WO2010074209A1 - Mutant lipase and use thereof - Google Patents

Mutant lipase and use thereof Download PDF

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WO2010074209A1
WO2010074209A1 PCT/JP2009/071553 JP2009071553W WO2010074209A1 WO 2010074209 A1 WO2010074209 A1 WO 2010074209A1 JP 2009071553 W JP2009071553 W JP 2009071553W WO 2010074209 A1 WO2010074209 A1 WO 2010074209A1
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mutation
amino acid
lipase
cryptococcus
replaced
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PCT/JP2009/071553
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French (fr)
Japanese (ja)
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國夫 中田
佳弘 臼田
信久 榛葉
亘 星野
榮一郎 鈴木
治幸 家藤
和夫 正木
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味の素株式会社
独立行政法人酒類総合研究所
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Publication of WO2010074209A1 publication Critical patent/WO2010074209A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
    • C12P13/227—Tryptophan

Definitions

  • the present invention relates to a method for producing a mutant lipase having excellent stability.
  • 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).
  • Non-Patent Documents 1 and 2 modification of Bacillus subtilis lipase A by Phage Display method
  • Non-Patent Document 3 improvement of activity and stability by DNA shuffling
  • Non-Patent Document 3 Examples include Patent Documents 4 and 5), modification of Pseudomonas aeruginosa lipase by the CAST method (Non-patent Document 6), and the like.
  • Patent Document 7 a method of suppressing enzyme inactivation by gradually dropping methanol into the reaction solution
  • Non-patent Document 7 a method of presenting lipase on the cell surface of yeast
  • 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.
  • 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.
  • the mutant lipase, wherein the lipase having no mutation is a protein of the following (I) or (II).
  • 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.
  • a transformed microorganism containing the DNA (9) The microorganism as described above, wherein the transformed microorganism is Escherichia coli.
  • 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.
  • 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 microorganism is selected from a coryneform bacterium and a microorganism belonging to the family Enterobacteriaceae.
  • the target substance is an L-amino acid.
  • the L-amino acid is selected from L-lysine, L-threonine, and L-tryptophan.
  • 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 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.
  • 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.
  • a lipase is an enzyme that has oil and fat degradability and acts on fats and oils to hydrolyze into glycerol and fatty acids. It 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 esters (Jaeger, K. E. and Eggert, T. 2002. Curr. Opin. Biotechnol. 13 : 390-397).
  • 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 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 may be a lipase having an amino acid sequence including substitution, deletion, insertion, addition, or inversion of one or several amino acids in SEQ ID NO: 7.
  • a lipase of the genus Cryptococcus or its related species Gibberella genus, or Ustilago genus, which is a yeast, is particularly preferable.
  • the genus Cryptococcus include the following microorganisms.
  • 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.
  • lipases produced by Cryptococcus spp. In particular Cryptococcus sp. S-2, have the following physicochemical properties.
  • Substrate specificity Decomposes tribtilin, tricaprylin, tripalmitin and triolein well. Triacetin, tricaprin, and trilaurin are moderately degraded. Degradability against trimyristin and tristearin is weak.
  • 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 decreases to 20% or less by heat treatment at 60 ° C for 20 hours. To do.
  • 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.
  • 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
  • the amino acid sequence of the lipase precursor encoded by this CS2 gene is shown in SEQ ID NO: 2.
  • positions -30 to -1 are predicted to correspond to a signal peptide
  • 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 in the center, 1-chome, 1-chome, 1st, 6th), transferred to an international deposit based on the Budapest Treaty on April 25, 2008, and given the accession number FERM BP-10961 .
  • the lipase may be a homologue of a lipase gene cloned from a yeast of the genus Cryptococcus or a related species or other microorganisms based on the 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.
  • the signal peptide corresponds to positions -19 to -1 and the mature protein corresponds to positions 1 to 204.
  • 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.
  • a protein encoding a protein having a lipase function can be confirmed by expressing these genes in a host cell and confirming the oil / fat resolution.
  • “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
  • 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.
  • clean are mentioned.
  • amino acid sequences of SEQ ID NOs: 2, 4 and 6 are 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.
