JP2014027908A - Method for producing biodiesel fuel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new method for producing a biodiesel fuel.SOLUTION: The method for producing a biodiesel fuel is provided by treating a phospholipid-containing biomass feedstock with an enzyme to esterify fatty acids FA to fatty acid esters ME along with hydrolyzing of the phospholipids in the biomass feedstock. As the enzyme, the enzyme exhibiting hydrolytic activity on the phospholipids, preferably at least one of the enzymes which contain polypeptides each having two kinds of specific amino acid sequences, is used. These polypeptides may optionally have the amino acid sequence with one or more amino acids substituted, inserted, deleted, and/or added, or with at least 75% homology. The biodiesel fuel can be easily produced in high productivity by subjecting the biomass feedstock to enzyme treatment.

Description

  The present invention relates to a method for producing biodiesel fuel using waste or the like.

  Conventionally, a method for producing biodiesel fuel (BDF) using glycerides contained in edible oil or waste edible oil as a raw material using an alkali, a solid acid catalyst, or lipase is known.

  For example, Patent Document 1 discloses a transesterification reaction between a raw oil / fat obtained by esterifying a free fatty acid of vegetable oil / fatty derived from a sorghum oil with sulfuric acid in advance and an alkyl alcohol having 2 or less carbon atoms in the presence of a calcium oxide catalyst. And a method for producing biodiesel fuel is disclosed.

  In Patent Document 2, fats and oils containing glycerides, water and lipase are mixed and subjected to a hydrolysis reaction until a predetermined decomposition rate is reached, and then alcohol is added to further continue the hydrolysis reaction. A method for producing biodiesel fuel is disclosed.

  A biodiesel fuel is a diesel fuel obtained from a biomass raw material. As a chemical substance, for example, fatty acid methyl ester is currently known.

JP 2011-012254 A JP 2010-106107 A

  However, when edible oil or waste edible oil is used as a raw material for biodiesel fuel, there is a problem that the balance between supply and demand is not established, and there is a problem that it competes with edible oil. Moreover, in the conventional manufacturing method of biodiesel fuel, the high manufacturing cost, the method of treating the waste liquid after the reaction, and coping with surplus of by-product glycerin are listed as practical problems. Because of these problems, it was difficult to put biodiesel fuel into practical use.

  The present invention has been made in view of the above problems, and an object of the present invention is to easily produce biodiesel fuel with high productivity.

  The method for producing biodiesel fuel according to the present invention produces biodiesel fuel by hydrolyzing phospholipids in biomass raw materials and esterifying fatty acids by treating biomass raw materials containing phospholipids with enzymes. Is.

  As the enzyme, an enzyme that exhibits hydrolytic activity for phospholipids is used.

  As the enzyme, preferably, at least one of the following enzymes A and B, or at least one of the following enzymes C and D is used.

Enzyme A: an enzyme comprising the following polypeptide (a1), (a2), or (a3),
(A1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1;
(A2) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 1 and exhibiting hydrolytic activity against phospholipids ;
(A3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 1 and exhibiting hydrolytic activity against phospholipids.

Enzyme B: an enzyme comprising a polypeptide of the following (b1), (b2), or (b3),
(B1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2;
(B2) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and exhibiting hydrolytic activity against phospholipids ;
(B3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting hydrolytic activity against phospholipids.

Enzyme C: an enzyme comprising the following polypeptide (c1), (c2), or (c3),
(C1) a polypeptide produced from a microorganism having the nucleotide sequence of SEQ ID NO: 3 as a polynucleotide;
(C2) a polypeptide produced from a microorganism having a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 3;
(C3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the base sequence set forth in SEQ ID NO: 3.

Enzyme D: an enzyme comprising a polypeptide of the following (d1), (d2), or (d3),
(D1) a polypeptide produced from a microorganism having the nucleotide sequence of SEQ ID NO: 4 as a polynucleotide;
(D2) a polypeptide produced from a microorganism having a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 4;
(D3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the base sequence set forth in SEQ ID NO: 4.

  In the present invention, the enzyme is preferably derived from a microorganism belonging to the genus Streptomyces. Moreover, it is preferable that the said biomass raw material is a waste material. The esterification is preferably methyl esterification.

  According to the present invention, biodiesel fuel can be produced by enzymatic treatment of a biomass raw material, so that biodiesel fuel can be easily produced with high productivity.

It is a photograph of TLC which shows the result of producing | generating biodiesel fuel with the enzyme of this invention. (A) And (b) is the photograph of TLC which shows the result of having produced | generated biodiesel fuel with the enzyme. It is a photograph of TLC which shows the result of having produced biodiesel fuel with an enzyme. It is a graph which shows the result of having produced | generated biodiesel fuel on condition of 25 mol% of methanol using enzyme PLA1. It is a graph which shows the result of having produced | generated biodiesel fuel on condition of 25 mol% of methanol using enzyme PLB. It is a graph which shows the comparison of enzyme PLA1 and enzyme PLB at the time of producing | generating biodiesel fuel on conditions of 25 mol% of methanol. It is a graph which shows the result of having produced | generated biodiesel fuel on condition of methanol 50mol% using enzyme PLA1. It is a graph which shows the result of having produced | generated biodiesel fuel on condition of 50 mol% of methanol using enzyme PLB. It is a graph which shows the comparison of enzyme PLA1 and enzyme PLB at the time of producing | generating biodiesel fuel on the conditions of 50 mol% of methanol. It is a graph which shows the result of having produced | generated biodiesel fuel on conditions of 75 mol% of methanol using enzyme PLA1. It is a graph which shows the result of having produced | generated biodiesel fuel on condition of 75 mol% of methanol using enzyme PLB. It is a graph which shows the comparison of enzyme PLA1 and enzyme PLB at the time of producing | generating biodiesel fuel on the conditions of 75 mol% of methanol. It is a graph which shows the tendency of enzyme PLA1 in the production | generation of biodiesel fuel. It is a graph which shows the tendency of enzyme PLB in the production | generation of biodiesel fuel. It is a graph which shows the comparison of the tendency of enzyme PLA1 and enzyme PLB in the production | generation of biodiesel fuel.

  In the method for producing biodiesel fuel according to the present invention, a biomass raw material containing phospholipid is hydrolyzed with respect to phospholipid, such as at least one enzyme selected from enzyme A, enzyme B, enzyme C, and enzyme D. Treat with indicated enzyme.

[enzyme]
In the present specification, the enzyme is not limited to a purified enzyme, but includes a crude product, an immobilized product, and the like. The enzyme is purified by a method well known to those skilled in the art, such as ammonium sulfate precipitation, ion exchange chromatography, hydrophobic chromatography, etc., using a microorganism culture solution. Thereby, enzymes with various degrees of purification (including enzymes purified to approximately one) can be obtained.

  In the present specification, microorganisms are wild strains, mutant strains (for example, those induced by ultraviolet irradiation, etc.), recombinants induced by genetic engineering techniques such as cell fusion or genetic recombination methods, etc. Any strain may be used. Genetically engineered microorganisms such as recombinants are known to those skilled in the art, as described, for example, in Molecular Cloning A Laboratory Manual, 2nd edition (Sambrook, J. et al., Cold Spring Harbor Laboratory Press, 1989). Can be easily created using various techniques. The culture solution of microorganisms means both a culture solution containing microbial cells and a culture solution from which microbial cells have been removed by centrifugation or the like.

  As the enzyme, an enzyme exhibiting hydrolytic activity with respect to phospholipid is used. It is an enzyme called so-called phospholipase (PL). As the phospholipase, PLA1, PLA2, PLB and the like can be used. Examples of such enzymes include commercially available enzymes such as PLA2 from porcine pancreas, mphejicobra, and Streptomyces violaceoruber, commercially available from Sigma Aldrich, PLA1 from Thermomyces lanuginosus, and PLA1 from Lecitase Ultra. Etc. Alternatively, PLA1 from Mitsubishi Chemical Foods, lecitase from Novozyme, PLA2 Lipomod 699L from Genencor Kyowa, PLA2 Nagase from Nagase ChemteX, PLA2 Maxa Pearl A2 from DSM Japan, PLA2 from Asahi Kasei Pharmatech, PLA2 from Asahi Kasei Pharmatech Industrial enzymes such as PLB may be used. Among these, from the viewpoint of production of biodiesel fuel, the enzyme preferably has alcohol resistance (particularly resistance to methanol).

  Furthermore, in the production of biodiesel fuel, enzyme A, enzyme B, enzyme C and enzyme D described below are preferable to these enzymes. The enzyme described below can eliminate the addition of an inorganic salt such as a calcium salt, can be reacted at a low temperature to a high temperature (20 to 70 ° C.), and can improve the efficiency of the enzyme treatment. Moreover, since it can be made to react by pH 3-11, reaction control is easy. Moreover, since the titer of the enzyme is extremely high, the reaction efficiency is remarkably high. Furthermore, since it acts on many types of phospholipids, the reaction yield is high.

[Enzyme A, Enzyme C]
Enzyme A is (a1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, or (a2) one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 1 are substituted, inserted, deleted and / or Or a polypeptide having an added amino acid sequence and exhibiting hydrolytic activity for phospholipids, or (a3) having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 1, It contains a polypeptide that exhibits hydrolytic activity.

  The enzyme C is (c1) a polypeptide produced from a microorganism having the base sequence described in SEQ ID NO: 3 as a polynucleotide, or (c2) a base sequence complementary to the base sequence described in SEQ ID NO: 3 and a stringent Produced from a microorganism having a polynucleotide that hybridizes under various conditions, or (c3) produced from a microorganism having a polynucleotide having at least 75% sequence identity to the base sequence set forth in SEQ ID NO: 3 Contains a polypeptide.

  The polynucleotide having the base sequence described in SEQ ID NO: 3 can encode the polypeptide (amino acid sequence) described in SEQ ID NO: 1. That is, in a preferred embodiment of the present invention, enzyme A and enzyme C match.

  Enzyme A and Enzyme C preferentially hydrolyze the fatty acid ester bond at the α-position (sn-1 position) of the glycerol group in the glycerol phospholipid (dominant over the sn-2 position), that is, phospholipase A1 ( It is a kind of PLA1). Accordingly, enzyme A and enzyme C, which are PLA1, are phospholipid to glycerol-3-phosphate compounds such as 2-lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphocholine (glycerol-3-phosphorus). Acid phosphodiester) is an enzyme that produces at least one, preferably all three.

