JP2012044869A - Mutant lipase and application thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a mutant lipase having excellent stability; to provide a method for producing an oils-and-fats degradation product by using the mutant lipase; and to provide a method for using the degradation product.SOLUTION: A mutant lipase produced by introducing a mutation for increasing the stability of an enzyme into a lipase produced by a microorganism belonging to the genus Cryptococcus, Gibberella, Ustilago or the like is allowed to act on oils-and-fats to produce glycerol and the like. An L-amino acid such as L-lysine, L-threonine or L-tryptophan is produced through a fermentation process using, as a carbon source, the glycerol or the like.

Description

  The present invention relates to a method for producing a mutant lipase having excellent stability.

  Currently, diesel oil is a representative example of petroleum fuel that is consumed in large quantities, and is used in industrial engines such as diesel cars, ships, and generators. However, in recent years, the balance between supply and demand for fossil fuels has been tight, and the price of crude oil has soared. Furthermore, as part of efforts to solve global environmental problems, the reduction of carbon dioxide originating from fossil fuels, which is a major cause of global warming, has become an urgent goal. Thus, efforts are being made to develop non-fossil fuels, and in particular in European countries, vegetable oil has been used for diesel engines or gasoline engines. A product obtained by reacting vegetable oil or animal fat with methanol to form a methyl ester is called biodiesel and is used as an alternative fuel such as light oil. In European countries, biodiesel oil is mainly produced mainly from rapeseed, while soybean is used as a raw material in the United States and palm oil is used in Southeast Asia. Waste oil and sunflower are also used.

  Currently, a chemical catalyst method using an alkali catalyst is used for biodiesel fuel production. However, it is difficult to recover glycerol, which is a by-product, and there are problems such as many impurities contained in biodiesel. Therefore, attempts have been made to produce biodiesel by enzymatic degradation and methyl esterification of fats and oils using lipase.

  Lipase is an enzyme that hydrolyzes its ester bond using lipid as a substrate. It is a digestive enzyme that digests lipids mainly in digestive juices (gastric juice and pancreatic juice), and is involved in lipid metabolism in cells of many organisms. Lipases are present in many organisms and their genes are also found in some viruses. There are various functions and three-dimensional structures, but many have serine, aspartic acid, and histidine at the active center. It is also used in artificial ester synthesis and exchange reactions because it breaks down the ester bond of the substrate and also works in reverse reactions (ester synthesis).

  As described above, it has already been reported that fats and oils are treated with lipase to produce biodiesel and glycerol, but the enzyme is expensive compared to the alkali catalyst method and has not been put into practical use. Therefore, in order to suppress the price of the enzyme, research has been conducted to improve the activity and stability by modifying the enzyme. For example, modification of Bacillus subtilis lipase A by Phage Display method (Non-patent Documents 1 and 2), improvement of activity and stability by DNA shuffling (Non-patent Document 3), modification of C. antactica Lipase B by CALB method (non-patent document 3) Examples thereof include Patent Documents 4 and 5), modification of Pseudomonas aeruginosa lipase by CAST method (Non-Patent Document 6), and the like. In addition, in order to improve the reaction process, a method of suppressing enzyme inactivation by gradually dropping methanol into the reaction solution (Non-patent Document 7), or a method of presenting lipase on the cell surface of yeast (Non-patent Document) Documents 7 and 8) have also been developed.

However, despite the accumulation of such technology, the production cost of biodiesel using enzymes is still not as high as that of the alkali catalyst method, and further improvement of the production method is required. Against this background, a new lipase was reported at the National Research Institute for Liquors, and it was proved that biodiesel was produced from fats and oils using the lipase (Patent Document 1).
Patent No. 3507860 Droge et al., ChemBioChem, 2006, 7: 149-157. Danielsen et al., Gene, 2001, 272: 267-274. Suen et al., Protein Eng. Design & Selection, 2004, 17: 133-140 Qian et al., J. Amer. Chem. Soc., 2005, 127: 13466-13467. Zhang et al., Protein Eng., 2003, 16: 599-605 Reets et al., Angew. Chem. Int. Ed., 2005, 44: 4192-4196. Fukuda et al., J. Biosci. Bioeng., 2001, 92: 405-416. Shiraga et al., Appl. Environ. Microbiol., 2005, 71: 4335-4338.

  An object of the present invention is to provide a lipase with excellent stability suitable for oil degradation, and to provide a method for producing a degradation product of fats and oils using the mutant lipase, and a method for using the degradation product. .

  The present inventors aimed to reduce the cost required for biodiesel production by preparing a modified form of the novel enzyme and improving its stability. In addition, if glycerol produced as a by-product is used as a carbon source for the production of amino acids and nucleic acids, and if fatty acids produced when oils and fats are decomposed can also be used for amino acid production at the same time, effective use of plant raw materials will be possible. I thought. As a result of extensive research, we succeeded in producing a modified lipase exhibiting higher stability than wild-type lipase.

  That is, the present invention is as follows.

(1) It has an amino acid sequence of SEQ ID NO: 7 or an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in SEQ ID NO: 7, and the following (a) to ( A mutant lipase comprising a mutation selected from q).
(A) Mutation in which glycine at position 32 is replaced with another amino acid residue (b) Mutation in which tyrosine at position 50 is replaced with another amino acid residue (c) Alanine at position 95 is replaced with another amino acid residue Mutation to be substituted (d) Mutation in which lysine at position 126 is substituted with other amino acid residues (e) Mutation in which cysteine at position 131 is substituted with other amino acid residues (f) Other valine at position 133 Mutation that is substituted with an amino acid residue (g) Mutation that replaces glycine at position 153 with another amino acid residue (h) Mutation that replaces leucine at position 164 with another amino acid residue (i) Position 7 Mutation in which X is alanine and this alanine is substituted with another amino acid residue (j) Mutation in which X at position 42 is alanine and this alanine is substituted with another amino acid residue (k) 83 X of the place is valine, and this valine is other Mutation to be substituted with amino acid residue (l) X at position 88 is glutamine, Mutation to which this glutamine is substituted with other amino acid residue (m) X at position 154 is serine, Mutation that is substituted with another amino acid residue (n) X at position 177 is alanine, and mutation that this alanine is substituted with another amino acid residue (o) X at position 203 is alanine, Mutation in which alanine is substituted with another amino acid residue (p) X at position 206 is lysine, Mutation in which this lysine is substituted with another amino acid residue (q) X at position 209 is glycine The mutation in which the glycine is substituted with another amino acid residue (2) The mutant lipase, wherein the mutations (a) to (q) are the mutations (A) to (Q) below, respectively.
(A) Mutation in which glycine at position 32 is replaced by aspartic acid (B) Mutation in which tyrosine at position 50 is replaced by asparagine (C) Mutation in which alanine at position 95 is replaced by aspartic acid (D) Position 126 Mutation in which lysine is replaced with isoleucine (E) Mutation in which cysteine at position 131 is replaced with an amino acid residue selected from serine, threonine, isoleucine, and leucine (F) Mutation in which valine at position 133 is replaced with lysine (G) Mutation in which glycine at position 153 is replaced with proline (H) Mutation in which leucine at position 164 is replaced with glutamine (I) Mutation in which alanine at position 7 is replaced with proline (J) Alanine at position 42 Mutation substituted with glutamine (K) Mutation where valine at position 83 is replaced with glutamine (L) Glutamine at position 88 is replaced with asparagine Mutation (M) mutation where serine at position 154 is replaced by isoleucine (N) mutation where alanine at position 177 is replaced by proline (O) mutation where alanine at position 203 is replaced by leucine (P) position 206 Mutation in which lysine is substituted with glutamine (Q) Mutation in which glycine at position 209 is substituted with proline (3) Mutations selected from (a) to (q) above are (d), (e), ( The mutant lipase, which is a mutation of f) or (o).
(4) The mutant lipase, wherein the mutation (d), (e), (f) or (o) is the mutation (D), (E), (F) or (O).
(5) The mutant lipase, wherein the lipase having no mutation is a protein of the following (I) or (II).
(I) A protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6.
(II) an amino acid sequence described in SEQ ID NO: 2, 4, or 6 in the sequence listing, consisting of an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions, and a lipase Protein with activity.
(6) DNA encoding the mutant lipase.
(7) The DNA having a base sequence of the following (i) or (ii) and having a mutation corresponding to the mutation in the amino acid sequence.
(I) The nucleotide sequence set forth in SEQ ID NO: 1, 3, or 5.
(Ii) A sequence that hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 5 in the Sequence Listing or a probe that can be prepared from the sequence.
(8) A transformed microorganism containing the DNA.
(9) The microorganism as described above, wherein the transformed microorganism is Escherichia coli.
(10) A method for producing a mutant lipase, wherein the transformed microorganism is cultured in a medium, and the mutant lipase is accumulated in the medium and / or in the microorganism.
(11) A method for producing glycerol, characterized in that glycerol is produced by allowing the mutant lipase to act on fats and oils.
(12) The mutated lipase is allowed to act on fats and oils to produce glycerol, and a microorganism having the ability to utilize glycerol and producing a target substance is cultured in a medium to which the produced glycerol is added as a carbon source. A method for producing a target substance, wherein the target substance is collected from the culture.
(13) A medium in which the mutated lipase is allowed to act on fats and oils to produce glycerol and fatty acids, and the produced glycerol and fatty acids are added as a carbon source. A method for producing a target substance, comprising culturing a microorganism that produces the target substance and collecting the target substance from the culture.
(14) The method as described above, wherein the microorganism is selected from a coryneform bacterium and a microorganism belonging to the family Enterobacteriaceae.
(15) The method as described above, wherein the target substance is an L-amino acid.
(16) The method as described above, wherein the L-amino acid is selected from L-lysine, L-threonine, and L-tryptophan.