  • amino acids surrounded by a rectangle indicate a mutation point possessed by the mutant lipase of the present invention.
  • the amino acid indicated by an asterisk (*) indicates another mutation point possessed by a mutant lipase derived from the lipase of Cryptococcus sp.
  • S-2 shown in SEQ ID NO: 2.
  • 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.
  • 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.
  • 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.
  • 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), etc., a method of treatment with mutagen that is usually used for mutagenesis, error prone PCR (Cadwell, RC PCR Meth.
  • NTG N-methyl -N'-nitro-N-nitrosoguanidine
  • EMS ethyl methanesulfonate
  • the activity of the mutant lipase can also be evaluated by measuring the decrease in the content of fat or oil, 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.
  • mutations that increase the stability of lipase include the following mutations (a) to (q). Each position is the position in SEQ ID NO: 7.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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, and His may be any amino acid. 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.
  • 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.
  • 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.
  • 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) A 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, 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.
  • (d), (e), (f), and (o) are preferable from the viewpoint of stability.
  • (k), (e), and (p) are preferable from the viewpoint of the specific activity (enzyme activity per protein amount) of the mutant lipase.
  • 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.
  • “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.
  • the mutant lipase of the present invention may have a mutation that improves other enzymatic properties in addition to the stability.
  • mutations include specific activity, pH characteristics, substrate specificity, and the like.
  • mutations possessed by the mutant lipase there are mutations that reduce the specific activity of the lipase.
  • 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.
  • mutations (i) to (vii) include the following mutations.
  • Phenylalanine at position 58 is selected from isoleucine, leucine, valine, and tryptophan Mutation substituted with amino acid residue
  • V Mutation where valine at position 160 is replaced with isoleucine
  • IV Mutation where valine at position 173 is replaced with isoleucine
  • 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. This is confirmed by the same method as in Examples 1 to 5 described later.
  • 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. .
  • 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
  • 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.
  • the vector into which the gene encoding the mutant lipase is incorporated is not particularly limited as long as it can replicate in the host.
  • Escherichia coli 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.
  • 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.
  • hosts include various bacteria including Escherichia coli such as Escherichia coli, Empedobacter bacteria, Sphingobacteria bacteria, Flavobacterium bacteria, and Bacillus subtilis.
  • Escherichia coli such as Escherichia coli
  • Empedobacter bacteria such as Escherichia coli
  • Sphingobacteria bacteria such as Escherichia coli
  • Flavobacterium bacteria such as Bacillus subtilis
  • Bacillus subtilis examples of hosts include various bacteria including Escherichia coli such as Escherichia coli, Empedobacter bacteria, Sphingobacteria bacteria, Flavobacterium bacteria, and Bacillus subtilis.
  • Various eukaryotic cells including prokaryotic cells, Saccharomyces cerevisiae, Pichia stipitis, Aspergillus oryzae can be used.
  • a unique promoter can be used if a promoter unique to the wild type gene functions in the host, but other promoters may be used.
  • a promoter that functions in the host is used.
  • 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.
  • mutant lipase may be expressed as a precursor protein containing a signal peptide, or the mature protein may be directly expressed.
  • a signal peptide suitable for a host and a mutant lipase precursor protein or mature protein linked thereto may be expressed.
  • 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.
  • fats and oils that act on lipase one or more of animal and fats (including fish) and plants can be used.
  • animal and fats including fish
  • plants can be used.
  • 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.
  • the fats and oils may be solid fats and oils.
  • the fat and oil raw material may be pure fat or oil, or a mixture containing substances other than fats and oils.
  • the plant extract containing fats and oils or its fraction is mentioned.
  • alcohols other than lower alcohols such as methanol
  • 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
  • various alcohol etc. are mentioned, it is not limited to these.
  • Glycerol and fatty acids produced 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.
  • the emulsification treatment When using oil and fat hydrolyzate containing glycerol and fatty acid, it is preferable to emulsify the oil and fat hydrolyzate.