  PLA1 activity can be confirmed, for example, as follows, but the confirmation method is not limited thereto. Specifically, for example, PLA1 activity can be confirmed by measuring the amount of free fatty acid produced as a result of the enzyme reaction.

  As a specific method, first, 1 g of egg yolk phosphatidylcholine (manufactured by Nacalai Tesque) was dissolved in 10 mL of 0.02% (w / v) Triton X-100 (manufactured by Nacalai Tesque), and 10% ( w / v) Prepare egg yolk phosphatidylcholine. 0.025 mL of this 10% (w / v) egg yolk phosphatidylcholine, 0.060 mL of 0.2 M Tris-HCl buffer (pH 8.0), 0.5 M EDTA disodium (manufactured by Wako Pure Chemical Industries, Ltd.) 0 Add 0.005 mL. And after preheating at 37 degreeC for 5 minute (s), 0.010 mL of samples which confirm enzyme activity are added, and it is made to react at 37 degreeC for 5 minutes. After the enzyme reaction, the reaction is stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution can be determined using, for example, “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.), which is a free fatty acid measurement kit, according to the instructions attached to the kit. Measure as described. The amount of enzyme that produces 1 μmol of free fatty acid per minute is defined as 1 unit (1 U).

  Enzyme A and enzyme C include, for example, acetic acid-sodium acetate buffer (pH 4.1-5.6), bistris-hydrochloric acid buffer (pH 5.6-7.2), tris-hydrochloric acid buffer ( pH 7.2 to 8.8) and glycine-sodium hydroxide buffer (pH 8.8 to 10.5), and when the above egg yolk phosphatidylcholine and the enzyme are put under reaction conditions, the pH range It can show PLA1 activity within (pH 4.1 to 10.5). The optimum pH is around pH 5.6, but exhibits substantially 100% activity at pH 5-8.

  Enzyme A and enzyme C are, for example, from pH 4.1 to pH 10 when the hydrolysis activity at pH 5.6 is 100% under the condition that egg yolk phosphatidylcholine and enzyme are reacted at 37 ° C. for 5 minutes as described above. It is preferable to show an activity of 50% or more within the range.

  Enzyme A and enzyme C can act at about 20 to 65 ° C., for example, under the reaction conditions of egg yolk phosphatidylcholine and the enzyme described above. The optimum temperature can be within this range. Preferably it is in the range of about 30-55 ° C, more preferably in the range of 40-55 ° C, even more preferably about 50 ° C.

  Enzyme A and enzyme C, for example, can be stable when treated with 120 mM acetic acid-sodium acetate buffer (pH 5.6) for 30 minutes, with almost no decrease in activity seen from 4 ° C. to 40 ° C., and Even at 45 ° C., an activity of about 80% (for example, 75%) or more remains.

Enzyme A and enzyme C are, for example, acetic acid-sodium acetate buffer solution (pH 5.6) as a buffer solution. When the above phospholipid and enzyme solution are placed under reaction conditions, 100 mM EDTA is present. Are not inhibited, and preferably exhibit almost the same activity as when EDTA is not added. Further, in the presence of 10 mM Ca ²⁺ and Zn ²⁺ , it is preferable to exhibit about 80% activity (for example, about 80 to 95% activity). On the other hand, 10 mM Fe ³⁺ and Fe ²⁺ can inhibit the activity.

  When enzyme A and enzyme C are reacted at 50 ° C. for 5 minutes as described above under the condition of pH 9.0, the hydrolysis activity with respect to the case where egg yolk phosphatidylcholine is a substrate is 100%. Then, 50% or less for DPPC (lower limit is 30%), 350% or more for PA (upper limit is 400%), 400% or more for PG (upper limit is 450%), 450% for PS Above (upper limit is 480%), 100% or more for PE (upper limit is 140%), 350% or more for PI (upper limit is 380%), 300% or more for soybean phosphatidylcholine (manufactured by SIGMA) (upper limit) 360%) and 75.0% or more (upper limit is 90%) with respect to soybean lecithin (manufactured by Wako Pure Chemical Industries, Ltd.).

  Enzyme A and Enzyme C are 100% or more (upper limit) with respect to DPPC, when egg yolk phosphatidylcholine is used as a substrate and the hydrolysis activity is 100% at 50 ° C. and pH 5.6 for 5 minutes. 140%), 100% or less for PA (lower limit is 60%), 150% or more for PG (upper limit is 190%), 150% or more for PS (upper limit is 190%), for PE 100% or more (upper limit is 120%), 250% or more for PI (upper limit is 290%), 250% or more for soybean phosphatidylcholine (manufactured by SIGMA, L-α-Phosphatidylcholine) (upper limit is 290%), Has an activity of 250% or more (upper limit is 280%) with respect to soybean lecithin (manufactured by Wako Pure Chemical Industries), and almost 0% (for example, 5% or less, 3% or less, or 1%) with respect to triglyceride (olive oil) Lower, or is preferably a detection limit or less). It is preferable to have such substrate specificity.

  DPPC is an abbreviation for 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine. PA is an abbreviation for 1,2-Dimyristoyl-sn-glycerol-3-phosphate. PG is an abbreviation for 1,2-Diacyl-sn-glycero-3-phospho- (1-rac-glycerol). PS is an abbreviation for L-α-Phosphatidylserine. PE is an abbreviation for 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine. PI is an abbreviation for L-α-Phosphatidylinositol).

  Enzyme A and Enzyme C are 1-palmitoyl-2-oleate when egg yolk lecithin (L) is used as a substrate and the hydrolysis activity is 100% at 50 ° C. for 5 minutes at pH 7.2. 50% or more (up to 200%) for oil-sn-glycero-3-phosphoethanolamine (POPE), 500% for 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate (POPA) Above (upper limit 600%), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) 150% or more (upper limit 350%), L-α-phosphatidylinositol (PI) 300% or more (upper limit 450%), 300% or more (upper limit 700%) with respect to L-α-phosphatidyl-L-serine (PS), 1-palmitoyl- More than 100% (up to 300%) for 2-oleoyl-sn-glycero-3-phospho-rac- (1-glycerol) (POPG), for soybean-derived L-α-phosphatidylcholine (SB-PC) 100% or more (upper limit 150%), 30% or more (upper limit 150%) for soybean lecithin (SBL), 250% or more (upper limit 350%) for L-α-lysophosphatidylcholine (LPC), 1- 50% or more (upper limit 90%), 1-hexadecyl-2- (9Z-) with respect to (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphoethanolamine (PlsPE (C18, 20: 4)) Octadecenoyl) -sn-glycero-3-phosphoethanolamine (PlsPE (C16, 18: 1)) 200% or more (upper limit 300 %), 1- (1Z-octadecenyl) -2-arachidonoyl-sn-glycero-3-phosphocholine (PlsPC (C18, 20: 4)), 0% for soybean oil and olive oil (lower limit) 0%), 4-nitrophenyl butyrate (pNPB), 4-nitrophenyl octanoate (pNPO), 4-nitrophenyl decanoate (pNPD), 4-nitrophenyl laurate (pNPL), 4-nitrophenyl It is preferable to have a relative activity of 15% or less (lower limit 0%) with respect to myristate (pNPM), 4-nitrophenyl palmitate (pNPP) and 4-nitrophenyl stearate (pNPS).

  Enzyme A and enzyme C may vary slightly depending on electrophoresis conditions and the like, but the molecular weight in SDS-PAGE is within the range of 25,000 to 30,000 (for example, about 28,000 or about 27,000). It is preferable to show. Moreover, it is preferable that the molecular weight computed from the amino acid composition of the enzyme A and the enzyme C exists in the range of 25,000-30,000. For example, a natural enzyme derived from Streptomyces albidoflavus NA297 strain has a molecular weight of about 28,000 in SDS-PAGE, specifically 28,000. The natural enzyme derived from this Streptomyces albidoflavus NA297 strain has a molecular weight of 27,199 calculated from its amino acid composition.

  Enzyme A and enzyme C preferably exhibit an isoelectric point in the range of 6.0 to 6.1 (for example, 6.06). The isoelectric point of the enzyme can be calculated from the amino acid sequence by GENETYX.

  One embodiment of enzyme A and enzyme C, namely phospholipase A1, consists of the amino acid sequence set forth in SEQ ID NO: 1. The phospholipase A1 preferably has an amino acid sequence from position 34 to position 269 of SEQ ID NO: 1 (also referred to herein as "amino acid sequence described in SEQ ID NO: 1").

  As long as enzyme A and enzyme C have PLA1 activity, one or more amino acids are substituted, deleted or inserted into the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1. And / or an enzyme having an added amino acid sequence. A person skilled in the art, for example, site-directed mutagenesis (Nucleic Acid Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; PCR: Atactical Approach, IRL Press, 1991, pp. 200), etc., as appropriate substitution, deletion, insertion, and / or addition mutation The structure of the protein can be modified by introducing. The number of amino acid residues that can be substituted, deleted, inserted, and / or added is usually 50 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, and even more preferably 0 to 3 It is. Further, amino acid mutations include not only artificially mutated enzymes but also naturally mutated enzymes in enzymes A and C as long as they have PLA1 activity.

  A protein having an amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 1 is also included in enzyme A and enzyme C as long as it has PLA1 activity. Enzyme A and enzyme C are preferably at least 75%, preferably at least 80%, more preferably at least 85% of the amino acid sequence set forth in SEQ ID NO: 1 or the amino acid sequence set forth in SEQ ID NO: 1. More preferably it may be a protein having an amino acid sequence with a homology of at least 90%, even more preferably at least 95%, even more preferably at least 99%.

  For protein homology (homology) search, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, DAD, or DNA databases such as DDBJ, EMBL, or Gene-Bank, etc. BLAST, FASTA, etc. This program can be used, for example, via the Internet. Confirmation of the activity of the protein can be performed using the procedure described above.

  Although the supply source of enzyme A and enzyme C is not particularly limited, enzyme A and enzyme C can be obtained from living cells such as microorganisms. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Streptomyces albidoflavus (Streptomyces albidoflavus) and Streptomyces albidoflavus and Streptomyces albidoflavus NA297 (accession number: NITE BP-1014, hereinafter referred to as “NA297 BP”) are preferred. Stock "). This Streptomyces albidoflavus NA297 has a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 3 in DNA.

  For example, the Streptomyces albidoflavus NA297 (accession number: NITE BP-1014) is secreted out of the cells by liquid culture in an appropriate nutrient medium, and the culture supernatant is lyophilized. Those treated with salting out, organic solvent, etc. can be produced as enzyme preparations.