  According to the present invention, a mutant lipase having excellent stability is provided. The mutant lipase of the present invention can be suitably used for the production of oil and fat degradation products. The decomposition product of fats and oils obtained by the present invention contains glycerol and fatty acids, and can be used as a raw material in fermentation production of amino acids and nucleic acids.

Hereinafter, the present invention will be described in detail.
Note that the various genetic engineering techniques listed ^{below, Molecular Cloning, 2 nd edition,} Cold Spring Harbor press (1989), Cell Technology Handbook, Toshio Kuroki et al., Eds., Yodosha (1992), New Genetic Engineering Handbook There are many standard experimental manuals such as the revised third edition, edited by Muramatsu et al., Yochisha (1999), and those skilled in the art can implement them by referring to these documents.

The abbreviations such as amino acids, peptides, nucleic acids, and base sequences in this specification are represented by IUPAC (International Union of Pure and Applied Chemistry) or IUBMB (International
Regulations by the Union of Biochemistry and Molecular Biology), “Guidelines for the creation of specifications including base sequences or amino acid sequences” (edited by the Japan Patent Office), “Standard for
The presentation of nucleotide and amino acid sequence listings in patent applications "(WIPO Standard ST.25), and common symbols in the field.

1. Lipase of the present invention The lipase of the present invention is a lipase derived from Cryptococcus genus, for example, a lipase produced by Cryptococcus sp. S-2, and a lipase having a primary structure similar to that, and a mutant lipase having a specific mutation. is there. Hereinafter, the lipase having no mutation of the mutant lipase of the present invention is sometimes referred to as a wild-type lipase, as distinguished from the mutant lipase of the present invention. The lipase having a mutation other than the specific mutation and having no specific mutation is the wild-type lipase referred to in the present invention.

  In the present invention, lipase is an enzyme that has oil-degradability and acts on oil to hydrolyze into glycerol and fatty acid, and is also called triacylglycerol lipase or triacylglyceride lipase. . Fats and oils are esters of fatty acids and glycerol, also called triglycerides. Lipase catalyzes the transesterification reaction between fat and alcohol in the presence of alcohol such as methanol to produce fatty acid ester (Jaeger, KE and Eggert, T. 2002. Curr. Opin. Biotechnol. 13: 390- 397). Here, the oil and fat decomposition activity includes the activity of acting on triglycerides to hydrolyze into glycerol and fatty acids as described above, and the activity of generating fatty acid esters and glycerol by transesterification. The length of the fatty acid that constitutes the fat and oil serving as the lipase substrate is not particularly limited, and the length of the fatty acid or fatty acid ester generated by decomposition varies depending on the length.

The mutant lipase of the present invention can be obtained by introducing the specific mutation into a wild type lipase. The wild-type lipase in the present invention is a lipase that catalyzes the above-mentioned reactions, and any lipase derived from any species can be used as long as the enzyme activity is increased by introducing the specific mutation. Is possible. More specifically, examples of the wild-type lipase include a lipase having the amino acid sequence shown in SEQ ID NO: 7. The wild-type lipase is a substitution, deletion, insertion of one or several amino acids in SEQ ID NO: 7,
It may be a lipase having an amino acid sequence containing an addition or inversion.

As the wild-type lipase as described above, a lipase of the genus Cryptococcus or its related species, Gibberella genus, or Ustilago genus, which is a yeast, is particularly preferable.
Examples of the genus Cryptococcus include the following microorganisms.
Cryptococcus neoformans JEC21
Cryptococcus neoformans B-3501A
Cryptococcus sp.S-2 (FERM P-15155) (Patent No. 3507860)
Filobasidiella depauperata
Cryptococcus albidus
Cryptococcus terreus
Cryptococcus uniguttulatus
Cryptococcus gastricus
Cryptococcus amylolentus
Cryptococcus bacillisporus
Cryptococcus sp. N6
Cryptococcus neoformans A
Cryptococcus neoformans A / D
Cryptococcus neoformans var.grubii H99
Cryptococcus allantoinivorans
Cryptococcus amylolyticus
Cryptococcus aff.amylolyticus AS 2.2398
Cryptococcus aff.amylolyticus AS 2.2439
Cryptococcus aff.amylolyticus AS 2.2501
Cryptococcus armeniacus
Cryptococcus aureus
Cryptococcus cf. aureus NRRL Y-30213
Cryptococcus cf. aureus NRRL Y-30215
Cryptococcus bestiolae
Cryptococcus carnescens
Cryptococcus cellulolyticus
Cryptococcus cistialbidi
Cryptococcus dejecticola
Cryptococcus dimennae
Cryptococcus fagi
Cryptococcus festucosus
Cryptococcus flavescens
Cryptococcus cf.flavescens CBS 4926
Cryptococcus flavus
Cryptococcus foliicola
Cryptococcus heimaeyensis
Cryptococcus heveanensis
Cryptococcus laurentii
Cryptococcus aff. Laurentii A48
Cryptococcus aff. Laurentii A57
Cryptococcus aff. Laurentii CBS 9007
Cryptococcus aff.laurentii D-072.1a.1
Cryptococcus aff. Laurentii SJ008
Cryptococcus luteolus
Cryptococcus cf. luteolus BSR92Y5
Cryptococcus marinus
Cryptococcus nemorosus
Cryptococcus nyarrowii
Cryptococcus paraflavus
Cryptococcus peneaus
Cryptococcus perniciosus
Cryptococcus podzolicus
Cryptococcus rajasthanensis
Cryptococcus skinneri
Cryptococcus statzelliae
Cryptococcus surugaensis
Cryptococcus taibaiensis
Cryptococcus tephrensis
Cryptococcus victoriae
Cryptococcus aff.victoriae CBS 8979
Cryptococcus aff.victoriae CBS 8993
Cryptococcus aff.victoriae CBS 8999
Cryptococcus aff.victoriae CBS 9012
Cryptococcus aff.victoriae CBS 9013
Cryptococcus aff.victoriae CBS 9023
Cryptococcus aff.victoriae CBS 9024
Cryptococcus aff.victoriae CBS 9025
Cryptococcus watticus
Cryptococcus zeae
Cryptococcus sp. 3-12
Cryptococcus sp. Aspoy5E
Cryptococcus sp. AspoyC
Cryptococcus sp.AST-2007a
Cryptococcus sp. BC23
Cryptococcus sp. BC25
Cryptococcus sp. BC28
Cryptococcus sp. BC43
Cryptococcus sp.BI20
Cryptococcus sp. BSR92 Y71
Cryptococcus sp. Car95 Y15
Cryptococcus sp. CBS 10258
Cryptococcus sp. CBS 2993
Cryptococcus sp. CBS 6024
Cryptococcus sp. CBS 6123
Cryptococcus sp. CBS 6578
Cryptococcus sp. CBS 681.93
Cryptococcus sp. CBS 7712
Cryptococcus sp. CBS 7713
Cryptococcus sp. CBS 7743
Cryptococcus sp. CBS 7890
Cryptococcus sp. CBS 8016
Cryptococcus sp. CBS 8024
Cryptococcus sp. CBS 8355
Cryptococcus sp. CBS 8356
Cryptococcus sp. CBS 8358
Cryptococcus sp. CBS 8363
Cryptococcus sp. CBS 8364
Cryptococcus sp. CBS 8365
Cryptococcus sp. CBS 8366
Cryptococcus sp. CBS 8367
Cryptococcus sp. CBS 8368
Cryptococcus sp. CBS 8369
Cryptococcus sp. CBS 8372
Cryptococcus sp. CECT 11955
Cryptococcus sp. CJDX4-Y23
Cryptococcus sp. CJDX4-Y8
Cryptococcus sp. CN2005a
Cryptococcus sp. CRUB 1230
Cryptococcus sp. CSDX3-Y1
Cryptococcus sp. F6
Cryptococcus sp. FYB-2007a
Cryptococcus sp. HB 1222
Cryptococcus sp.HB1042
Cryptococcus sp.HB1048
Cryptococcus sp.HB1052
Cryptococcus sp.HB1104
Cryptococcus sp.HB1122
Cryptococcus sp.HB1134
Cryptococcus sp.HB1144
Cryptococcus sp.HB1155
Cryptococcus sp.HB1160
Cryptococcus sp.HB1163
Cryptococcus sp.HB946
Cryptococcus sp.HB953
Cryptococcus sp. HX-2006a
Cryptococcus sp. HX-2006b
Cryptococcus sp. HX-2006c
Cryptococcus sp. JCM 11356
Cryptococcus sp.KCTC 17061
Cryptococcus sp.KCTC 17062
Cryptococcus sp.KCTC 17063
Cryptococcus sp.KCTC 17064
Cryptococcus sp.KCTC 17065
Cryptococcus sp.KCTC 17066
Cryptococcus sp.KCTC 17067
Cryptococcus sp.KCTC 17068
Cryptococcus sp.KCTC 17069
Cryptococcus sp.KCTC 17070
Cryptococcus sp.KCTC 17071
Cryptococcus sp.KCTC 17072
Cryptococcus sp.KCTC 17073
Cryptococcus sp.KCTC 17074
Cryptococcus sp.KCTC 17075
Cryptococcus sp.KCTC 17076
Cryptococcus sp.KCTC 17077
Cryptococcus sp.KCTC 17078
Cryptococcus sp.KCTC 17079
Cryptococcus sp. LoBar95 Y19
Cryptococcus sp. LoBar95-Y3
Cryptococcus sp.RC93-2B-Y12
Cryptococcus sp. SJ196
Cryptococcus sp. SM13S04
Cryptococcus sp.SS-1727
Cryptococcus sp. SS1
Cryptococcus sp.ST-111
Cryptococcus sp. ST-201
Cryptococcus sp. ST-71
Cryptococcus sp. ST-73
Cryptococcus sp. T-11
Cryptococcus sp. T-26
Cryptococcus sp. YS NB5
Cryptococcus sp. YY1