  • the emulsification treatment include emulsification accelerator addition, stirring, homogenization, ultrasonic treatment and the like. It is considered 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 L-amino acid-producing ability make it easy to assimilate the mixture of fatty acid and glycerol.
  • an emulsification accelerator or a surfactant may be added as an emulsification method.
  • examples of the emulsification promoter include phospholipids and sterols.
  • the surfactant in the nonionic surfactant, polyoxyethylene sorbitan fatty acid ester such as poly (oxyethylene) sorbitan monooleate (Tween ⁇ ⁇ 80), alkyl glucoside such as n-octyl ⁇ -D-glucoside, Examples thereof include sucrose fatty acid esters such as sucrose stearate and polyglycerin fatty acid esters such as polyglycerin stearate.
  • the zwitterionic surfactant include N, N-dimethyl-N-dodecylglycine betaine which is an alkylbetaine.
  • Triton X-100 Triton X-100
  • polyoxyethylene (20) cetyl ether Brij-58
  • nonylphenol ethoxylate Tegitol NP-40
  • This operation may be any operation that promotes emulsification and homogenization of a mixture of fatty acid and glycerol.
  • 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.
  • 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 fatty acids are more stable.
  • the alkaline condition is preferably pH 9 or higher, more preferably pH 10 or higher.
  • 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.
  • 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, Senb
  • 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 genera such as Escherichia, Enterobacter, Erbinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, and Yersinia.
  • 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.
  • 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 can be sold, for example, from 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 microbiologists. Certain types of Enterobacter agglomerans have recently been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewarty and others (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 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.
  • coryneform bacteria examples 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
  • strains can be exemplified.
  • Corynebacterium acetoacidophilum ATCC13870 Corynebacterium acetoglutamicum ATCC15806 Corynebacterium alkanolyticum ATCC21511 Corynebacterium carnae ATCC15991 Corynebacterium glutamicum ATCC13020, ATCC13032, ATCC13060
  • 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 ATCC138
  • 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.
  • microorganisms that produce L-amino acids include microorganisms and strains described in JP-A-2005-261433 (US Patent Application Publication No. 20050214911A1). Incidentally, methods for imparting or enhancing L-amino acid producing ability are also disclosed in these publications.
  • L-amino acid-producing bacteria examples include L-lysine-producing bacteria , L-threonine-producing bacteria, and L-tryptophan-producing bacteria are exemplified below.
  • L-lysine producing bacteria belonging to the genus Escherichia examples 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.
  • L-lysine analogs include, but are not limited to, oxalysine, lysine hydroxamate, S- (2-aminoethyl) -L-cysteine (AEC), ⁇ -methyllysine, ⁇ -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.
  • WC196 strain can be used as an L-lysine-producing bacterium of Escherichia coli.
  • This strain was obtained from the W3110 strain derived from E. coli K-12, and encodes aspartokinase III in which feedback inhibition by L-lysine was released by replacing threonine at position 352 with isoleucine.
  • the stock was named Escherichia coli AJ13069.
  • L-lysine-producing bacteria or parent strains for inducing them include strains in which one or more activities of L-lysine biosynthetic enzymes are enhanced.
  • L-lysine biosynthetic enzymes include dihydrodipicolinate synthase (dapA), aspartokinase (lysC), dihydrodipicolinate reductase (dapB), diaminopimelate decarboxylase (lysA), diaminopimelate dehydrogenase (ddh) (US Pat.No. 6,040,160).
  • ppc Phosphoenolpyruvate carboxylase
  • ppc Phosphoenolpyruvate carboxylase
  • dapF diaminopimelate epimerase
  • dapD tetrahydrodipicolinate succinylase
  • dapE succinyl diaminopimelate deacylase
  • aspartase aspA
  • the parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Patent No. 5,830,716), ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased.
  • L-lysine-producing bacteria or parent strains for deriving the same include reduction or loss of the activity of enzymes that catalyze reactions that branch off from the L-lysine biosynthetic pathway to produce compounds 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 WC196 ⁇ cadA ⁇ ldc / 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 that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine.
  • 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.