  The microorganism that can be used for the production of the enzyme preparation is not limited to Streptomyces albidoflavus NA297, and may be a microorganism that belongs to the genus Streptomyces and can produce enzyme A or enzyme C. . In addition, natural or artificial mutant strains of these species or gene fragments necessary for the expression of PLA1 activity are artificially extracted, and other species that incorporate them are also used to produce enzymes A and C. Can be used. Moreover, even if it does not belong to Streptomyces genus, if it is a microorganism which can produce said enzyme A or enzyme C, it can also be used.

  An example of an enzyme preparation using Streptomyces albidoflavus NA297 will be described.

  Since this bacterium is secreted out of the bacterium by liquid culture in a nutrient medium, the culture supernatant is treated with lyophilization, salting out, an organic solvent, or the treated product is immobilized. Thus, an enzyme preparation can be produced. More specifically, first, this bacterium is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete the enzyme. Here, examples of the carbon source include starch and starch hydrolysate, sugars such as glucose and sucrose, alcohols such as glycerol, and organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt). Can be mentioned. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean flour, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. The concentration of the carbon source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v). The concentration of the nitrogen source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v). The culture temperature is preferably a temperature at which the enzyme A and the enzyme C are stable and the microorganism to be cultured can sufficiently grow, and is preferably 20 to 37 ° C, for example. The culture time is preferably a time during which the enzyme is sufficiently produced, and is preferably about 1 to 7 days, for example. Culturing can be preferably performed under aerobic conditions, for example, with aeration stirring or shaking.

  Polypeptides contained in enzyme A and enzyme C are fractionated according to protein solubility (precipitation with organic solvents, salting out with ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelate, Purification can be performed by appropriately combining known methods such as affinity chromatography using dyes, antibodies, and the like. For example, after recovering the culture supernatant of the microorganism, it can be purified by ammonium sulfate precipitation, further anion exchange chromatography, hydrophobic chromatography, and / or cation exchange chromatography. Thereby, in polyacrylamide gel electrophoresis (SDS-PAGE), it can refine | purify to a substantially single band. That is, the polypeptide which comprises the enzyme A and the enzyme C can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Enzyme B and Enzyme D]
Enzyme B is (b1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2, or (b2) one or more amino acids in the amino acid sequence set forth in SEQ ID NO: 2 are substituted, inserted, deleted and / or Or a polypeptide having an added amino acid sequence and exhibiting hydrolytic activity for phospholipids, or (b3) having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2, It contains a polypeptide that exhibits hydrolytic activity.

  Enzyme D is (d1) a polypeptide produced from a microorganism having the base sequence described in SEQ ID NO: 4 as a polynucleotide, or (d2) a base sequence complementary to the base sequence described in SEQ ID NO: 4 and a stringent Produced from a microorganism having a polynucleotide that hybridizes under various conditions, or (d3) produced from a microorganism having a polynucleotide having at least 75% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 Contains a polypeptide.

  The polynucleotide having the base sequence described in SEQ ID NO: 4 can encode the polypeptide (amino acid sequence) described in SEQ ID NO: 2. That is, enzyme B and enzyme D match in a preferred embodiment of the present invention.

  Enzyme B and enzyme D hydrolyze both the fatty acid ester groups of the fatty acid ester bond at the α-position (sn-1 position) of the glycerol group and the fatty acid ester group at the β-position (sn-2 position) of the glycerol group. Has enzyme activity. That is, the enzyme B and the enzyme D are enzymes having both phospholipase A1 activity and phospholipase A2 activity, and are one kind of enzymes called so-called phospholipase B (PLB). Accordingly, enzyme B and enzyme D, which are PLBs, are phospholipid to lysophospholipid, glycerol-3-phosphate, and glycerol-3-phosphate ester compounds such as glycerol-3-phosphocholine (glycerol-3-phosphate phospho An enzyme that produces at least one, preferably all three of the diesters.

  The PLB activity can be confirmed, for example, as follows, but the confirmation method is not limited to this. Specifically, for example, PLB activity can be confirmed by measuring the amount of free fatty acid produced as a result of the enzyme reaction.

  As a specific method, first, 1 g of dimyristoylphosphatidic acid (manufactured by Funakoshi) was dissolved in 10 mL of 10% (w / v) Triton X-100 (manufactured by Nacalai Tesque), and 10% (w / V) Prepare dimyristoyl phosphatidic acid. To 0.005 mL of 10% (w / v) dimyristoyl phosphatidic acid, 0.025 mL of 0.2 M tris-hydrochloric acid buffer (pH 8.4) and 0.5 M EDTA disodium (Wako Pure Chemical Industries, Ltd.) ) Add 0.002 mL and 0.063 mL of distilled water. And after preheating at 37 degreeC for 5 minute (s), 0.005 mL of samples which confirm enzyme activity are added, and it is made to react at 37 degreeC for 5 minutes. After the enzyme reaction, the reaction is stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution can be determined using, for example, “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.), which is a free fatty acid measurement kit, according to the instructions attached to the kit. Measure as described. The amount of enzyme that produces 1 μmol of free fatty acid per minute is defined as 1 unit (1 U).

  Enzyme B and enzyme D are, for example, acetic acid-sodium acetate buffer (pH 4.1-5.6), bis-tris-hydrochloric acid buffer (pH 5.6-7.2), tris-hydrochloric acid buffer ( pH 7.2 to 8.8) and glycine-sodium hydroxide buffer (pH 8.8 to 10.5), and the above phosphatidic acid and the enzyme are subjected to reaction conditions. May exhibit PLB activity within (pH 4.1 to 10.5). The optimum pH can be around pH 8 to 8.8.

  Enzyme B and Enzyme D are, for example, pH 7.2 when the hydrolysis activity at pH 8.4 is 100% under the condition that dimyristoylphosphatidic acid and enzyme are reacted at 37 ° C. for 5 minutes as described above. To 50% or more in the range of pH 10.0.

  Enzyme B and enzyme D can act at about 20-65 ° C., for example, under the reaction conditions of dimyristoyl phosphatidic acid and the enzyme as described above. The optimum temperature can be within this range. Preferably, it is within the range of about 37-60 ° C, more preferably within the range of 45-55 ° C, and even more preferably about 50 ° C.

  Enzyme B and enzyme D, for example, can be stable with almost no decrease in activity from 4 ° C. to 45 ° C. when treated with 160 mM Tris-HCl buffer (pH 8.4) for 30 minutes, and preferably Has an activity of about 80% (for example, 75%) or more even at 50 ° C.

Enzyme B and enzyme D are not inhibited by 10 mM EDTA when, for example, Tris-HCl buffer (pH 8.4) is used as a buffer and the above phospholipid and enzyme solution are placed under reaction conditions. It is preferable that the activity is almost the same as when EDTA is not added. Further, in the presence of 10 mM Ca ²⁺ , it is preferable to exhibit about 90% activity (for example, about 85 to 95% activity). On the other hand, 10 mM Mg ²⁺ , Mn ²⁺ , Co ²⁺ , Cu ²⁺ , Zn ²⁺ , Fe ³⁺ , and Fe ²⁺ can inhibit the activity.

  Enzyme B and Enzyme D have a hydrolysis activity of 100 when dimyristoylphosphatidic acid is the substrate when the enzyme and the substrate are reacted at 50 ° C. for 5 minutes under the condition of pH 8.4 as described above. %, The activity is 95% or more for phosphatidylcholine (PC), 20% or more for phosphatidylinositol (PI) and phosphatidylglycerol (PG), and 25% or more for phosphatidylserine (PS). It is preferable. Under the same conditions, the activity against sphingomyelin, phosphatidylethanolamine (PE), tristearin and dipalmitoyl glyceride is almost 0% (for example, 5% or less, 3% or less, or 1% or less, or below the detection limit). It is preferable. It is preferable to have such substrate specificity.

  Enzyme B and enzyme D may vary slightly depending on electrophoresis conditions and the like, but the molecular weight in SDS-PAGE is within the range of 38,000 to 40,000 (for example, about 39,000 or 38,900). It is preferable. Moreover, it is preferable that the molecular weight computed from the amino acid composition of the enzyme B and the enzyme D exists in the range of 41,000-43,000. For example, a natural enzyme derived from Streptomyces sp. NA684 strain has a molecular weight of about 39,000, specifically 38,900 in SDS-PAGE. The natural enzyme derived from this Streptomyces sp. NA684 strain has a molecular weight of about 42,000 calculated from its amino acid composition.

  Enzyme B and enzyme D preferably exhibit an isoelectric point in the range of 6.2 to 6.6 (for example, 6.4). The isoelectric point of the enzyme can be calculated from the amino acid sequence by GENETYX.

  One embodiment of enzyme B and enzyme D, ie, phospholipase B, consists of the amino acid sequence set forth in SEQ ID NO: 2. Phospholipase B preferably has an amino acid sequence from position 31 to position 412 of SEQ ID NO: 2 (also referred to herein as “amino acid sequence described in SEQ ID NO: 2”).

  As long as enzyme B and enzyme D have PLB activity, one or more amino acids are substituted, deleted, or inserted into the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 2. And / or an enzyme having an added amino acid sequence. A person skilled in the art, for example, site-directed mutagenesis (Nucleic Acid Res., 1982, 10, pp. 6487; Methods in Enzymol., 1983, 100, pp. 448; Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; PCR: Atactical Approach, IRL Press, 1991, pp. 200), etc., as appropriate substitution, deletion, insertion, and / or addition mutation The structure of the protein can be modified by introducing. The number of amino acid residues that can be substituted, deleted, inserted, and / or added is usually 50 or less, such as 30 or less, or 20 or less, preferably 16 or less, more preferably 5 or less, and even more preferably 0 to 3 It is. In addition, amino acid mutations include not only artificially mutated enzymes but also naturally mutated enzymes in enzymes B and D as long as they have PLB activity.

  A protein having an amino acid sequence shown in SEQ ID NO: 2 or an amino acid sequence having homology to the amino acid sequence shown in SEQ ID NO: 2 is also included in enzyme B and enzyme D as long as it has PLB activity. The PLB is preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 75% of the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in SEQ ID NO: 2. It may be a protein having an amino acid sequence with 90%, even more preferably at least 95%, even more preferably at least 99% homology.

  For protein homology (homology) search, for example, databases related to amino acid sequences of proteins such as SWISS-PROT, PIR, DAD, or DNA databases such as DDBJ, EMBL, or Gene-Bank, etc. BLAST, FASTA, etc. This program can be used, for example, via the Internet. Confirmation of the activity of the protein can be performed using the procedure described above.