  For example, a lipase produced by Cryptococcus spp., Particularly Cryptococcus sp. S-2, has the following physicochemical properties.

(1) Action It has fat and oil degradability, acts on triglycerides, and hydrolyzes into glycerol and fatty acids.

(2) Substrate specificity Decomposes tribtilin, tricaprylin, tripalmitin and triolein well. Triacetin, tricaprin, and trilaurin are moderately degraded. Degradability against trimyristin and tristearin is weak.

(3) Regiospecificity When it acts on triolein, oleic acid and a small amount of 1,2 (2,3) -diolein are produced, and 1,3-diolein and monoolein are not detected.

(4) Optimal pH and stable pH range Optimal pH: 7.0
Stable pH range: 5-9

(5) Reaction optimum temperature and temperature deactivation conditions Reaction optimum temperature: 37 ° C
Conditions of deactivation due to temperature: Deactivation of activity due to temperature rise is moderate, and the activity is maintained even after heat treatment at 60 ° C for 30 minutes, but the activity is less than 20% after heat treatment at 60 ° C for 20 hours descend.

(6) Stability to organic solvent, stable to activated organic solvent, and further increased transesterification activity by dimethyl sulfoxide and diethyl ether.

(7) Molecular mass
21kDa (Expasy Compute pI / Mw)

  A lipase of the genus Cryptococcus having such properties or a lipase having a structure similar to that can be obtained by a method similar to the method described in Japanese Patent No. 3507860, and the homology of the gene sequence encoding the lipase described below. You can also get it using.

As a gene encoding a lipase derived from the genus Cryptococcus, the lipase gene CS2 gene of Cryptococcus sp. S-2 (FERM P-15155) is known (Japanese Patent Laid-Open No. 2004-73123). The base sequence of this CS2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the lipase precursor encoded by this CS2 gene is shown in SEQ ID NO: 2. In the amino acid sequence shown in SEQ ID NO: 2, positions -30 to -1 are expected to correspond to a signal peptide, and positions 1 to 250 are expected to correspond to a mature protein. Cryptococcus sp.S-2 is the National Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center) on September 5, 1995 (Tsukuba, Ibaraki, Japan 305-5466) Deposited at FERM P-15155 under No. 1 at 1-chome, 1-chome, City East, and transferred to an international deposit based on the Budapest Treaty on April 25, 2008, and assigned the accession number FERM BP-10961.

  In the present invention, the lipase may be a homologue of a lipase gene cloned from a yeast of a genus Cryptococcus or a close species, or other microorganisms based on homology with the sequence of SEQ ID NO: 1 or 2. The homology between the amino acid sequence and the base sequence is, for example, the algorithm BLAST by Karlin and Altschul (Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)) and FASTA by Pearson (Methods Enzymol., 183, 63 (1990)). Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed (see http://www.ncbi.nlm.nih.gov). The homology between amino acid sequences and base sequences can also be calculated by CLUSTALW (Thompson J.D., Higgins D.G. and Gibson T.J. Nucleic Acids Res. 1994, 22: 4673-4680). For example, as a gene having high homology with the CS2 gene of Cryptococcus sp. S-2, Gibberella zeae PH-1 gene (Genbank Accession No. gi | 42549908 | gb | EAA72751.1 | [42549908] Nucleotide sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4), gene derived from Ustilago maydis (Genbank Accession No.gi | 46096123 | gb | EAK81356.1 | [46096123], nucleotide sequence: SEQ ID NO: 5. Amino acid sequence: SEQ ID NO: 6) can be used.

In SEQ ID NO: 4, the signal peptide corresponds to positions -19 to -1 and the mature protein corresponds to positions 1 to 204. In SEQ ID NO: 6, the signal peptide corresponds to positions -20 to -1 and the mature protein corresponds to positions 1 to 204. Is expected to correspond to
In each of the above lipases, the positions of the signal peptide and the mature protein were predicted using SignalP (http://www.cbs.dtu.dk/services/SignalP/).

The lipase gene homolog is a gene derived from other microorganisms or a natural or artificial mutant gene, which shows a high similarity in structure to the CS2 gene of cryptococcus and exhibits lipase activity. The homologue of the lipase gene has a homology of 80% or more, preferably 90% or more, more preferably 95%, particularly preferably 98% or more with respect to the entire amino acid sequence at positions 1 to 205 of SEQ ID NO: 2. And a protein encoding a protein having a lipase function. In addition, having a lipase function can be confirmed by expressing these genes in a host cell and confirming the oil / fat resolution.
In the present specification, “homology” may refer to “identity”.

The lipase that can be used in the present invention is a DNA that hybridizes under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence of SEQ ID NO: 1, 3, or 5 or a probe that can be prepared from the nucleotide sequence. It may be a DNA encoding a protein having a function as Here, “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Although it is difficult to clearly quantify this condition, for example, DNAs with high homology, for example, 80%, preferably 90% or more, more preferably 95%, particularly preferably 98% or more are present. DNAs having homology hybridize to each other, DNAs having lower homology to each other do not hybridize, or normal Southern hybridization washing conditions at 60 ° C., 1 × SSC, 0.1% SDS, Preferably, 0.1 × SSC, 0.1% SDS, more preferably 68 ° C., salt concentration and temperature corresponding to 0.1 × SSC, 0.1% SDS, more preferably 1 to 3 times. The conditions to wash | clean are mentioned.

  An alignment of the amino acid sequences of SEQ ID NOs: 2, 4 and 6 (mature protein portion) is shown in FIG. A consensus sequence is shown below these sequences. The amino acid indicated by “X” in the common sequence indicates that it is not common to the three lipases. “-” Indicates that no amino acid is present at that position. In addition, amino acids surrounded by a rectangle indicate a mutation point possessed by the mutant lipase of the present invention. Furthermore, the amino acid indicated by an asterisk (*) indicates another mutation point possessed by the mutant lipase derived from the lipase of Cryptococcus sp. S-2 shown in SEQ ID NO: 2.

As described above, the three lipases have high homology. Further, as described later, in the lipase of Cryptococcus sp. S-2, 8 out of 17 mutations in which lipase activity is increased, the amino acid is common among the three lipases. Therefore, in the present invention, wild-type lipase can be identified as a protein having the amino acid sequence shown in SEQ ID NO: 7.
When SCORE representing homology between lipase mature proteins was determined by CLUSTALWversion1.83, it was 64 for SEQ ID NO: 2 and SEQ ID NO: 4, 58 for SEQ ID NO: 2 and SEQ ID NO: 6, and for SEQ ID NO: 4 and SEQ ID NO: 6. 59.

2. Mutant lipase of the present invention The mutant lipase of the present invention means a lipase that is more stable than the wild-type lipase by introducing a mutation into the wild-type lipase as described above. Stability means thermal stability. The mutant lipase preferably has a residual activity of 20% or more, more preferably 30% or more after treatment at 60 ° C. for 20 hours.

  As a method for introducing a mutation, a method of treating the coding region sequence of a wild-type lipase gene in vitro with hydroxylamine or the like, and a microorganism carrying the gene, for example, Escherichia coli into which the lipase gene has been introduced, with ultraviolet light or N-methyl -N'-Nitro-N-nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS), a method of treatment with mutagen that is usually used for mutagenesis, error prone PCR (Cadwell, RC PCR Meth. Appl 2, 28 (1992)), DNA shuffling (Stemmer, WP Nature 370, 389 (1994)), StEP-PCR (Zhao, H. Nature Biotechnol. 16, 258 (1998)), etc. In particular, there is a method of introducing a mutation into the lipase. The lipase activity of these mutant lipase genes can be confirmed, for example, by measuring the esterase activity by spectrophotometry using p-nitrophenylbutyric acid or p-nitrophenylpalmitic acid as a substrate.