  • WC196 ⁇ cadA ⁇ ldcC was named AJ110692, and on October 7, 2008, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology (AIST), Patent Biological Deposit Center, 305-8566, Tsukuba City East, Ibaraki Prefecture, Japan It is deposited internationally at 1st Street, 1st Floor, Central 6th), and is given the accession number FERM BP-11027.
  • L-threonine-producing bacteria examples include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371) E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli. coli FERM BP-3519 and FERM BP-3520 (US Pat.No.
  • E. coli MG442 Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)
  • E. coli VL643 and VL2055 Examples include, but are not limited to, strains belonging to the genus Escherichia, such as (EP 1149911 A).
  • the TDH-6 strain lacks the thrC gene, is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine.
  • the B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine.
  • E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it.
  • the B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter.
  • VKPM B-5318 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318. It has been deposited.
  • the bacterium used in the present invention is further modified so that expression of one or more of the following genes is increased.
  • -A thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine-A thrB gene encoding homoserine kinase-A thrC gene encoding threonine synthase-A rhtA gene encoding a putative transmembrane protein-Aspartate- asd gene encoding ⁇ -semialdehyde dehydrogenase-aspC gene encoding aspartate aminotransferase (aspartate transaminase)
  • the thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990).
  • the thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12.
  • the thrB gene encoding E. ⁇ ⁇ ⁇ coli homoserine kinase has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 99049175990).
  • the thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12.
  • the thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990).
  • the thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon.
  • the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).
  • mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.
  • the rhtA gene is present on the 18th minute of the E. ⁇ coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system.
  • the rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene.
  • the unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant to homoserine and threonine).
  • the E. coli asd gene has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene. (See White, T. J., Arnheim, N., and Erlich, H. A. 1989. Trends Genet. 5: 185-189). The asd gene of other microorganisms can be obtained similarly.
  • the aspC gene of E. coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC — 000913.1, gi: 16128895), and can be obtained by PCR.
  • the aspC gene of other microorganisms can be obtained similarly.
  • L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking the tryptophanyl-tRNA synthetase encoded by the mutant trpS gene (DSM10123) (U.S. Pat.No. 5,756,345), E.
  • coli having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase not subject to feedback inhibition by tryptophan.
  • SV164 pGH5 (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614)
  • E. coli AGX17 / pGX50, pACKG4-pps WO9708333, U.S. Pat.No.
  • L-tryptophan-producing bacteria or parent strains for inducing the same examples include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and a kind of activity of an enzyme selected from tryptophan synthase (trpAB) Also included are strains with increased above. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E.
  • coli SV164 carrying a desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase with desensitized feedback inhibition
  • Examples include a transformant obtained by introducing the plasmid pGH5 (WO 94/08031) into E.coli SV164.
  • L-tryptophan-producing bacteria or parent strains for deriving the same examples include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (Japanese Patent Laid-Open Nos. 57-71397 and 57). 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of ⁇ and ⁇ subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO2005 / 103275).
  • Example 1 Expression of lipase gene in Escherichia coli A gene encoding a mature lipase (CS gene) excluding the secretion signal of Cryptococcus sp. S-2 (FERM BP-10961) is expressed in pET-22b (+) vector (Novagen Cleavage was performed with MscI (near pelB leader) and NotI (upstream of His • Tag) in the cloning / expression region column, and the mature lipase gene was inserted directly under pelBleader. 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- ⁇ -D-thiogalactopyranoside.
  • Escherichia coli OrigamiB (DE3) is transformed, inoculated into an LB agar medium containing 100 ⁇ g / ml ampicillin, and having a target plasmid having ampicillin resistance as an indicator. Selected.
  • 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 ⁇ g / ml ampicillin at 25 ° C. for 18 hours.
  • An appropriate amount of this culture solution is added to LB medium containing 100 ⁇ g / 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- ⁇ -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.
  • 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.
  • each primer in Table 2 was used by replacing it with the corresponding codon sequence according to the type of amino acid residue to be introduced.
  • Table 3 shows each codon corresponding to an amino acid residue.