  Although the supply source of the enzyme B and the enzyme D is not particularly limited, the enzyme B and the enzyme D can be obtained from a living cell such as a microorganism. Examples of such microorganisms include microorganisms belonging to the genus Streptomyces. Preferably, Streptomyces sp., Streptomyces chattanoogensis and Streptomyces lydicus are used. Since these strains are closely related, it is considered that enzymes B and D having the same activity can be obtained.

  For example, the Streptomyces sp. NA684 (Accession Number: NITE BP-1015) secretes the enzyme out of the cells by liquid culture in an appropriate nutrient medium, so the culture supernatant is frozen. What was processed with drying, salting out, an organic solvent, etc. can be manufactured as an enzyme formulation. Streptomyces sp. NA684 has a polynucleotide represented by the nucleotide sequence of SEQ ID NO: 4 in DNA.

  In addition, it has been confirmed by experiments that Streptomyces schattanoogensis NBRC12754 has the same activity as the NA684 strain. Therefore, Streptomyces sp. NA684 and Streptomyces chattanogensis show similar activities.

  Microorganisms that can be used for the production of enzyme preparations are not limited to Streptomyces sp. NA684, even if they belong to the genus Streptomyces and can produce enzyme B or enzyme D. Good. In addition, natural or artificial mutants of these species or gene fragments necessary for the expression of PLB activity can be artificially extracted and used to produce PLB even in other species incorporating them. . Moreover, even if it does not belong to Streptomyces genus, if it is microorganisms which can produce said PLB, it can also be used.

  The production of the enzyme preparation using Streptomycessp. NA684 will be described as an example.

  Since this bacterium is secreted out of the bacterium by liquid culture in a nutrient medium, the culture supernatant is treated with lyophilization, salting out, an organic solvent, or the treated product is immobilized. Thus, an enzyme preparation can be produced. More specifically, first, this bacterium is cultured in a suitable medium, for example, a medium containing a suitable carbon source, nitrogen source, and inorganic salts to secrete the enzyme. Here, examples of the carbon source include starch and starch hydrolysate, sugars such as glucose and sucrose, alcohols such as glycerol, and organic acids (for example, acetic acid and citric acid) or salts thereof (for example, sodium salt). Can be mentioned. Examples of the nitrogen source include organic nitrogen sources such as yeast extract, peptone, meat extract, corn steep liquor, soybean flour, and inorganic nitrogen compounds such as ammonium sulfate, ammonium nitrate, and urea. Examples of inorganic salts include sodium chloride, monopotassium phosphate, magnesium sulfate, manganese chloride, calcium chloride, and ferrous sulfate. The concentration of the carbon source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v). The concentration of the nitrogen source is, for example, in the range of 1 to 20% (w / v), preferably 1 to 10% (w / v). The culture temperature is preferably a temperature at which the enzymes B and D are stable and the cultured microorganisms can sufficiently grow, and is preferably 20 to 37 ° C, for example. The culture time is preferably a time during which the enzyme is sufficiently produced, and is preferably about 1 to 7 days, for example. Culturing can be preferably performed under aerobic conditions, for example, with aeration stirring or shaking.

  Polypeptides contained in Enzyme B and Enzyme D are fractionated by protein solubility (precipitation with organic solvents, salting out by ammonium sulfate, etc.); cation exchange, anion exchange, gel filtration, hydrophobic chromatography; chelate, Purification can be performed by appropriately combining known methods such as affinity chromatography using dyes, antibodies, and the like. For example, after recovering the culture supernatant of the microorganism, it can be purified by ammonium sulfate precipitation, further anion exchange chromatography, hydrophobic chromatography, and / or cation exchange chromatography. Thereby, in polyacrylamide gel electrophoresis (SDS-PAGE), it can refine | purify to a substantially single band. That is, the polypeptide which comprises the enzyme B and the enzyme D can be estimated as a monomer by HPLC analysis and gel filtration chromatography analysis.

[Microorganisms]
Enzyme A, enzyme B, enzyme C, and enzyme D can be produced by microorganisms. Here, a polypeptide can be artificially generated from the amino acid sequence information of enzyme A and enzyme B, and the base sequence information of enzyme B and enzyme D, and BDF can be produced by artificial synthesis. The obtained enzyme (polypeptide) may be used. However, when an enzyme is produced by a microorganism, the above enzyme can be easily obtained.

  As the microorganism, the aforementioned Streptomyces bacterium can be used, but a microorganism into which the above-described polynucleotide is introduced may be used. That is, a polynucleotide having a base sequence described in SEQ ID NO: 3 or SEQ ID NO: 4 and a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence described in SEQ ID NO: 3 or SEQ ID NO: 4 And a microorganism into which at least one polynucleotide selected from the polynucleotide having at least 75% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 has been introduced. The microorganism into which the above polynucleotide is introduced can be obtained by a vector or a transformant. That is, a transformant having the ability to produce enzyme A, enzyme B, enzyme C, or enzyme D can be prepared by introducing a polynucleotide or a vector into a host.

  Here, the polynucleotide encoding the polypeptide shown by enzyme A (PLA1) is at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably the nucleotide sequence shown in SEQ ID NO: 3. Includes a polynucleotide having a base sequence having at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity, and encoding a protein having PLA1 activity.

  In addition, the polynucleotide encoding the polypeptide (PLB) represented by enzyme B is at least 75%, preferably at least 80%, more preferably at least 85%, and still more preferably the base sequence shown in SEQ ID NO: 4. Polynucleotides having a base sequence having at least 90%, even more preferably at least 95%, even more preferably at least 99% sequence identity and encoding a protein having PLA1 activity.

  In addition, the polynucleotide that hybridizes under stringent conditions is at least 20, preferably at least 30, for example, 40, 60, or 100 in the base sequence described in SEQ ID NO: 3 or SEQ ID NO: 4. A probe is designed by selecting one or a plurality of contiguous sequences, and using, for example, ECL direct acid labeling and detection system (manufactured by GE Healthcare) (for example, washing conditions: 42 ° C., 0 (Primary wash buffer) containing 5 × SSC. More specifically, “stringent conditions” are, for example, usually 42 ° C., 2 × SSC, 0.1% SDS, preferably 50 ° C., 2 × SSC, 0.1% SDS. More preferably, the conditions are 65 ° C., 0.1 × SSC, and 0.1% SDS, but are not particularly limited to these conditions. Factors affecting the stringency of hybridization include a plurality of factors such as temperature and salt concentration, and those skilled in the art can realize optimum stringency by appropriately selecting these factors.

  The procedure for producing a transformant and the construction of a recombinant vector suitable for the host can be performed according to techniques commonly used in the fields of molecular biology, biotechnology, and genetic engineering (for example, Sambrook et al. , Molecular Cloning: Laboratory Manual 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). Particularly for actinomycetes, it can be performed with reference to “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000)”.

  In order to express a polynucleotide encoding the above-mentioned enzyme in a microorganism, this DNA is first introduced into a plasmid vector or a phage vector that is stably present in the microorganism, and the genetic information is transcribed and translated. Therefore, it is preferable to incorporate a promoter corresponding to a unit for controlling transcription / translation 5 'upstream of the DNA strand. Further, it is preferable to incorporate a terminator, which is a unit for controlling transcription / translation, downstream of the 3 ′ side of the DNA strand. More preferably, both the promoter and terminator are incorporated at each site. As the promoter and terminator, a promoter and terminator known to function in microorganisms used as hosts are used. Regarding vectors, promoters, terminators and the like that can be used in these various microorganisms, “Microbiology Basic Course 8 Genetic Engineering, Kyoritsu Shuppan”, especially regarding actinomycetes, “PRACTICAL STREPTOMYCES GENETICS (Kieser et al., John Inns Foundation, 2000) It is possible to use this method. Moreover, it can be efficiently secreted and produced extracellularly by using a signal sequence as necessary. The signal sequence used at this time may be a phospholipase or another.

  The host to be transformed is not particularly limited as long as it is an organism that can be transformed with a vector containing a polynucleotide encoding the enzyme and express the enzyme activity. For example, bacteria, actinomycetes, Bacillus subtilis, Escherichia coli, yeast, mold and the like can be mentioned. More specifically, for example, genus Escherichia, genus Bacillus, genus Pseudomonas, genus Serratia, genus Brevibacterium, genus Corynebacterium, genus Corynebacterium Bacteria for which host vector systems such as Streptococcus and Lactobacillus have been developed; Actinomycetes for which host vector systems such as Rhodococcus and Streptomyces have been developed; Saccharomyces (Saccharomyces) Saccharomyces), Kluyveromyces, Shizosa A genus of the genus Schizosaccharomyces, the genus Zygosaccharomyces, the genus Yarrowia, the genus Trichosporon, the genus Rhodosporidium, the genus Rhodaspodium, and the genus C And yeasts that have been developed for host vector systems such as Neurospora genus, Aspergillus genus, Cephalospolium genus, Trichoderma genus. Escherichia coli is preferred for ease of gene recombination, and actinomycetes are preferred for ease of gene expression.

  In addition to microorganisms, various host / vector systems have been developed in plants and animals. For example, insects such as moths (Nature 315, 592-594 (1985)), rapeseed, corn, potatoes, etc. Systems for expressing heterologous proteins have been developed, and these may be used.

  The obtained transformant can be used for enzyme production as described above. Specifically, the transformant is liquid-cultured in an appropriate nutrient medium, the expressed polypeptide is secreted outside the cell, and the culture supernatant is lyophilized, salted out, treated with an organic solvent, etc. Can be manufactured.

  Although the culture conditions can vary depending on the host cell, the culture can be performed under conditions commonly used by those skilled in the art. For example, when an actinomycete such as Streptomyces is used as a host, a tryptic soy medium containing thiostrepton (for example, manufactured by Becton Dickinson) can be used. The enzyme produced by the transformant can be further purified as described above.