  The activity of the mutant lipase can also be evaluated by measuring the decrease in the content of fat or oil as a raw material or the production of fatty acid ester or glycerol after mixing and incubating the mutant lipase, fat and alcohol, and alcohol. Alternatively, after mixing and incubating the mutant lipase, fats and oils, and water, the decrease in the content of fats and oils as raw materials or the production of fatty acids or glycerol may be measured.

Specific examples of the mutation that increases the stability of the lipase include the following mutations (a) to (q). Each position is the position in SEQ ID NO: 7.

(A) Mutation in which Gly at position 32 is substituted with another amino acid residue Here, the other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu, Thr, Val, Leu, Ile , Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, and His may be any amino acid. Of these, substitution to Asp is most preferable.

(B) Mutation in which Tyr at position 50 is substituted with another amino acid residue Here, the other amino acid residue may be any amino acid as long as it is a natural amino acid. Lys, Glu, Thr, Val, Leu, Ile , Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Gly, Ala, Pro, and His may be any amino acid. Of these, substitution to Asn is most preferable.

(C) Mutation in which Ala at position 95 is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid. Lys, Glu, Thr, Val, Leu, Ile , Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, and His may be any amino acid. Of these, substitution to Asp is most preferable.

(D) Mutation in which Lys at position 126 is substituted with another amino acid residue The other amino acid residue may be any amino acid as long as it is a natural amino acid. Glu, Thr, Val, Leu, Ile, Ser Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, and His may be any amino acid. Of these, substitution to Ile is most preferable.

(E) Mutation in which Cys at position 131 is substituted with another amino acid residue Here, the other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu, Thr, Val, Leu, Ile , Ser, Asp, Asn, Gln, Arg, Met, Phe, Trp, Tyr, Gly, Ala, Pro, and His may be any amino acid. Of these, substitution to Ser, Thr, Leu, or Ile is most preferable.

(F) Mutation in which Val at position 133 is substituted with another amino acid residue Here, the other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu, Thr, Leu, Ile, Ser Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, and His may be any amino acid. Of these, substitution to Lys is most preferable.

(G) Mutation in which Gly at position 153 is substituted with another amino acid residue Here, the other amino acid residue may be any amino acid as long as it is a natural amino acid. Lys, Glu, Thr, Val, Leu, Ile , Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, and His may be any amino acid. Of these, substitution to Pro is most preferable.

(H) Mutation in which Leu at position 164 is substituted with another amino acid residue Here, the other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu, Thr, Val, Ile, Ser Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, and His may be any amino acid. Of these, substitution to Gln is most preferable.

(I) A mutation in which X at position 7 is Ala, and this Ala is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys,
Glu, Thr, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro,
Any amino acid selected from His may be used. Of these, substitution to Pro is most preferable.

(J) A mutation in which X at position 42 is Ala, and this Ala is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, and Lys, Glu , Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, and His may be any amino acid. Of these, substitution to Gln is most preferable.

(K) A mutation in which X at position 83 is Val and this Val is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, and Lys, Glu , Thr, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, His may be any amino acid. Of these, substitution to Gln is most preferable.

(L) Mutation in which X at position 88 is Gln, and this Gln is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu , Thr, Val, Leu, Ile, Ser, Asp, Asn, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, His may be any amino acid. Of these, substitution to Asn is most preferable.

(M) Mutation in which X at position 154 is Ser, and this Ser is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu , Thr, Val, Leu, Ile, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, His may be any amino acid. Of these, substitution to Ile is most preferable.

(N) A mutation in which X at position 177 is Ala, and this Ala is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, Glu , Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, and His may be any amino acid. Of these, substitution to Pro is most preferable.

(O) A mutation in which X at position 203 is Ala, and this Ala is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, and Lys, Glu , Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, and His may be any amino acid. Of these, substitution with Leu is most preferable.

(P) Mutation in which X at position 206 is Lys, and this Lys is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, Glu, Thr , Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, and His may be any amino acid. Of these, substitution to Gln is most preferable.

(Q) Mutation in which X at position 209 is Gly, and this Gly is substituted with another amino acid residue. The other amino acid residue may be any amino acid as long as it is a natural amino acid, Lys, G
Any amino acid selected from lu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, and His may be used. Of these, substitution to Pro is most preferable.

  Among the above mutations, (d), (e), (f), and (o) are preferable from the viewpoint of stability. Moreover, (k), (e), and (p) are preferable from the viewpoint of the specific activity (enzyme activity per protein amount) of the mutant lipase.

  7th, 32nd, 42nd, 50th, 83rd, 88th, 95th, 126th, 131st, 133th, 153rd, 154th, 164th, 164th, 177th of (a) to (q) above 203, 206, and 209 are the 1st, 26th, 36th, 44th, 77th, 82th, 89th, 120th, 125th, 127th positions in the amino acid sequence of SEQ ID NO: 2, respectively. , 147, 148, 158, 171, 197, 200, 203. The positions of mutations in SEQ ID NOs: 4 and 6 correspond to the positions described in (a) to (q) in the alignment shown in FIG.

  In this specification, the mutation possessed by the mutant lipase is specified from the abbreviations of amino acid residues as shown in Table 1 and the site in the amino acid sequence shown in the Sequence Listing. For example, regarding the mutation in SEQ ID NO: 2, “C125I” of variant 1 indicates that the amino acid residue cysteine at position 125 in the sequence of SEQ ID NO: 2 was substituted with isoleucine. That is, the notation of the mutant type is the type of amino acid residue of the wild type (amino acid specified in SEQ ID NO: 2); the position of the amino acid residue in the amino acid sequence described in SEQ ID NO: 2; Indicates the type. The same applies to other variants.

  The mutant lipase of the present invention has excellent stability. That is, it has higher resistance to heat than wild-type lipase. More specifically, each of the mutant lipases of the present invention has improved performance over the wild type protein in terms of properties such as temperature stability.

  In addition to stability, the mutant lipase of the present invention may have a mutation that improves other enzymatic properties. Examples of such mutations include specific activity, pH characteristics, substrate specificity, and the like. Among the mutations possessed by the mutant lipase, there are mutations that reduce the specific activity of the lipase. When the mutant lipase has such a mutation, it is possible to achieve both stability and specific activity by introducing a mutation that further increases the specific activity.

The present inventors have found the following mutations as mutations that increase the specific activity of lipase. These mutations are mutations in the wild-type lipase having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions in SEQ ID NO: 7.

(I) Mutation in which glycine at position 22 is replaced with another amino acid residue (ii) Mutation in which valine at position 53 is replaced with another amino acid residue (iii) Phenylalanine at position 58 is replaced with another amino acid residue Mutation to be substituted (iv) Mutation in which cysteine at position 131 is substituted with other amino acid residues (v) Mutation in which valine at position 160 is substituted with other amino acid residues (vi) Other valine at position 173 Mutation to be substituted with an amino acid residue (vii) Mutation in which leucine at position 187 is substituted with another amino acid residue (viii) X at position 180 is phenylalanine, and this phenylalanine is substituted with isoleucine or valine ( ix) Mutation in which X at position 185 is glutamine and glutamine is substituted with glutamic acid or leucine

More specific examples of the mutations (i) to (vii) include the following mutations.
(I) Mutation in which glycine at position 22 is substituted with alanine (II) Mutation in which valine at position 53 is replaced with leucine or arginine (III) Phenylalanine at position 58 is selected from isoleucine, leucine, valine, and tryptophan Mutation substituted with amino acid residue (IV) Mutation where cysteine at position 131 is substituted with alanine (V) Mutation where valine at position 160 is replaced with isoleucine (IV) Mutation where valine at position 173 is replaced with isoleucine (VII) Mutation in which leucine at position 187 is substituted with phenylalanine The specific activity of the mutant lipase having the mutation (I) to (VII), or (viii) or (ix) is higher than that of the wild-type lipase. It has been confirmed by the same method as in Examples 1 to 5 described later.

2. Method for Producing Mutant Lipase of the Present Invention A method for producing the mutant lipase of the present invention, and a method for producing recombinants and transformants used therefor will be described.
The mutant lipase can be produced by introducing the mutation into DNA encoding wild-type lipase, cloning it into an appropriate vector, introducing it into an appropriate host, and expressing it. .

  When designing and preparing a mutant lipase, a DNA encoding the amino acid sequence of SEQ ID NO: 7, for example, a gene encoding the amino acid sequence of SEQ ID NO: 2, 4, 6 is located in the codon corresponding to the mutation What is necessary is just to modify | change by a specific variation | mutation method, introduce | transduce the obtained modified gene in a suitable vector, introduce | transduce into a host, and to culture | cultivate a transformant. The target mutant lipase can be recovered from cultured cells of the transformant. It is preferable to confirm that the obtained mutant lipase has higher stability than wild-type lipase.