  • the PCR product obtained by treating with DpnI and digesting the template double-stranded DNA (pET-22b (+) _ CLE plasmid) was transformed into Escherichia coli JM109 strain, with 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 riOrigamiB (DE3) pET-22b (+) _ CLE_M / OrigamiB (DE3) strain carrying pET-22b (+) _ CLE_M.
  • mutant types may be separated by “/” and each mutant type may be listed.
  • 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 4 Preparation of crude extract from lipase-expressing cells 1 mL of the culture solution obtained in Example 3 was placed in a 1.5 mL tube and centrifuged at 14000 g for 1 minute. The supernatant was discarded, and 400 ⁇ l 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.
  • nonionic surfactant CelLytic B, Sigma
  • 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.
  • 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 ⁇ L of the reaction solution was dispensed into 96-well plates (Nunc-Immuno Plate, 475094), and 5 ⁇ L 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 ⁇ L 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.
  • 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.
  • an ultrasonic crusher ISONATOR 201M, KUBOTA
  • the resulting supernatant is filtered with a 0.22 ⁇ m 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.
  • the column used was a hydrophobic interaction column (HiLoad 16/10 Phenyl Sepharose High Performance, GE Healthcare Bio-Sciences), and as eluents solution A (100 mM phosphate buffer (pH 7.0)) and solution B (ethylene glycol 80%, 100 mM phosphate buffer (pH 7.0) 20%) was used.
  • 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.
  • 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 9 L-lysine production culture using fat and oil degradation product as a carbon source Escherichia coli WC196 ⁇ cadA ⁇ ldc / 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.
  • 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).
  • 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.
  • 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.
  • 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.

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Abstract

A mutant lipase produced by introducing a mutation into a lipase produced by a microorganism belonging to the genus Cryptococcus, Gibberella, Ustilago or the like so as to increase the stability of the enzyme. An L-amino acid such as L-lysine, L-threonine or L-tryptophan can be produced through a fermentation process using, as a carbon source, glycerol or the like that is produced by reacting the mutant lipase with an oil-and-fat.

Description

変異型リパーゼとその応用Mutant lipase and its application
　本発明は、優れた安定性を有する変異型リパーゼの製造方法等に関する。 The present invention relates to a method for producing a mutant lipase having excellent stability.
　現在、大量に消費されている石油燃料の代表的なものとしてディーゼルオイルがあり、ディーゼル車、船舶、発電機等の産業用のエンジンに使用されている。しかし、近年、化石燃料の需要と供給のバランスが逼迫し、原油の価格が高騰している。更に、国際的な地球環境問題解決の取り組みの一環として、地球温暖化の主な原因とされる化石燃料起源の二酸化炭素の削減が、危急的な目標となっている。そこで、非化石燃料の開発に力が注がれ、特にヨーロッパ諸国では、ディーゼルエンジン用、もしくはガソリンエンジン用に植物油が用いられるようになってきている。植物性油や動物性脂をメタノールと反応させメチルエステル化したものは、バイオディーゼルと呼ばれ、軽油などの代替燃料として使用されている。ヨーロッパ諸国では、主に菜種を中心にバイオディーゼルオイルが製造されている一方、米国では大豆、東南アジアではパーム油などが原料として用いられている。また、廃油やひまわりなども活用されている。 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.
　バイオディーゼル燃料生産には現在、アルカリ触媒を用いた化学触媒法が利用されている。しかし、副産物であるグリセロールの回収が困難である上、バイオディーゼルに含まれる不純物が多いなど課題を抱えている。そこで、リパーゼを用いた油脂の酵素分解とメチルエステル化によってバイオディーゼルを生成させる試みが行われている。 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.
　リパーゼとは、脂質を基質としてそのエステル結合を加水分解する酵素である。主に消化液（胃液、膵液）に含まれ脂質の消化を行う消化酵素であり、多くの生物の細胞で脂質の代謝に関与する。リパーゼは多くの生物に存在し、その遺伝子は一部のウイルスにもある。機能も立体構造もさまざまであるが、活性中心にセリン、アスパラギン酸、ヒスチジンを持つものが多い。基質のエステル結合を分解すると同時に逆反応（エステル合成）にも働くことから、人工的なエステル合成・交換反応にも用いられている。 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).