[Biomass raw material]
Biomass raw materials used for the production of biodiesel fuel (BDF) are not particularly limited as long as they contain phospholipids, including vegetable oils, animal oils, fish oils, microbial oils, biological wastes, biowastes, Food waste, sewage sludge, activated sludge, surplus sludge, human waste, livestock waste, raw garbage, weeds and waste crops, algae (freshwater algae or seaweed algae, microalgae), etc. Various wastes such as biological corpses can be used. In particular, when waste is used as a raw material, biodiesel fuel can be produced using waste, so that waste can be reused as an energy source, and waste processing can be facilitated. It is possible to obtain fuel that takes into consideration energy resources and the environment. In addition, for example, if sewage sludge that is discarded inexhaustably or greasy (phospholipid) or seeds that are discarded when edible oil is produced, a large amount of energy can be continuously extracted. In addition, since biodiesel fuel can be produced by enzyme treatment, it is possible to easily or almost eliminate the treatment of waste liquid such as alkali and acid. In addition, since glycerin is supplemented as a glycerol-phosphate compound (glycerol-3-phosphate phosphodiester), it is also possible to prevent a large amount of by-product glycerol from being discharged.

  The biomass raw material may be in the form of a dispersion or slurry that is easy to be enzymatically treated by adding an organic solvent such as water or alcohol and adjusting the viscosity, solids concentration, phospholipid concentration, etc. as appropriate. Moreover, the solid mixture mixed in the state which can mix a solid substance may be sufficient. In short, if the phospholipid and the enzyme can be in contact with each other, the enzyme reaction can proceed.

[Hydrolysis]
In the production of biodiesel fuel, phospholipids in the biomass raw material are hydrolyzed and fatty acids are esterified by enzymatic treatment of the biomass raw material with the above enzyme. The obtained fatty acid ester becomes a biodiesel fuel. The fatty acid ester is preferably a methyl ester. The fatty acid methyl ester can be easily approved as a biodiesel fuel, and a fuel with high fuel efficiency can be obtained. In addition, hydrocarbon esters such as ethyl ester, propyl ester, isopropyl ester, and butyl ester may be used as long as they can be used as biodiesel fuel.

  The hydrolysis reaction and esterification reaction proceed according to the following <Reaction Formula 1>.

  <Reaction Formula 1>

  In the above reaction formula 1, the site indicated by arrow “A1” is a site hydrolyzed by phospholipase A1 (PLA1), and the site indicated by arrow “A2” is a site hydrolyzed by phospholipase A2 (PLA2). . Phospholipase B (PLB) hydrolyzes both A1 and A2. The upper part of Reaction Formula 1 is a pathway in which PLA1 acts and then PLA2 acts to produce a fatty acid ester and a glycerol-3-phosphate compound (glycerol-3-phosphate phosphodiester). The lower part of Reaction Formula 1 is a pathway in which PLA1 acts and then PLA1 acts to produce a fatty acid ester and a glycerol-3-phosphate compound (glycerol-3-phosphate phosphodiester). In PLB, the upper and lower reactions proceed simultaneously. The above enzymes A and C have PLA1 activity, and in such PLA1, in order to obtain more fatty acids, they may be used in combination with PLA2 enzyme or PLB enzyme.

  In the hydrolysis reaction, the reaction is preferably performed in the presence of an alcohol that reacts with a fatty acid by esterification. For example, when producing a fatty acid methyl ester, it is preferable to perform a hydrolysis reaction in the presence of methanol (MeOH). Thereby, esterification can be advanced simultaneously with a hydrolysis reaction, and fatty acid ester can be manufactured easily. That is, a biodiesel fuel can be produced more easily than a two-step reaction in which a free fatty acid is once generated and then esterified.

  The fatty acid to be esterified is not limited as long as it is a fatty acid contained in a raw material and bound to a phospholipid. Saturated fatty acids can be exemplified. Specifically, butyric acid (butyric acid), valeric acid (valeric acid), caproic acid, enanthic acid (heptylic acid), caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, pentadecylic acid, palmitic acid, palmitic train Acid, margaric acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, eleostearic acid, tuberculostearic acid, arachidic acid, arachidonic acid, behenic acid, lignoceric acid, nerbon Acid, serotic acid, montanic acid, melicic acid and the like can be mentioned.

  The conditions for the hydrolysis reaction can be the conditions under which the enzyme exhibits a hydrolytic action.

  For example, the reaction conditions are preferably temperature and pH conditions at which the enzyme acts in the presence of alcohol that does not deactivate the enzyme. More preferably, the reaction is carried out in the presence of methanol of about 1 to 80 mol%, preferably 5 to 75 mol%, more preferably about 10 to 55 mol%, at the optimum temperature and pH of the enzyme. As temperature, 10-70 degreeC is preferable from a viewpoint of enzyme activity, 20-65 degreeC is more preferable, and 35-55 degreeC is further more preferable. Moreover, it is also preferable to make it react at room temperature (for example, 20-25 degreeC) as temperature. In the case of reaction at room temperature, biodiesel fuel can be obtained without heating. This pH is preferable because the enzyme reaction can proceed at pH 4 to 11. From the viewpoint of enzyme activity, in enzyme A and enzyme C, pH 4.1 to 10.5 is more preferable, pH 5 to 9 is more preferable, and pH 5.5 to 8 is still more preferable. In enzyme B and enzyme D, pH 5.6 to 10.4 is more preferable, pH 7 to 10 is more preferable, and pH 7.5 to 9 is still more preferable. Moreover, it is also preferable to make it react by pH of neutral region (about pH 6-8) from a viewpoint of mild conditions.

  The amount of the enzyme may vary depending on the purity and titer of the enzyme, the content of the active ingredient in the enzyme preparation, etc. For example, from the viewpoint of enzyme handling and uniformity of the enzyme reaction, On the other hand, the enzyme can be 0.000001 parts by mass or more, the enzyme 0.00001 parts by mass or more, the enzyme 0.0001 parts by mass or more, the enzyme 0.001 parts by mass or more, or the enzyme 0.01 parts by mass or more. . Further, from the viewpoint of improving the efficiency of enzyme treatment, 10 parts by mass or less of enzyme, 1 part by mass or less of enzyme, 0.1 part by mass or less of enzyme, 0.01 part by mass or less of enzyme, or enzyme 0 0.001 part by mass or less. The amount of the enzyme can be adjusted, for example, by adjusting the enzyme titer with an auxiliary agent such as an excipient.

  In addition, as an enzyme, it seems that a certain level of titer is sufficient. In addition, those having a titer against phospholipid of 10,000 U / mL or less, 1000 U / mL or less, or 500 U / mL or less can be used. By making it an appropriate titer, the processability of the enzyme reaction can be improved.

  The amount of alcohol for esterifying a fatty acid such as methanol is preferably 1 to 100 parts by weight of alcohol with respect to 100 parts by weight of phospholipid, and 5 to 75 parts by weight. Is more preferable, and it is still more preferable to set it as the quantity used as 10-60 mass parts. Moreover, in order not to inactivate enzyme activity as much as possible, you may make low the alcohol concentration of the processed material containing polypeptide.

  The reaction time is preferably longer from the viewpoint of proceeding with the enzyme reaction, and shorter from the viewpoint of increasing the processing efficiency. For example, the reaction time is preferably about 1 to 72 hours, preferably about 12 to 72 hours. More preferably, it is more preferably about 24 to 48 hours.

  Moreover, you may add sequentially alcohol (for example, methanol) for esterifying a fatty acid during an enzyme treatment. For example, when methanol is added sequentially, the added methanol can be sequentially esterified, and biodiesel fuel can be produced without deactivating the enzyme. Moreover, an enzyme may be added sequentially as needed, and an immobilized enzyme may be used as the enzyme. By sequentially adding enzymes or using immobilized enzymes, it may be possible to suppress inactivation of enzymes.

  By the way, in the production of biodiesel fuel, fatty acid esters are obtained by adding an enzyme containing a polypeptide to the raw material and subjecting it to an enzyme treatment. For example, a microorganism that produces the enzyme (polypeptide) is used as a raw material. In addition to producing enzymes from microorganisms, fatty acid esters can be produced from raw materials by the produced enzymes. In this case, the fatty acid ester can be obtained simply by adding microorganisms to the raw material to obtain predetermined reaction conditions, and biodiesel fuel can be easily produced. The predetermined condition is a condition in which an enzyme is produced from a microorganism and the enzyme undergoes a hydrolysis reaction, and since both are derived from a living body, the condition can be easily set.

  The produced fatty acid ester can be used as a biodiesel fuel. In order to use it as a biodiesel fuel, it is preferable to purify the fatty acid ester used as a biodiesel fuel from a composition containing a fatty acid ester obtained by an enzymatic reaction. For the purification, various separation methods such as precipitation separation, column separation, liquid phase separation, specific gravity separation, and distillation separation can be used. As the refining method, the same method as the method for producing biodiesel fuel from edible oil can be used. As the degree of purification, it is not necessary to purify the fatty acid ester completely. That is, as long as it can be used as a biodiesel fuel, it may have a certain degree of purification and may be a mixture (crude composition) containing a fatty acid ester. However, from the viewpoint of safety, it is preferable to remove components that generate toxic gas when burned. From the viewpoint of handling, it is preferably liquid or solid at room temperature (25 ° C.), and further preferably liquid at room temperature (25 ° C.).

  As an example of the purification method, for example, first, a solvent capable of solubilizing a fatty acid ester is added to the composition, and solids that do not dissolve are removed by precipitation or filtration. As the solvent, water or an organic solvent can be used. As the organic solvent, alcohols such as methanol and ethanol can be used. Next, utilizing the difference in specific gravity, the product is centrifuged or allowed to stand to separate and recover the fatty acid ester layer. The standing time may be 1 hour or longer, preferably 12 hours or longer, more preferably about 18 hours, and even more preferably about 24 hours. Next, the solvent contained in the fatty acid ester layer is removed by distillation or the like. When the solvent is removed by distillation, it is preferable to use a solvent having a low boiling point (for example, 100 ° C. or lower). If the boiling point is low, it can be easily distilled off, and there is an advantage that much energy is not required for heating. Examples of the distillation method include normal pressure or vacuum distillation.

  In this way, fatty acid esters that can be used as biodiesel fuel can be obtained. The preferred use of biodiesel fuel may be similar to biodiesel fuel produced from edible oil. Biodiesel fuel can be extracted as energy by combustion. For example, as biodiesel fuel, it can be used as fuel for boilers and diesel engines.

  By the way, the glycerol 3-phosphate compound (glycerol-3-phosphate phosphodiester) obtained as a by-product can be used as a phosphate component (fertilizer) for soil improvement materials and the like. That is, fraction components other than the biodiesel fuel obtained by the above enzyme treatment can be used more effectively.