As a site-specific mutagenesis method for causing a target mutation at a target site of DNA, a method using PCR shown in Examples [R. Higuchi, PCR for DNA manipulation, in PCR technology (Higuchi, R., Using PCR to engineer DNA, in PCR technology). A. Erich (Erlich, HA, Stockton press), p. 61, (1989))]; [P. In addition to Carter, P., Meth. In Enzymol, Vol. 154, p. 382 (1987)], a method using phage (W. Kramer and H. J. Fritz (Kramer , W. and Frits, HJ), Methods in Enzymology, Volume 154, 350 (1987); T. A Kunkel et al. (Kunkel, TA et al.) Methods in Enzymology, 15
Volume 4, Page 367 (1987).

  The vector into which the gene encoding the mutant lipase is incorporated is not particularly restricted providing it can be replicated in the host. When Escherichia coli is used as a host, a plasmid capable of autonomous replication in the bacterium can be exemplified. For example, pUC19, pET, pGEMEX, etc. can be used. Preferred hosts include Escherichia coli strains. In addition to this, the origin of replication of the constructed recombinant DNA and the mutant lipase gene function, and the recombinant DNA can replicate and the mutant lipase gene. Any microorganism can be used as a host if it can be expressed. Examples of hosts include various Escherichia bacteria such as Escherichia coli, Empedobacter bacteria, Sphingobacterium bacteria, Flavobacterium bacteria, and various species including Bacillus subtilis. Various eukaryotic cells, including prokaryotic cells, Saccharomyces cerevisiae, Pichia stipitis, Aspergillus oryzae, can be used.

There is no particular limitation on the method for introducing a recombinant DNA obtained by ligating a DNA fragment containing a gene encoding a mutant lipase and a vector into a host, and it can be performed by a usual method. When Escherichia coli is used as a host, the calcium chloride method [J. Mol. Biol., Vol. 53, pp. 159 (1970)], the Hanahan method [Journal of Molecular Bio Roth, 166, 557 (1983)], SEM method (Gene, 96, 23 (1990)), Chung et al. [Proceedings of the National Academy of Science of the USA, Vol. 86, p. 2172 (1989)].

  As a promoter for expressing a gene encoding a mutant lipase, a native promoter can be used when a promoter unique to the wild type gene functions in the host, but other promoters may be used. When a unique promoter does not function in the host, a promoter that functions in the host is used. For example, promoters that function in Escherichia coli include lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, tet promoter and the like.

  The mutant lipase may be expressed as a precursor protein containing a signal peptide, or a mature protein may be expressed directly. Further, a signal peptide suitable for a host and a mutant lipase precursor protein or mature protein linked thereto may be expressed. For example, when the host is Escherichia coli, signal peptides of genes such as pelB and ompT can be mentioned.

  The transformant introduced with the recombinant DNA containing the gene encoding the mutant lipase obtained as described above is cultured in a suitable medium containing a carbon source, nitrogen source, inorganic ions, and if necessary, an organic nutrient source. By doing so, a mutant lipase can be expressed.

3. Utilization of Mutant Lipase By reacting the mutant lipase of the present invention, fats and oils and water, fatty acid and glycerol are produced (hydrolysis reaction). In addition, a fatty acid ester and glycerol are produced by reacting mutant lipase, fats and oils (transesterification reaction). Since the mutant lipase of the present invention is more stable than the wild-type lipase, it is possible to efficiently produce glycerol and fatty acids obtained by hydrolyzing fats and oils.

As fats and oils that act on lipase, one or more of animal and fats (including fish) and plants can be used. For example, palm oil, olive oil, rapeseed oil, soybean oil, rice bran oil, walnut oil , Vegetable oils such as sesame oil, camellia oil, peanut oil, animal oils such as butter, pork fat, beef tallow, sheep fat, chicken oil, fish oil such as whale oil, sardine oil, herring oil, cod liver oil, etc. However, it is not limited to these. The fats and oils may be solid fats and oils. Furthermore, the fat and oil raw material may be pure fat or oil, or a mixture containing substances other than fats and oils. For example, when fats and oils are a plant origin, the plant extract containing fats and oils or its fraction is mentioned.

  As the alcohol, various alcohols other than lower alcohols such as methanol can be used. For example, methyl alcohol, ethyl alcohol, propyl alcohol, isopropyl alcohol, butyl alcohol, isobutyl alcohol, S-butyl alcohol, t-butyl alcohol, Aliphatic alcohols such as aluminum alcohol and hexyl alcohol; unsaturated aliphatic alcohols such as allyl alcohol and propargyl alcohol; alicyclic alcohols such as cyclohexanol and cyclopentanol; aromatic alcohols such as benzyl alcohol and cinnamyl alcohol; Although various alcohol etc. are mentioned, it is not limited to these.

  The produced glycerol and fatty acid can be used as a carbon source for a medium for culturing bacteria belonging to the family Enterobacteriaceae. The reaction product of fats and oils with lipase does not contain impurities that greatly impair the growth of bacteria, and can be used for culture without purifying the fatty acids and glycerol produced. The hydrolysis reaction of fats and oils is a reaction for producing fatty acids and glycerol from fats and oils and water. At temperatures near room temperature, a lower layer that is an aqueous phase in which glycerol is dissolved and an upper layer that is an oil phase containing fatty acids. It is general that they are separated. Both glycerol produced in the aqueous phase and fatty acids produced in the oil phase can be used as fermentation raw materials. Also,

  When using the thing containing glycerol and a fatty acid as a hydrolyzate of fats and oils, it is preferable to emulsify the hydrolyzate of fats and oils. Examples of the emulsification treatment include emulsification accelerator addition, stirring, homogenization, ultrasonic treatment and the like. It is thought that the emulsification treatment makes it easier for bacteria to assimilate glycerol and fatty acids, and L-amino acid fermentation becomes more effective. The emulsification treatment may be any treatment as long as the bacteria having the ability to produce L-amino acids can easily assimilate the mixture of fatty acid and glycerol. For example, as an emulsification method, an emulsification accelerator or a surfactant may be added. Here, examples of the emulsification promoter include phospholipids and sterols. As the surfactant, nonionic surfactants include polyoxyethylene sorbitan fatty acid esters such as poly (oxyethylene) sorbitan monooleate (Tween 80), alkyl glucosides such as n-octyl β-D-glucoside, Examples thereof include sucrose fatty acid esters such as sucrose stearate and polyglycerin fatty acid esters such as polyglycerin stearate. Examples of the zwitterionic surfactant include N, N-dimethyl-N-dodecylglycine betaine which is an alkylbetaine. Other than this, Triton X-100 (Triton X-100), polyoxyethylene (20) cetyl ether (Brij-58), nonylphenol ethoxylate (Tergitol NP-40), etc. are generally used in the field of biology Available surfactants are available.

  Furthermore, an operation for promoting emulsification and homogenization of a hardly soluble substance such as a fatty acid is also effective. This operation may be any operation that promotes emulsification and homogenization of a mixture of fatty acid and glycerol. Specifically, stirring treatment, homogenizer treatment, homomixer treatment, ultrasonic treatment, high pressure treatment, high temperature treatment and the like can be mentioned, and stirring treatment, homogenizer treatment, ultrasonic treatment and combinations thereof are more preferable.

It is particularly preferable to combine the treatment with the above emulsification accelerator with the stirring treatment, the homogenizer treatment and / or the ultrasonic treatment, and these treatments are desirably performed under alkaline conditions where the fatty acid is more stable. The alkaline condition is preferably pH 9 or higher, more preferably pH 10 or higher.

  Examples of target substances that can be produced by fermentation using glycerol or fatty acid as a carbon source include L-amino acids (EP1715056, EP1715055, International Publication No. 2007/013695), organic acids (Dharmadi Y, Murarka A, Gonzalez R. 2006. Biotechnol Bioeng 94: 821-829.), Ethanol (Ito T, Nakashimada Y, Senba K, Matsui T, Nishio N. 2005. J Biosci Bioeng. 100: 260-255., Cheng KK, Liu DH, Sun Y, Liu WB 2004. Biotechnol Lett. 26: 911-915.), Hydrogen (to T, Nakashimada Y, Senba K, Matsui T, Nishio N. 2005. J Biosci Bioeng. 100: 260-255) and the like. In addition, when the target substance can form a salt, the salt is also included in the target substance.

  L-amino acids are L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L- Examples include lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine and L-valine. In particular, L-threonine, L-lysine, L-tryptophan and L-glutamic acid are preferable.

Examples of the organic acid include succinic acid, citric acid, fumaric acid, malic acid and the like, and succinic acid is particularly preferable.
Examples of ethanol include ethanol, propanol, 1,3-propanediol and the like.
Examples of microorganisms that can be used for fermentation production include microorganisms belonging to the family Enterobacteriaceae, microorganisms belonging to coryneform bacteria, and the like.

  Microorganisms belonging to the family Enterobacteriaceae include bacteria belonging to the genera such as Escherichia, Enterobacter, Erbinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, Yersinia and the like. In particular, Enterobacteriaceae according to the taxonomy used in NCBI (National Center for Biotechnology Information) database (http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347) Bacteria classified in the family are preferred.

