chGL fusion protein described in Example 7 with FactorXa for 1 hour was used as an enzyme solution. After adding 25 μl of enzyme solution to 225 μl of enzyme reaction solution (175 μl of 200 mM Tris-HCl solution (pH 7.1), 25 μl of “1% DGDG, 1% TritonX-100 mixture”, 25 μl of 100 mM CaCl ₂ solution), Incubated at 37 ° C for 1 hour. Then Hitachi autosampler L-7200, pump
The HPLC analysis of the reaction sample was performed using an HPLC analyzer equipped with L-7100 and a differential refractive index detector L-2490 and a sugar analysis column SH-1011 (Showa Denko). Water was used as the mobile phase. As a result, as shown in FIG. 15, a peak of digalactosylglycerol, a kind of sugar glycerol, was detected at a retention time of 7.01 minutes.

<Explanation of Sequence Listing>
SEQ ID NO: 1: N-terminal amino acid sequence of mature chGL SEQ ID NO: 2: Internal amino acid sequence of chGL SEQ ID NO: 3: Internal amino acid sequence of chGL SEQ ID NO: 4: Base sequence of DNA encoding the N-terminal amino acid sequence of mature chGL No. 5: nucleotide sequence of 3′-RACE primer SEQ ID NO: 6: nucleotide sequence of 5′-RACE primer SEQ ID NO: 7: nucleotide sequence of cDNA encoding chGL SEQ ID NO: 8: amino acid sequence of chGL SEQ ID NO: 9: chGL Nucleotide sequence of DNA encoding the internal amino acid sequence of SEQ ID NO: 10: nucleotide sequence of cDNA encoding mature chGL SEQ ID NO: 11: amino acid sequence of mature chGL SEQ ID NO: 12: primer for cloning the cDNA encoding chGL SEQ ID NO: 13: Primer nucleotide sequence for cloning cDNA encoding chGL SEQ ID NO: 14: Primer nucleotide sequence for pCold TF DNA DNA fragment amplification No. 15: base sequence of primer for amplifying DNA fragment of pCold TF DNA SEQ ID NO: 16: base sequence of primer for amplifying cDNA fragment encoding mature chGL SEQ ID NO: 17: for amplifying cDNA fragment encoding mature chGL Primer base sequence SEQ ID NO: 18: amino acid sequence of galactolipase derived from Aspergillus japonicus SEQ ID NO: 19: amino acid sequence of galactolipase derived from Chlamydomonas reinhardtii SEQ ID NO: 20: base sequence of primer for amplification of DNA fragment encoding a part of chGL SEQ ID NO: 21: nucleotide sequence of primer for amplification of DNA fragment encoding part of chGL SEQ ID NO: 22: nucleotide sequence of primer for amplification of DNA fragment encoding part of chGL SEQ ID NO: 23: encoding part of chGL Sequence of primers for amplification of DNA fragments

Claims (13)

Lipases having the following properties (1) to (3):
(1) having an activity of catalyzing a reaction of hydrolyzing a carboxyl ester bond of glycerolipid;
(2) the molecular weight is from 50,000 to 60,000;
(3) Derived from the genus Chlorella.   The lipase according to claim 1, wherein the glycerolipid is a glyceroglycolipid.   The lipase according to claim 1 or 2, wherein the chlorella algae is Chlorella kessleri. Lipase, which is a protein described in the following (a) or (b):
(A) a protein comprising the amino acid sequence shown in SEQ ID NO: 8 or 11;
(B) the amino acid sequence shown in SEQ ID NO: 8 or 11, comprising an amino acid sequence containing substitution, deletion, insertion, or addition of 1 to 10 amino acid residues, and hydrolyzing the carboxyl ester bond of glyceroglycolipid A protein having an activity of catalyzing a decomposing reaction.   A polynucleotide encoding the lipase according to any one of claims 1 to 4. The polynucleotide according to (A) or (B) below:
(A) a polynucleotide comprising the base sequence represented by SEQ ID NO: 7 or 10;
(B) encodes a protein comprising a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 7 or 10 and having an activity of catalyzing a reaction of hydrolyzing a carboxyl ester bond of glyceroglycolipid Polynucleotide.   A recombinant vector comprising the polynucleotide according to claim 5 or 6.   A transformant into which the recombinant vector according to claim 7 has been introduced.   A transformant according to claim 7 is cultured in a medium to produce the lipase according to any one of claims 1 to 4, and the lipase is collected from the culture. Manufacturing method.   The method to hydrolyze a glycerolipid including making the lipase of any one of Claims 1-4 act on a glycerolipid.   The method according to claim 10, wherein the glycerolipid is a food or drink containing glycerolipid or a raw material thereof.   The method to modify food / beverage products or its raw material including making the lipase of any one of Claims 1-4 act on the food / beverage products containing glycerolipid, or its raw material.   A method for producing glycoglycerol, comprising causing the lipase according to any one of claims 1 to 4 to act on a glyceroglycolipid.