  As described above, in the method for producing biodiesel fuel using the above enzyme, biodiesel fuel (fatty acid methyl ester) can be produced simply by mixing raw material waste, enzyme, and methanol. Since heating is not required, the manufacturing location is not limited, and it is simple and does not require complicated equipment and technology. For example, it is possible to produce fuel by mixing enzyme and methanol in garbage.

[Enzyme A: Purification of polypeptide A]
(A) Microbial culture Streptomyces albidoflavus NA297 (accession number: NITE BP-1014) was used as the cells.

  First, 500 mL of tryptic soy medium (manufactured by Becton Dinkinson) is prepared, and 50 ml each is dispensed into a 500 mL baffled Erlenmeyer flask, and 1% soybean lecithin and 0.1% Tween 80 are further added. After the addition, steam sterilization was performed at 121 ° C. for 15 minutes.

  Then, an appropriate amount of the above-mentioned colonies of the cells grown on the plate medium in advance is taken and inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of tryptic soy medium (Becton Dinkinson) at 28 ° C. Shake culture until good growth was obtained. 0.5 ml of this culture solution was inoculated into 50 mL of the previously sterilized medium and cultured with shaking at 28 ° C. for 55 hours. The supernatant was recovered from this culture using a centrifuge.

(B) Ammonium sulfate fraction To the culture supernatant collected in (a) above, ammonium sulfate was added so as to be 80% (w / v) saturated, and the resulting precipitate was centrifuged (15,000 rpm, 30 minutes, 4 minutes). C). This precipitate was solubilized with 30 ml of 20 mM Tris-HCl buffer (pH 9.0) and dialyzed with 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(C) Toyopearl Phenyl-650M column chromatography To the crude enzyme solution obtained in (b) above, 1.5 M ammonium sulfate was added at a final concentration, and 20 mM Tris-HCl buffer (pH 8.0) containing 1.5 M ammonium sulfate. ), And applied to a Toyopearl Phenyl-650M column (inner diameter 26 mm, height 38 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of ammonium sulfate (from 1.5 M to 0 M).

(D) HiTrap SP HP column chromatography The active fractions obtained in (c) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this, 20 mM MES-sodium hydroxide buffer (pH 6.0) was added. This was applied to a HiTrap SP (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM MES-sodium hydroxide buffer (pH 6.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

(E) HiTrap Q HP column chromatography The active fractions obtained in (d) above were collected and concentrated and desalted using Viva spin. To this, 20 mM Tris-HCl buffer (pH 9.0) was added. This was applied to a HiTrap Q HP (5 ml) column (GE Healthcare Bioscience) pre-equilibrated with 20 mM Tris-HCl buffer (pH 9.0), washed with the same buffer, and then sodium chloride (0 M The active fraction was eluted with a linear gradient from 1 to 1M.

(F) SDS-PAGE
The active fraction eluted in (e) above was collected and analyzed by SDS-PAGE (15% (w / v) polyacrylamide gel). As a result, in the eluted fraction, a single band with a molecular weight of about 28,000 Da was obtained. Observed. As a result, a polypeptide (referred to as polypeptide A) that was electrophoretically purified from Streptomyces albidoflavus NA297 strain was obtained. This polypeptide A was estimated to be a monomer by HPLC analysis and gel filtration chromatography analysis.

(G) Analysis of amino acid sequence Using polypeptide A, the N-terminal amino acid sequence was analyzed by a protein sequencer. The internal amino acid sequence was analyzed by nanoLC-MS / MS. The analysis confirmed that the N-terminal amino acid sequence of the purified polypeptide obtained from Streptomyces albidoflavus NA297 was as shown in SEQ ID NO: 1. Therefore, it was confirmed that polypeptide A is enzyme A.

  As a result of predicting the isoelectric point based on the amino acid sequence of the enzyme using Genetyx, the isoelectric point of polypeptide A was 6.06.

[Enzyme B: Purification of polypeptide B]
(A) Microbial culture Streptomycessp. NA684 (accession number: NITE BP-1015) was used as the cells.

  First, NB medium “1% peptone (Becton Dinkinson Co., Ltd.), 1% meat extract (Kyokuto Pharmaceutical Co., Ltd.), 0.5% sodium chloride (Wako Pure Chemical Industries, Ltd.), pH 7 .2 "300 mL was prepared, and 100 mL each was dispensed into a 500 mL Erlenmeyer flask. Further, 1% soybean lecithin and 0.1% Tween 80 were added, followed by steam sterilization at 121 ° C for 15 minutes. .

  Then, an appropriate amount of the above-mentioned colonies of the cells grown on the plate medium in advance is taken and inoculated into a φ18 test tube (18 × 180 mm) containing 5 mL of tryptic soy medium (Becton Dinkinson) at 28 ° C. Shake culture until good growth was obtained. 1 ml of this culture solution was inoculated into 100 mL of the previously sterilized medium and cultured with shaking at 28 ° C. for 108 hours. The supernatant was recovered from this culture using a centrifuge.

(B) Ammonium sulfate fraction To the culture supernatant collected in (a) above, ammonium sulfate was added so as to be 80% (w / v) saturated, and the resulting precipitate was centrifuged (10,000 rpm, 30 minutes, 4 minutes). C). This precipitate was solubilized and dialyzed against 20 mM Tris-HCl buffer (pH 9.0) to obtain a crude enzyme solution.

(C) DEAE-Toyopearl column chromatography The “DEAE-Toyopearl 650M column” (inner diameter 26 mm, high height) obtained by previously equilibrating the crude enzyme solution obtained in (b) above with 20 mM Tris-HCl buffer (pH 9.0). To 55 mm, manufactured by Tosoh Corporation). After washing the column with the same buffer, the active fraction was eluted with a linear gradient of sodium chloride (from 0 M to 1 M).

(D) HiTrap Q column chromatography The active fractions obtained in (c) above were collected and concentrated and desalted using Viva spin (manufactured by Sartorius). To this, 20 mM Tris-HCl buffer (pH 9.0) was added. This was applied to a “HiTrap Q” (5 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM Tris-HCl buffer (pH 9.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium chloride (0M to 1M).

(E) RESOURCE PHE column chromatography The active fractions obtained in (d) above were collected and concentrated and desalted using Viva spin. To this, 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate was added. After applying to a “RESOURCE PHE” (1 ml) column (manufactured by GE Healthcare Biosciences) previously equilibrated with 20 mM Tris-HCl buffer (pH 8.0) containing 1 M ammonium sulfate, the column was washed with the same buffer. The active fraction was eluted with a linear gradient of ammonium sulfate (1M to 0M).

(F) Mono S column chromatography The active fractions obtained in (e) above were collected and concentrated and desalted using Viva spin. To this, 20 mM female-sodium hydroxide buffer (pH 6.0) was added. It was applied to a “Mono S” (1 ml) column (manufactured by GE Healthcare Bioscience) previously equilibrated with 20 mM female-sodium hydroxide buffer (pH 6.0), and the column was washed with the same buffer. The active fraction was eluted with a linear gradient of sodium (0M to 0.5M).

(G) SDS-PAGE
When the active fraction eluted in (e) above was collected and analyzed by SDS-PAGE (12% (w / v) polyacrylamide gel), a single band was found in the eluted fraction with a molecular weight of about 39,000 Da. Observed. As a result, a polypeptide (referred to as polypeptide B) purified by electrophoresis was obtained. This polypeptide B was estimated to be a monomer by HPLC analysis and gel filtration chromatography analysis.

(H) Analysis of amino acid sequence Using polypeptide B, an N-terminal amino acid sequence was analyzed by a protein sequencer. The internal amino acid sequence was analyzed by nanoLC-MS / MS. The analysis confirmed that the N-terminal amino acid sequence of the purified polypeptide obtained from Streptomyces sp. NA684 is as shown in SEQ ID NO: 2. Therefore, it was confirmed that polypeptide B is enzyme B.

  As a result of predicting the isoelectric point based on the amino acid sequence of the enzyme using Genetyx, the isoelectric point of polypeptide B was 6.4.

[Microbial DNA]
The base sequence encoding the polypeptide of SEQ ID NO: 1 is the base sequence of SEQ ID NO: 3, and therefore, Streptomyces albidoflavus NA297 is presumed to have the polynucleotide of SEQ ID NO: 3 as DNA. And when the DNA of the core region in this Streptomyces albidoflavus was amplified by PCR and analyzed, the polynucleotide of SEQ ID NO: 3 was confirmed. Therefore, it was confirmed that Streptomyces albidoflavus NA297 has the polynucleotide of SEQ ID NO: 3 as part of the DNA.

  The base sequence encoding the polypeptide of SEQ ID NO: 2 is the base sequence of SEQ ID NO: 4, and therefore, Streptomyces sp. NA684 is presumed to have the polynucleotide of SEQ ID NO: 4 as DNA. And when the DNA of the core region in this Streptomyces sp was amplified by PCR and analyzed, the polynucleotide of SEQ ID NO: 4 was confirmed. Therefore, it was confirmed that Streptomyces sp. NA684 has the polynucleotide of SEQ ID NO: 4 as part of the DNA.

[Phospholipase activity]
About said polypeptide A and polypeptide B, the phospholipase activity and activity conditions were confirmed by the following method.

(A) Polypeptide A
0.02% (w / v) Triton X-100 (manufactured by Nacalai Tesque) 10 g yolk phosphatidylcholine (Nacalai, L-α-phosphatidylcholine (egg yolk)) dissolved in 10 mL (10%) w / v) Egg yolk phosphatidylcholine was prepared. 0.025 mL of this 10% (w / v) egg yolk phosphatidylcholine, 0.060 mL of 0.2 M Tris-HCl buffer (pH 8.0), 0.5 M EDTA disodium (manufactured by Wako Pure Chemical Industries, Ltd.) 0 0.005 mL was added. And after preheating at 37 degreeC for 5 minute (s), 0.010 mL of the sample containing polypeptide A was added, and it was made to react at 37 degreeC for 5 minutes. Here, the mass of the polypeptide A with respect to 100 parts by mass of the phospholipid is 4 parts by mass when the 0.01 ml is an enzyme and the specific gravity is 1. However, depending on the purity of the polypeptide A in the sample to be used, Since the mass changed, attention was paid to the adjustment of the addition amount of the polypeptide A-containing sample. After the enzyme reaction, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution is described in the instructions attached to the kit using “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.) which is a free fatty acid measurement kit. It measured as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute was defined as 1 unit.

  This enzyme test confirmed that polypeptide A exhibits PLA1 activity.