  The bacterium belonging to the genus Escherichia means that the bacterium is classified into the genus Escherichia according to the classification known to microbiologists. Examples of bacteria belonging to the genus Escherichia used in the present invention include, but are not limited to, Escherichia coli (E. coli).

The bacteria belonging to the genus Escherichia that can be used in the present invention are not particularly limited. For example, Neidhardt et al. (Neidhardt, FC Ed. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology / Second Edition pp. 2477- 2483. Table 1. Includes those described in the American Society for Microbiology Press, Washington, DC. Specific examples include Escherichia coli W3110 (ATCC 27325) and Escherichia coli MG1655 (ATCC 47076) derived from the prototype wild type K12 strain.
These strains are for example the American Type Culture Collection (address PO
Box 1549 Manassas, VA 20108, United States of America). That is, the registration number corresponding to each strain is given, and it can receive distribution using this registration number. The registration number corresponding to each strain is described in the catalog of American Type Culture Collection.

  The bacterium belonging to the genus Pantoea means that the bacterium is classified into the genus Pantoea according to the classification known to experts in microbiology. Certain types of Enterobacter agglomerans have recently been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewarty, etc. (Int. J. Syst. Bacteriol., 43, 162173 (1993)). In the present invention, the bacteria belonging to the genus Pantoea include bacteria that have been reclassified to the genus Pantoea in this way.

  Coryneform bacteria are a group of microorganisms defined in the Bergey's Manual of Determinative Bacteriology, 8th edition, page 599 (1974), aerobic, Gram-positive, Non-acidic, microorganisms classified as gonococci having no sporulation ability can be used. Coryneform bacteria were previously classified as genus Brevibacterium but are now integrated as Corynebacterium (Int.J. Syst. Bacteriol., 41, 255 (1991)), and coryneform bacteria. Includes Brevibacterium and Microbatterium bacteria that are very closely related to the genus Bacteria.

Examples of such coryneform bacteria include the following.
Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium alkanolyticum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lylium Corynebacterium merasecola Corynebacterium thermoaminogenes ( Corynebacterium efficiens
Corynebacterium herculis Brevibacterium divaricatam Brevibacterium flavum Brevibacterium inmariophilum Brevibacterium lactofermentum Brevibacterium roseum Brevibacterium saccharolyticum Brevibacterium thiogenitalis Corynebacterium Umm ammoniagenes Brevibacterium album Brevibacterium cerinum Microbacterium ammonia film

Specifically, the following strains can be exemplified.
Corynebacterium acetoacidophilum ATCC13870
Corynebacterium acetoglutamicum ATCC15806
Corynebacterium alkanolyticum ATCC21511
Corynebacterium carnae ATCC15991
Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
Corynebacterium lilium ATCC15990
Corynebacterium melasecola ATCC17965
Corynebacterium efficiens AJ12340 (FERM BP-1539)
Corynebacterium herculis ATCC13868
Brevibacterium divaricatam ATCC14020
Brevibacterium flavum ATCC13826, ATCC14067, AJ12418 (FERM BP-2205)
Brevibacterium immariophilum ATCC14068
Brevibacterium lactofermentum ATCC13869 (Corynebacterium glutamicum ATCC13869)
Brevibacterium rose ATCC13825
Brevibacterium saccharolyticum ATCC14066
Brevibacterium thiogenitalis ATCC19240
Corynebacterium ammoniagenes ATCC6871, ATCC6872
Brevibacterium album ATCC15111
Brevibacterium cerinum ATCC15112
Microbacterium ammonia film ATCC15354

These can be obtained, for example, from the American Type Culture Collection (address PO Box 1549, Manassas, VA 2010812301 Parklawn Drive, Rockville, Maryland 20852, United States of America). That is, a corresponding registration number is assigned to each strain, and distribution can be received using this registration number. In addition, AJ12340 shares were issued on October 27, 1987 at the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center) (Ibaraki, 305-5466, Japan) It is deposited under the Budapest Treaty under the accession number of FERM BP-1539 in 1st, 1st East, 1st Street, Tsukuba City. In addition, AJ12418 stock was deposited on January 5, 1989 at the Institute of Biotechnology, Ministry of International Trade and Industry under the contract number FERM BP-2205 under the Budapest Treaty.

  In order to enhance the utilization of glycerol, the bacterium used in the present invention has a reduced expression of glpR gene (EP1715056), glpA, glpB, glpC, glpD, glpE, glpF, glpG, glpK, glpQ, glpT, Expression of glycerol metabolism genes (EP1715055A, WO 2007/013695 pamphlet) such as glpX, tpiA, gldA, dhK, dhaL, dhaM, dhaR, fsa and talC genes may be enhanced.

  The microorganism producing the target substance is not particularly limited as long as it produces the target substance when cultured in a medium containing glycerol or fatty acid as a carbon source. Examples of microorganisms that produce L-amino acids include microorganisms and strains described in JP-A-2005-261433 (US Patent Application Publication No. 20050214911A1). In addition, methods for imparting or enhancing L-amino acid producing ability are also disclosed in these publications.

As an example of L-amino acid-producing bacteria, L-lysine-producing bacteria are exemplified below.
Examples of L-lysine producing bacteria belonging to the genus Escherichia include mutants having resistance to L-lysine analogs. L-lysine analogues inhibit the growth of bacteria belonging to the genus Escherichia, but this inhibition is completely or partially desensitized when L-lysine is present in the medium. Examples of L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), γ-methyllysine, α-chlorocaprolactam, and the like. . Mutant strains resistant to these lysine analogs can be obtained by subjecting bacteria belonging to the genus Escherichia to normal artificial mutation treatment. Specific examples of bacterial strains useful for the production of L-lysine include Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; see US Pat. No. 4,346,170) and Escherichia coli VL611. In these microorganisms, feedback inhibition of aspartokinase by L-lysine is released.

The WC196 strain can be used as an L-lysine-producing bacterium of Escherichia coli. This strain was bred by conferring AEC resistance to the W3110 strain derived from Escherichia coli K-12. This strain was named Escherichia coli AJ13069. On December 6, 1994, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Center, Ibaraki, 305-8566, Japan Deposited as FERM P-14690 in Tsukuba City Higashi 1-chome 1 1 Central 6), transferred to the international deposit under the Budapest Treaty on 29 September 1995, and assigned the accession number FERM BP-5252 (US Pat. No. 5,827,698).

  Examples of L-lysine-producing bacteria or parent strains for deriving the same also include strains in which one or more activities of L-lysine biosynthetic enzymes are enhanced. Examples of such enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat.No. 6,040,160). ), Phosphoenolpyruvate carboxylase (ppc), aspartate semialdehyde dehydrogenase gene, diaminopimelate epimerase (dapF), tetrahydrodipicolinate succinylase (dapD), succinyl diaminopimelate deacylase (dapE) and aspartase (aspA) ( EP 1253195 A), but is not limited to these. Among these enzymes, dihydrodipicolinate reductase, diaminopimelate decarboxylase, diaminopimelate dehydrogenase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, diaminopimelate epimerase, aspartate semialdehyde dehydrogenase, tetrahydrodipicolinate succinylase, and Succinyl diaminopimelate deacylase is particularly preferred. In addition, the parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Pat.No. 5,830,716), a ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased.

  Examples of L-lysine producing bacteria or parent strains for deriving the same include a decrease or loss of the activity of an enzyme that catalyzes a reaction that branches off from the biosynthetic pathway of L-lysine to produce a compound other than L-lysine. There are also stocks. Examples of enzymes that catalyze reactions that branch off from the biosynthetic pathway of L-lysine to produce compounds other than L-lysine include homoserine dehydrogenase, lysine decarboxylase (US Pat. No. 5,827,698), and malate enzyme ( WO2005 / 010175).

  A preferred L-lysine-producing bacterium is Escherichia coli WC196ΔcadAΔldc / pCABD2 (WO2006 / 078039). This strain is obtained by introducing the plasmid pCABD2 described in US Pat. No. 6,040,160 into the WC196 strain in which the cadA and ldcC genes encoding lysine decarboxylase are disrupted. pCABD2 is a mutant dapA gene encoding dihydrodipicolinate synthase (DDPS) derived from Escherichia coli having a mutation in which feedback inhibition by L-lysine is released, and a mutation in which feedback inhibition by L-lysine is released. A mutant lysC gene encoding aspartokinase III derived from Escherichia coli, dapB gene encoding dihydrodipicolinate reductase derived from Escherichia coli, and ddh encoding a diaminopimelate dehydrogenase derived from Brevibacterium lactofermentum Contains genes.

  The following examples further illustrate the present invention. The present invention is not limited in any way by this example.

[Example 1] Expression of lipase gene in E. coli
The gene encoding the mature lipase excluding the secretion signal of Cryptococcus sp.S-2 (FERM BP-10961) (CS gene) is placed in the cloning / expression region column of the pET-22b (+) vector (Novagen) Cut with a certain MscI (near pelB leader) and NotI (upstream of His / Tag), and pe
The gene for mature lipase was inserted directly under lBleader.
The CS gene was inserted directly under the pelB leader of the pET-22b (+) vector (Novagen) to prepare plasmid pET-22b (+) _ CLE. The pelB gene is controlled by the T7lac promoter and expression is induced with isopropyl-β-D-thiogalactopyranoside. Using this pET-22b (+) _ CLE, Escherichia coli OrigamiB (DE3) is transformed, inoculated into an LB agar medium containing 100 μg / ml ampicillin, and having a target plasmid having ampicillin resistance as an indicator. Selected. Note that Escherichia coli OrigamiB (DE3) carrying pET-22b (+) _ CLE is also referred to as pET-22b (+) _ CLE / OrigamiB (DE3) strain.

pET-22b (+) _ CLE / OrigamiB (DE3) was cultured with shaking in LB medium containing 100 μg / ml ampicillin at 25 ° C. for 18 hours. An appropriate amount of this culture solution is added to LB medium containing 100 μg / ml ampicillin, and after shaking culture at 37 ° C. until the turbidity at 600 nm (OD ₆₀₀ ) of the culture solution reaches 1.0, the final concentration is 0.5 mM. Isopropyl-β-D-thiogalactopyranoside was added, and the mixture was cultured with shaking at 25 ° C. for 18 hours. The esterase activity of the crude extract from the microbial cells contained in the obtained culture broth was measured according to the procedure of Example 5, and it was confirmed that the crude extract had esterase activity. The crude cell extract obtained by culturing in the same manner using Escherichia coli OrigamiB (DE3) without transformation instead of pET-22b (+) _ CLE / OrigamiB (DE3) has esterase activity. It was confirmed that it does not have.

[Example 2] Construction of mutant lipase In order to construct mutant lipase, pET-22b (+) _ CLE plasmid was used as a template for site-directed mutagenesis. Mutagenesis was performed using a commercially available polymerase (PrimeSTAR HS Polymerase, TaKaRa Bio, Inc.) using a PCR reaction according to the manufacturer's protocol. In order to introduce site-specific mutations into each target residue, a primer containing a codon corresponding to each target residue in the center and about 15 mer each corresponding to the wild type lipase sequence was used. The sequence of each primer is shown in Table 2.

  The central portion “XXX” of each primer in Table 2 was used by replacing it with the corresponding codon sequence depending on the type of amino acid residue to be introduced. Table 3 shows each codon corresponding to an amino acid residue.

  After mutagenesis PCR, using a PCR product digested with double-stranded DNA (pET-22b (+) _ CLE plasmid) treated with DpnI and transformed into Escherichia coli JM109 strain, using ampicillin resistance as an index, A strain having the desired plasmid containing the mutant lipase gene was selected. The target plasmid containing the mutant lipase gene was generically named pET-22b (+) _ CLE_M. PET-22b (+) _ CLE_M was isolated from the transformed strain, and Escherichia coli OrigamiB (DE3) was transformed. It is also referred to as Escherichia coli OrigamiB (DE3) pET-22b (+) _ CLE_M / OrigamiB (DE3) strain carrying pET-22b (+) _ CLE_M.

In addition, when pET-22b (+) _ CLE_M indicates a specific mutant type, the part “M” may be replaced with the content of the mutant type and displayed as in pET-22b (+) _ CLE_L181F. When two or more types of mutations are included, the mutant types may be separated by “/” and each mutant type may be listed. For example, pET-22b (+) _ CLE_A50P / I125S is obtained by introducing A50P and C125S mutations into the lipase gene carried in pET-22b (+) _ CLE. It was confirmed by nucleotide sequencing that only the target mutation was introduced into each plasmid.

[Example 3] Expression of lipase
pET-22b (+) _ CLE / OrigamiB (DE3) and pET-22b (+) _ CLE_M / OrigamiB (DE3) were cultured with shaking at 37 ° C. for 16 hours in LB medium containing 100 μg / ml ampicillin. An appropriate amount of this culture solution is added to LB medium containing 100 μg / ml ampicillin, and after shaking culture at 37 ° C. until the turbidity at 600 nm (OD ₆₀₀ ) of the culture solution reaches 1.0, the final concentration is 0.5 mM. Isopropyl-β-D-thiogalactopyranoside was added, and the mixture was cultured with shaking at 25 ° C. for 18 hours.

[Example 4] Preparation of crude extract from lipase-expressing cells
1 mL of the culture solution obtained in Example 3 was taken in a 1.5 mL tube and centrifuged at 14000 g for 1 minute. The supernatant was discarded, and 400 μl of cell lysate containing nonionic surfactant (CelLytic B, Sigma) was added to the precipitated cells and vortexed for 2 minutes. After incubation at room temperature for 10 minutes, the mixture was centrifuged at 14000 g for 5 minutes to obtain a supernatant as a crude extract. The presence of lipase in the crude extract was confirmed by the presence of a band of the corresponding molecular weight by SDS-PAGE and the fact that the crude extract had esterase activity by the method described later. When measuring the esterase activity, the crude extract was diluted 10-fold with PBS (-) or 100 mM sodium acetate pH 5.5.

[Example 5] Measurement of esterase activity of crude extract from lipase-expressing bacterial cells The esterase activity was measured by spectrophotometry using p-nitrophenylbutyric acid as a substrate. The reaction solution used for the measurement is 4 (w / w)% Triton X-100 52.6% by volume, 1M acetate buffer (pH 5.5) 10.5%, and ultra-pure water 36.9%. The final concentration is 5.26 mM. A solution in which the substrate was dissolved was prepared so that 95 μL of the reaction solution was dispensed into 96-well plates (Nunc-Immuno Plate, 475094), and 5 μL of the crude extract obtained by the method described in Example 4 was added.

The reaction solution was incubated at 37 ° C. for 20 minutes, and 150 μL of acetone was added to stop the reaction. After the above operation, the absorbance at 410 nm was measured with an absorptiometer (Micro plate reader).
The crude extract diluted 10-fold with 100 mM sodium acetate pH 5.5 was heat-treated at 60 ° C. for 20 hours, and the esterase activity was measured in the same manner as described above.

  FIG. 2 shows the residual rate of esterase activity before and after heat treatment of the lipase crude extracts of variants 1 to 20. After the heat treatment, all mutant lipases retained esterase activity at a higher rate than wild-type lipase using p-nitrophenylbutyric acid as a substrate. Among them, the mutant type 11 (C125S) showed a high activity retention even after 20 hours of heat treatment at 60 ° C.

  FIG. 2 shows the remaining esterase activity of wild type and mutant after heat treatment at 60 ° C. for 20 hours. Mutant lipase, particularly C125S, showed higher residual esterase activity than wild-type lipase.

  Moreover, the relative hydrolysis activity of these mutant lipases with respect to wild-type lipase is shown in FIG.

  Since both wild-type lipase and mutant-type lipase have esterolytic activity, the following oil-and-oil decomposition carried out with wild-type lipase and L-lysine fermentation using the decomposed product show fat and oil using the mutant-type lipase. It is thought that a decomposition product is also possible.

[Example 6] Purification of wild-type lipase Cells were collected from the culture solution of pET-22b (+) _ CLE / OrigamiB (DE3) obtained in Example 3 using a centrifuge, and 100 mM phosphate buffer (pH After the cells were suspended in 7.0), the cells were collected again. The collected cells were suspended in an appropriate amount of 100 mM phosphate buffer (pH 7.0), and then subjected to ultrasonic crushing at 180 W for 25 minutes using an ultrasonic crusher (INSONATOR 201M, KUBOTA). Centrifuge the resulting solution at 15000 rpm for 20 minutes, collect the supernatant, and then centrifuge again at 15000 rpm for 20 minutes to collect the supernatant. The resulting supernatant is filtered with a 0.22 μm filter (MILLEX-GV, MILLIPORE). Filtered. Thereafter, the obtained filtrate was purified by hydrophobic interaction chromatography using an FPLC system (Akta explorer 10S, GE Healthcare Bio-Sciences). The specific procedure is shown below.

A hydrophobic interaction column (HiLoad 16/10 Phenyl Sepharose High Performance, GE Healthcare Bio-Sciences) was used as the column, and solution A (100 mM phosphate buffer (pH) was used as the eluent.
7.0)) and solution B (ethylene glycol 80%, 100 mM phosphate buffer (pH 7.0) 20%). The column was equilibrated by flowing 20.1 ml of the eluate with the composition of 75% of A liquid and 25% of B liquid, and the above filtrate was injected into the FPLC system. Furthermore, the composition of 75% of A liquid and 25% of B liquid The eluate was run through 90.5 ml. After flowing the eluate so that the composition of the eluate changes linearly from 75% of solution A and 25% of solution B to 42% of solution A and 58% of solution B while flowing 265.5 ml of solution. In addition, while flowing 20.1 ml of eluate, flow the eluate so that the composition of the eluate changes linearly from 42% of solution A and 58% of solution B to 0% of solution A and 100% of solution B. 36.2 ml of the eluate was flowed with a composition of B solution 100%. Each obtained fraction was analyzed by SDS-PAGE, and the fraction in which only the target lipase was confirmed was collected and confirmed to have esterase activity according to the procedure of Example 5.