  Further, 1-palmitoyl-2-oleoylphosphatidinecholine, which is a phospholipid, was subjected to an enzyme reaction in the same manner as described above. When the reaction solution after the enzyme reaction was analyzed by gas chromatography, the molar ratio (1st: 2nd) was about 71.5: 28.5 after 5 minutes, and 62.7: 37.3 after 10 minutes. As a result, it was confirmed that palmitic acid was obtained predominately and the activity was almost equal to or higher than that of known PLA1 (Mitsubishi Kasei Foods). Thereby, it was confirmed that polypeptide A is PLA1 which hydrolyzes sn-1 position predominantly.

  As a pH condition, it was confirmed that polypeptide A exhibits strong PLA1 activity at least in the range of pH 4.0 to 10.5.

  As a temperature condition, it was confirmed that polypeptide A exhibits strong PLA1 activity in the range of at least 20 to 60 ° C.

(B) Polypeptide B
1% dimyristoyl phosphatidic acid (manufactured by Funakoshi) was dissolved in 10 mL of 10% (w / v) Triton X-100 (manufactured by Nacalai Tesque) to prepare 10% (w / v) dimyristoyl phosphatidic acid. did. To 0.005 mL of 10% (w / v) dimyristoyl phosphatidic acid, 0.025 mL of 0.2 M tris-hydrochloric acid buffer (pH 8.4) and 0.5 M EDTA disodium (Wako Pure Chemical Industries, Ltd.) ) 0.002 mL and 0.063 mL of distilled water were added. Then, after preheating at 37 ° C. for 5 minutes, 0.005 mL of a sample containing polypeptide B was added and reacted at 37 ° C. for 5 minutes. Here, the mass of polypeptide B with respect to 100 parts by mass of phospholipid is 2 parts by mass if 0.005 ml is an enzyme with a specific gravity of 1. However, depending on the purity of polypeptide B in the sample to be used, Since the mass changed, attention was paid to the adjustment of the addition amount of the polypeptide B-containing sample. After the enzyme reaction, the enzyme reaction was stopped by heating at 100 ° C. for 5 minutes. After stopping the reaction, the amount of free fatty acid contained in 5 μL of the reaction solution is described in the instructions attached to the kit using “NEFA C Test Wako” (manufactured by Wako Pure Chemical Industries, Ltd.) which is a free fatty acid measurement kit. It measured as follows. The amount of enzyme that produces 1 μmol of free fatty acid per minute was defined as 1 unit.

  This enzyme test confirmed that polypeptide B exhibits PLB activity.

  Further, 1-palmitoyl-2-oleoylphosphatidinecholine, which is a phospholipid, was subjected to an enzyme reaction in the same manner as described above. When the reaction solution after the enzyme reaction was analyzed by gas chromatography, it was confirmed that palmitic acid and oleic acid were obtained at about 2: 3. Thereby, it was confirmed that polypeptide B is PLB which hydrolyzes sn-1 position and sn-2 position to the same extent.

  As a pH condition, it was confirmed that polypeptide B exhibits strong PLB activity at least in the range of pH 6.0 to 10.5.

  As a temperature condition, it was confirmed that polypeptide B exhibits strong PLB activity in the range of at least 20 to 65 ° C.

[Examples 1 and 2]
Soybean lecithin (manufactured by Wako Pure Chemical Industries) was used as a raw material containing phospholipids. As an enzyme, an enzyme preparation (Example 1) containing the above polypeptide A and having PLA1 activity of 100 U / mL (lower limit 60 U / mL), or 100 U / mL (lower limit 60 U / mL) containing the above polypeptide B An enzyme preparation having a PLB activity of (Example 2) was used. These enzyme preparations are expression supernatants.

  First, 4.85 g of soybean lecithin, 0.3 g of methanol, and 0.25 mL (pH 8) of 1M Tris-HCL buffer were mixed. Next, 0.25 ml of the enzyme preparation of Example 1 or Example 2 was added, and this mixture was stirred and mixed with a stirrer at room temperature (about 25 ° C.) for 24 hours.

  After enzyme treatment, the mixture was analyzed by TLC (Thin Layer Chromatography). As the TLC conditions, Merck silica gel 60 was used as a TLC carrier, and a mixed solvent of n-hexane: ethyl acetate: acetic acid (9: 1: 0.1 volume ratio) was used as a developing solvent.

  FIG. 1 is a TLC photograph showing the results after enzyme treatment in Example 2 (enzyme: polypeptide B).

  Lane 1 is an oleic acid spot and is a fatty acid (FA) comparative control. Lane 2 is a soybean oil spot and is a comparative control of phospholipid (triglyceride: TG). Lane 3 is a spot for raw lecithin. However, in lane 3, the mixture of lecithin, buffer solution and methanol is developed before the reaction. In addition, the same result as in Lane 3 was obtained when the same treatment as the enzyme treatment was performed without using the enzyme. Lane 4 is a spot of the enzyme-treated product obtained as described above. Lane 5 is a spot of fatty acid ester (ME) obtained by chemically producing waste edible oil as a raw material with alkali (KOH).

  As shown in FIG. 1, when lane 3 (raw material) and lane 4 (enzyme-treated product) are compared, a spot of fatty acid methyl ester appears in lane 4, and soy lecithin is expressed by enzyme (polypeptide B). It was confirmed that when reacted, fatty acid methyl ester as biodiesel fuel was produced.

  Similarly, it was confirmed that fatty acid methyl ester was obtained also in Example 1 (polypeptide A).

  Furthermore, when the mixture after the enzyme treatment of Example 1 and Example 2 was ignited with a lighter, it burned. Thereby, it was confirmed that it can be used as biodiesel fuel.

[Enzyme Examples 1 to 7]
In the same manner as in Examples 1 and 2, soy lecithin was enzymatically treated according to Enzyme Examples 1-7. The reaction conditions were about 37 ° C., 24 hours (no calcium chloride added) or 18 hours (calcium chloride added), and stirring with a stirrer. The TLC conditions were Merck silica gel 60 as the TLC carrier, n-hexane: ethyl acetate: acetic acid (9: 1: 0.1 volume ratio) mixed solvent as the developing solvent, and the sample was hexane. 2 μl was applied after 10-fold dilution.

  Table 1 shows the details of the samples of enzyme examples 1-7.

  The enzyme treatment was performed under the conditions of adding calcium chloride and without adding calcium chloride. Table 2 shows the reaction solution composition under the calcium chloride-free condition, and Table 3 shows the reaction solution composition under the calcium chloride-added condition. When calcium chloride was added, the final calcium chloride concentration was 10 mM. Enz represents an enzyme.

  FIG. 2 shows the results under the condition where calcium chloride is not added.

  FIG. 2A is a TLC photograph showing the results after enzyme treatment in enzyme example 1 (enzyme containing polypeptide A) and enzyme example 2 (enzyme containing polypeptide B). In FIG. 2A, lane 1 shows enzyme example 1 (Enz1), lane 2 shows enzyme example 2 (Enz2), and lane 3 shows a fatty acid ester (BDF) obtained by a chemical production method.

  FIG. 2 (b) is a TLC photograph showing the result after treatment with an enzyme exhibiting phospholipase activity (enzyme examples 3, 4, and 5). In FIG. 2B, lane 1 is a fatty acid ester (BDF) obtained by a chemical production method, lane 2 is enzyme example 3 (Enz3), lane 3 is enzyme example 5 (Enz5), lane 4 is enzyme example 4 ( Enz4), Lane 5 is a mixture (a mixture of lecithin, buffer solution and methanol: Con) stirred with a stirrer under the same conditions.

  2 (a) and 2 (b), enzyme example 1 (enzyme containing polypeptide A), enzyme example 2 (enzyme containing polypeptide B), enzyme example 3 (with PLA1 activity), enzyme example 5 (with PLA1 activity and PLA2 activity), it was shown that biodiesel fuel is being produced. In addition, enzyme example 4 (with PLA2 activity) had weak enzyme activity under these conditions, and BDF was not produced. As shown in Table 1, BDF is not produced even though the enzyme activity is present, if the Ca salt is not added, the enzyme is not activated, has low methanol resistance, or does not act on soybean lecithin It may be due to multiple factors.

  FIG. 3 is a TLC photograph showing the results after enzyme treatment in enzyme examples 4, 6, and 7 under the conditions of calcium chloride addition. In FIG. 3, lane 1 is enzyme example 7 (with Enz7: PLA2 activity), lane 2 is enzyme example 4 (with Enz4: PLA2 activity), lane 3 is enzyme example 6 (with Enz6: PLB activity), and lane 3 is chemical. The fatty acid ester (BDF) obtained by the manufacturing method is shown.

  In Enzyme Examples 4, 6, and 7, it was confirmed that the enzyme activity was weak under this condition, and no fatty acid ester was produced even under the condition of adding calcium chloride. As a cause, it is thought that methanol tolerance is low and it is hard to act on crude lecithin.

[BDF generation activity]
Differences in BDF activity (BDF production, products) due to differences in alcohol concentration and enzyme were tested.

  Soybean lecithin (manufactured by Wako Pure Chemical Industries) was used as a raw material containing phospholipids. As an enzyme, an enzyme preparation containing the above polypeptide A and having a PLA1 activity of 207.21 U / mL (85.085 U / mg) (enzyme A, hereinafter referred to as “PLA1” in this test), or the above polypeptide B An enzyme preparation (enzyme B, hereinafter referred to as “PLB” in this test) having a PLB activity of 270.15 U / mL (134.928 U / mg) was used. These enzyme preparations are obtained by dialyzing the culture supernatant of the recombinant expression strain after ammonium sulfate precipitation. In the enzyme reaction, the methanol concentration was set to three concentration conditions of 25 mol%, 50 mol%, and 75 mol%.

  First, 4.2 g of soybean lecithin, a predetermined amount of methanol, a predetermined amount of distilled water (DW: Distilled Water), and 0.25 mL (pH 8) of 1M Tris-HCL buffer were mixed. The blending amount of methanol was 1.55 g under the condition of 25 mol% methanol, 2.8 g under the condition of 50 mol% methanol, and 3.2 g under the condition of 75 mol% methanol. The blending amount of distilled water (DW) was 2 g under the condition of 25 mol% of methanol, 1 g under the condition of 50 mol% of methanol, and 0 g (not added) under the condition of 75 mol% of methanol. Next, 0.25 ml of PLA1 or PLB enzyme preparation was added, and this mixture was stirred and mixed with a stirrer under the conditions of 37 ° C. and rotation speed of 200 rpm to carry out an enzyme reaction. Table 4 shows the reaction solution composition.