[Example 7] Preparation of olive oil hydrolysis reaction product The solvent of the purified enzyme solution obtained in Example 6 was used using a centrifugal concentration filter unit (Amicon Ultra-15 10k, MILLIPORE) with a molecular weight cut off of 10,000 Da. The enzyme solution was replaced with 10 mM acetate buffer (pH 5.5) containing 100 mM NaCl to prepare a 0.37 mM enzyme solution. 11 ml of this enzyme solution, 120 ml of olive oil (Sigma-Aldrich) and 120 ml of ultrapure water were put into a 1 L Erlenmeyer flask and shaken for 168 hours at 30 ° C. and 180 rpm.

[Example 8] Preparation of transesterification product of olive oil 11 ml of the same enzyme solution as in Example 7, 120 ml of olive oil (Sigma-Aldrich) and 1.64 ml of methanol (Pure Chemical Co., Ltd., special grade reagent) were placed in a 1 L Erlenmeyer flask. The mixture was shaken at 180 ° C. for 10 hours. Thereafter, 1.64 ml of methanol was added and shaken under the same conditions for 24 hours, and further 1.64 ml of methanol was added and shaken under the same conditions for 134 hours.

[Example 10] L-lysine production culture using fat and oil degradation product as a carbon source Escherichia coli WC196ΔcadAΔldc / pCABD2 (hereinafter referred to as "WC196LC / pCABD2") described in International Publication No. 2006/078039 as an L-lysine producing bacterium Used). WC196LC / pCABD2 was cultured at 37 ° C. for 24 hours in an LB agar medium (tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 15 g / L) containing 20 mg / L of streptomycin sulfate. Cells on the agar medium were scraped off, inoculated into a 500 ml Sakaguchi flask with 20 ml of the main culture medium, and cultured at 37 ° C. for 48 hours. In the main culture medium, a wild-type lipase is used as a carbon source of the hydrolysis reaction product of olive oil of Example 7, or the transesterification product of olive oil of Example 8, or glycerol (special grade reagent of Nacalai Tesque). Using.
Glycerol in the olive oil hydrolysis reaction product and transesterification reaction product was measured with an immobilized enzyme electrode biosensor (BF-5, Oji Scientific Instruments). Each carbon source was added to the medium so that the measured amount of glycerol was 40 g / L. The composition of the main culture medium is shown below.

[Main culture medium composition]
Carbon source 40g / L
Yeast extract 2g / L
FeSO ₄・ 7H ₂ O 10mg / L
MnSO ₄・ 4H ₂ O 10mg / L
KH ₂ PO ₄ 1.0g / L
MgSO ₄・ 7H ₂ O 1g / L
(NH ₄ ) ₂ SO ₄ 24g / L
Streptomycin sulfate 20mg / L

After completion of the culture, the concentration of glycerol remaining in the medium was measured with an immobilized enzyme electrode biosensor, and the degree of growth was measured with turbidity (OD ₆₀₀ ) at ₆₀₀ nm. The amount of L-lysine was measured with a Biotech Analyzer (AS210, Sakura Seiki). Two flasks were cultured for each carbon source. The average value of the results is shown in Table 4.

The figure which shows the alignment of the amino acid sequence (mature protein part) of sequence number 2, 4, and 6. FIG. “X” indicates that the three lipases are not common. “-” Indicates that no amino acid is present at that position. Amino acids surrounded by a rectangle indicate a mutation point. The amino acid indicated by an asterisk (*) indicates another mutation point possessed by the mutant lipase derived from the lipase of Cryptococcus sp. S-2 shown in SEQ ID NO: 2. The figure which shows the residual esterase activity of wild-type lipase and each mutant-type lipase before and behind heat processing at 60 degreeC for 20 hours. The figure which shows the esterase activity of a wild-type lipase and each variant lipase before heat processing.

Claims (16)

It has the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence containing substitution, deletion, insertion, addition, or inversion of one or several amino acids in SEQ ID NO: 7, and from the following (a) to (q) A mutant lipase containing a selected mutation.
(A) Mutation in which glycine at position 32 is replaced with another amino acid residue (b) Mutation in which tyrosine at position 50 is replaced with another amino acid residue (c) Alanine at position 95 is replaced with another amino acid residue Mutation to be substituted (d) Mutation in which lysine at position 126 is substituted with other amino acid residues (e) Mutation in which cysteine at position 131 is substituted with other amino acid residues (f) Other valine at position 133 Mutation that is substituted with an amino acid residue (g) Mutation that replaces glycine at position 153 with another amino acid residue (h) Mutation that replaces leucine at position 164 with another amino acid residue (i) Position 7 Mutation in which X is alanine and this alanine is substituted with another amino acid residue (j) Mutation in which X at position 42 is alanine and this alanine is substituted with another amino acid residue (k) 83 X of the place is valine, and this valine is other Mutation to be substituted with amino acid residue (l) X at position 88 is glutamine, Mutation to which this glutamine is substituted with other amino acid residue (m) X at position 154 is serine, Mutation that is substituted with another amino acid residue (n) X at position 177 is alanine, and mutation that this alanine is substituted with another amino acid residue (o) X at position 203 is alanine, Mutation in which alanine is substituted with another amino acid residue (p) X at position 206 is lysine, Mutation in which this lysine is substituted with another amino acid residue (q) X at position 209 is glycine , Mutations that replace this glycine with another amino acid residue The mutant lipase according to claim 1, wherein the mutations (a) to (q) are the following mutations (A) to (Q), respectively.
(A) Mutation in which glycine at position 32 is replaced by aspartic acid (B) Mutation in which tyrosine at position 50 is replaced by asparagine (C) Mutation in which alanine at position 95 is replaced by aspartic acid (D) Position 126 Mutation in which lysine is replaced with isoleucine (E) Mutation in which cysteine at position 131 is replaced with an amino acid residue selected from serine, threonine, isoleucine, and leucine (F) Mutation in which valine at position 133 is replaced with lysine (G) Mutation in which glycine at position 153 is replaced with proline (H) Mutation in which leucine at position 164 is replaced with glutamine (I) Mutation in which alanine at position 7 is replaced with proline (J) Alanine at position 42 Mutation substituted with glutamine (K) Mutation where valine at position 83 is replaced with glutamine (L) Glutamine at position 88 is replaced with asparagine Mutation (M) mutation where serine at position 154 is replaced by isoleucine (N) mutation where alanine at position 177 is replaced by proline (O) mutation where alanine at position 203 is replaced by leucine (P) position 206 Mutation in which lysine is replaced with glutamine (Q) Mutation in which glycine at position 209 is replaced with proline The mutant lipase according to claim 1, wherein the mutation selected from (a) to (q) is the mutation of (d), (e), (f), or (o).   The mutant lipase according to claim 3, wherein the mutation (d), (e), (f) or (o) is the mutation (D), (E), (F) or (O). . The mutant lipase according to any one of claims 1 to 5, wherein the lipase having no mutation is a protein of the following (I) or (II).
(I) A protein having the amino acid sequence set forth in SEQ ID NO: 2, 4, or 6.
(II) an amino acid sequence described in SEQ ID NO: 2, 4, or 6 in the sequence listing, consisting of an amino acid sequence containing one or several amino acid substitutions, deletions, insertions, additions, or inversions, and a lipase Protein with activity.   DNA encoding the mutant lipase according to any one of claims 1 to 5. The DNA according to claim 6, which has the following nucleotide sequence (i) or (ii) and has a mutation corresponding to the mutation in the amino acid sequence.
(I) The nucleotide sequence set forth in SEQ ID NO: 1, 3, or 5.
(Ii) A sequence that hybridizes under stringent conditions with the nucleotide sequence set forth in SEQ ID NO: 1, 3, or 5 in the Sequence Listing or a probe that can be prepared from the sequence.   A transformed microorganism comprising the DNA according to claim 6 or 7.   The microorganism according to claim 6, wherein the transformed microorganism is Escherichia coli.   The transformed microorganism according to claim 8 or 9 is cultured in a medium, and the mutant lipase according to any one of claims 1 to 5 is accumulated in the medium and / or the microorganism. A method for producing a mutant lipase.   A method for producing glycerol, characterized in that glycerol is produced by allowing the mutant lipase according to claim 1 to act on oils and fats.   A mutant lipase according to any one of claims 1 to 5 is allowed to act on fats and oils to produce glycerol, and a medium to which the produced glycerol is added as a carbon source has glycerol utilization ability and produces a target substance. A method for producing a target substance, comprising culturing a microorganism and collecting the target substance from the culture.   The lipase of claim 1 to 5 is allowed to act on fats and oils to produce glycerol and fatty acids, and the produced glycerol and fatty acids are added as a carbon source, and has the ability to assimilate glycerol and fatty acids, A method for producing a target substance, comprising culturing a microorganism that produces the target substance and collecting the target substance from the culture.   The method according to claim 13, wherein the microorganism is a bacterium selected from a coryneform bacterium and a microorganism belonging to the family Enterobacteriaceae.   The method according to claim 13 or 14, wherein the target substance is an L-amino acid.   16. The method of claim 15, wherein the L-amino acid is selected from L-lysine, L-threonine, and L-tryptophan.

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