  Over time (0, 3, 6, 12, 24, 48 hours), the enzyme-treated mixture (reaction solution) was sampled and analyzed by GC (gas chromatography). The reaction solution was collected by collecting 0.5 g at each time. Then, 500 μL of hexane was added, stirred by vortexing for 30 s, and centrifuged at 15,000 rpm for 10 min. After centrifugation, the upper hexane layer was taken out and used as a sample for GC measurement. At the time of GC measurement, appropriate dilution was performed so that the value of ΔArea was within the calibration curve.

  As the GC column, DB-WAX (manufactured by J & W, 30 m × 0.25 mm ID, 0.25 μm) was used. Using commercially available FAME (palmitic acid methyl ester (molecular weight 270.4), stearic acid methyl ester (molecular weight 298.5), oleic acid methyl ester (molecular weight 296.49), linoleic acid methyl ester (molecular weight 294.48), linolenic acid methyl ester (molecular weight 292.46) A calibration curve was created and quantitative analysis of each component of the reaction product was performed.

  The results are shown in FIGS. 4 to 15 are graphs showing changes with time in the amount of fatty acid ester (BDF) produced.

  FIG. 4 shows a graph when PLA1 is used under the condition of 25 mol% of methanol. FIG. 5 shows a graph obtained when PLB is used under the condition of 25 mol% of methanol. FIG. 6 shows a comparison between PLA1 and PLB when performed under the condition of methanol at 25 mol%.

  FIG. 7 shows a graph when PLA1 is used under the condition of 50 mol% of methanol. FIG. 8 shows a graph when PLB is used under the condition of 50 mol% of methanol. FIG. 9 shows a comparison between PLA1 and PLB in the case of methanol 50 mol%.

  FIG. 10 shows a graph when PLA1 is used under the condition of 75 mol% of methanol. FIG. 11 shows a graph when PLB is used under the condition of 75 mol% of methanol. FIG. 12 shows a comparison between PLA1 and PLB when performed under the condition of 75 mol% of methanol.

  FIG. 13 is a graph showing the methanol concentration tendency of PLA1 that summarizes the results of PLA1. FIG. 14 is a graph showing the methanol concentration tendency of PLB that summarizes the results of PLB. FIG. 15 is a graph showing a comparison of tendencies between PLA1 and PLB that summarizes the results of FIGS. 13 and 14.

  4, 5, 7, 8, 10, 11, “16: 0”, “18: 0”, “18: 1”, “18: 2”, “18: 3” represent fatty acids, and “16 : 0 "represents palmitic acid," 18: 0 "represents stearic acid," 18: 1 "represents oleic acid," 18: 2 "represents linoleic acid, and" 18: 3 "represents linolenic acid. “—Me” represents a methyl ester. “Total FAME” represents the total amount of fatty acid methyl ester produced.

  As can be seen from the graphs of FIGS. 4 to 15, PLA1 (enzyme A) and PLB (enzyme B) both produce fatty acid methyl ester (BDF) and have BDF production activity. Here, as shown in FIG. 6, at 25 mol% of methanol, PLA1 has a higher BDF generation activity than PLB. On the other hand, as shown in FIGS. 9 and 12, PLB has higher BDF generation activity than PLA1 at 50 mol% and 75 mol% of methanol. Therefore, it is confirmed that PLA1 is advantageous under conditions where the methanol concentration is low, and PLB is advantageous under conditions where the methanol concentration is high. From FIG. 15, the level of BDF production activity is such that PLA1 under the condition of 25 mol% of methanol and PLB under the condition of 50 mol% of methanol tend to have high production activity, and PLA1 under the conditions of 50 mol% and 75 mol% of methanol is produced. It can be seen that the activity tends to be slightly low. Further, both PLA1 and PLB have a lower production activity when the methanol concentration is higher, but PLB has less fluctuation in production activity with respect to changes in methanol concentration than PLA1. For this reason, for example, when the methanol content is 30 mol% or less, it is preferable to generate BDF with PLA1. In addition, when the methanol content is 40 mol% or more, it is preferable to generate BDF by PLB. Moreover, in the range where the methanol content is larger than 30 mol% and smaller than 40 mol%, it is preferable to select one from PLA1 and PLB and use them in combination.

  For the production of BDF, the yield was calculated from FAME Total at the time of reaction 48h. Table 5 shows a table for calculating the yield. The FAME amount (mg) and the FAME concentration (mg / ml) in the upper hexane after hexane extraction from 0.5 g of the reaction solution are as shown in the table because the upper layer volume was 500 to 600 μl. Thus, the molar concentration of soy lecithin charged was calculated from “FAME concentration in reaction solution (mM)” and “molar yield (%, average molecular weight of phosphatidylcholine which is the main component of soybean lecithin) 765. From 1 mol of glycerophospholipid, 2 mol of fatty acid is generated in principle, so that the molar yield was calculated with the fatty acid concentration (M) in the case of 100% reaction as 100%), “weight yield (%, lecithin concentration (mg / Ml) was taken as 100%) and "BDF-mg / reaction solution 1g", the results shown in the table were obtained.

[Enzymatic treatment of biomass waste]
A method for enzyme treatment of biomass waste will be described.

  Crude lecithin can be obtained by extracting lecithin from living waste such as plants and animals, sewage surplus sludge and the like by the Holch method, etc., and distilling off under reduced pressure using an evaporator. Biodiesel fuel can be produced by subjecting this crude lecithin as a raw material to enzyme treatment with the above enzyme.

  We studied Aedes albacus as a biomass raw material. 15.8 mg of phospholipid was obtained from 20 g (dry weight: 9.04 g) of Solidago radish leaves by mechanical crushing and solvent extraction, and the yield was 0.17% by mass. Moreover, the moisture content of the leaf was 54.8 mass%. As a result of TLC analysis of the obtained phospholipid, phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SPM) are mainly contained in phospholipids extracted from the leaves of Solidago radish. Soybean oil It was found to have almost the same composition as the manufactured by-product crude lecithin. Therefore, it was confirmed that phospholipids can be obtained from plants such as Aedes albopictus and BDF can be produced using this phospholipid.

  We also tried to extract phospholipids from sewage sludge. Digested cake, digested sludge, excess sludge, and concentrated sludge discarded from the sewage treatment plant were used as samples. As a result of solvent extraction from 100 g (wet weight) of these sludges, the largest amount of phospholipids could be extracted from excess sludge. The obtained phospholipid was 12.7 mg, and the yield was 0.0127% by mass. The yield was about 1/10 compared to the above-mentioned plant leaves, but when the moisture content was calculated as 99%, it was 1.27% by mass. If this conversion was appropriate, phospholipids were extracted from sewage sludge. It can be said that it is more efficient. When the phospholipid extracted from the sewage sludge was analyzed by TLC, it was found that the phospholipid extracted from the excess sludge mainly contains phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Therefore, it was confirmed that phospholipid can be obtained from sludge such as excess sludge, and it was found that BDF can be produced using this phospholipid.

[Streptomyces albidoflavus NA297 (Accession number: NITE BP-1014)]
Accession Number: NITE BP-1014
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: January 26, 2011.
[Streptomyces sp. NA684 (Accession number: NITE BP-1015)]
Accession Number: NITE BP-1015
Name of depositary institution: National Institute for Product Evaluation Technology Patent Microorganism Depositary (NPMD)
Name of depositary institution: Japan 2-5-8 Kazusa Kamashitsu, Kisarazu City, Chiba Prefecture 292-0818, Japan
Date of deposit: January 26, 2011.

SEQ ID NO: 1 polypeptide A
Sequence number 2: Polypeptide B
SEQ ID NO: 3: expressed gene of polypeptide A SEQ ID NO: 4: expressed gene of polypeptide B

Claims (6)

  Biodiesel fuel is produced by hydrolyzing phospholipids in biomass raw materials and esterifying fatty acids by treating biomass raw materials containing phospholipids with an enzyme that exhibits hydrolytic activity on phospholipids. A method for producing diesel fuel. By treating the biomass raw material containing phospholipid with at least one of the following enzymes A and B, hydrolyzing the phospholipid in the biomass raw material and esterifying the fatty acid to produce biodiesel fuel, A method for producing biodiesel fuel.
Enzyme A: an enzyme comprising the following polypeptide (a1), (a2), or (a3),
(A1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1;
(A2) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 1 and exhibiting hydrolytic activity against phospholipids ;
(A3) a polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 1 and exhibiting hydrolytic activity against phospholipids;
Enzyme B: an enzyme comprising a polypeptide of the following (b1), (b2), or (b3),
(B1) a polypeptide having the amino acid sequence set forth in SEQ ID NO: 2;
(B2) a polypeptide having an amino acid sequence in which one or more amino acids are substituted, inserted, deleted and / or added in the amino acid sequence shown in SEQ ID NO: 2 and exhibiting hydrolytic activity against phospholipids ;
(B3) A polypeptide having at least 75% homology with the amino acid sequence set forth in SEQ ID NO: 2 and exhibiting hydrolytic activity against phospholipids. By treating a biomass material containing phospholipids with at least one of the following enzymes C and D, hydrolyzing phospholipids in the biomass material and esterifying fatty acids to produce biodiesel fuel, A method for producing biodiesel fuel.
Enzyme C: an enzyme comprising the following polypeptide (c1), (c2), or (c3),
(C1) a polypeptide produced from a microorganism having the nucleotide sequence of SEQ ID NO: 3 as a polynucleotide;
(C2) a polypeptide produced from a microorganism having a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 3;
(C3) a polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the base sequence set forth in SEQ ID NO: 3;
Enzyme D: an enzyme comprising a polypeptide of the following (d1), (d2), or (d3),
(D1) a polypeptide produced from a microorganism having the nucleotide sequence of SEQ ID NO: 4 as a polynucleotide;
(D2) a polypeptide produced from a microorganism having a polynucleotide that hybridizes under stringent conditions with a base sequence complementary to the base sequence set forth in SEQ ID NO: 4;
(D3) A polypeptide produced from a microorganism having a polynucleotide having at least 75% sequence identity with the base sequence set forth in SEQ ID NO: 4.   The method for producing biodiesel fuel according to any one of claims 1 to 3, wherein the enzyme is derived from a microorganism belonging to the genus Streptomyces.   The manufacturing method of the biodiesel fuel of any one of Claims 1-4 whose said biomass raw material is a waste material.   The method for producing biodiesel fuel according to any one of claims 1 to 5, wherein the esterification is methyl esterification.