　前記したように、リパーゼを用いて油脂を処理し、バイオディーゼルとグリセロールを生成させることはすでに報告されているが、アルカリ触媒法に比べ酵素の価格が高く実用化に至っていない。そこで、酵素の価格を抑制するために、酵素を改質し活性や安定性を向上させる研究が行われている。例えば、Phage Display法によるBacillus subtilis lipase Aの改変（非特許文献１、２)、DNA shufflingによる活性と安定性の改善(非特許文献３)、CALB法によC.antactica Lipase Bの改変(非特許文献４、５)、CAST法によるPseudomonas aeruginosa lipaseの改変（非特許文献６)などが挙げられる。また、反応プロセスを改善するために、メタノールを反応液に徐々に滴下することによって酵素の失活を抑制する方法(非特許文献７)や、酵母の細胞表面にリパーゼを提示させる方法(非特許文献７、８)なども開発されている。 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 alkaline 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 include Patent Documents 4 and 5), modification of Pseudomonas aeruginosa lipase by the 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.
　ところが、このような技術蓄積にも関わらず、酵素を用いたバイオディーゼルの生産コストは未だアルカリ触媒法には及ばず、更なる製法の改善が求められている。このような背景のもと、独立行政法人酒類総合研究所にて新規のリパーゼが報告され、同リパーゼを用いて油脂からバイオディーゼルを生成させることが立証された（特許文献１）。 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).
特許第3507860号Patent No. 3507860
　本発明の目的は、油脂分解に適した安定性の優れたリパーゼを提供すること、およびこの変異型リパーゼを用いた油脂の分解物の製造法、および分解物の利用方法を提供することである。 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. .
　本発明者らは、前記新規酵素の改変体を作製し、安定性を向上させることによって、バイオディーゼル生成に要するコストの削減を目指した。また、副生するグリセロールを炭素源としてアミノ酸や核酸の生産へと転用したり、更には油脂を分解したときに生成する脂肪酸をも同時にアミノ酸生産に活用することができれば、植物原料の有効利用となると考えた。そして、鋭意研究を重ねた結果、野生型リパーゼよりも高い安定性を示すリパーゼ改変体を作製することに成功した。
　すなわち、本発明は以下のとおりである。 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）配列番号７のアミノ酸配列、又は配列番号７において１若しくは数個のアミノ酸の置換、欠失、挿入、付加、又は逆位を含むアミノ酸配列を有し、かつ、下記（ａ）～（ｑ）から選択される変異を含む、変異型リパーゼ。
（ａ）３２位のグリシンが他のアミノ酸残基に置換される変異
（ｂ）５０位のチロシンが他のアミノ酸残基に置換される変異
（ｃ）９５位のアラニンが他のアミノ酸残基に置換される変異
（ｄ）１２６位のリジンが他のアミノ酸残基に置換される変異
（ｅ）１３１位のシステインが他のアミノ酸残基に置換される変異
（ｆ）１３３位のバリンが他のアミノ酸残基に置換される変異
（ｇ）１５３位のグリシンが他のアミノ酸残基に置換される変異
（ｈ）１６４位のロイシンが他のアミノ酸残基に置換される変異
（ｉ）７位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
（ｊ）４２位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
（ｋ）８３位のＸがバリンであって、このバリンが他のアミノ酸残基に置換される変異
（ｌ）８８位のＸがグルタミンであって、このグルタミンが他のアミノ酸残基に置換される変異
（ｍ）１５４位のＸがセリンであって、このセリンが他のアミノ酸残基に置換される変異
（ｎ）１７７位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
（ｏ）２０３位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
（ｐ）２０６位のＸがリジンであって、このリジンが他のアミノ酸残基に置換される変異
（ｑ）２０９位のＸがグリシンであって、このグリシンが他のアミノ酸残基に置換される変異
（２）前記（ａ）～（ｑ）の変異が、それぞれ下記（Ａ）から（Ｑ）の変異である、前記変異型リパーゼ。
（Ａ）３２位のグリシンがアスパラギン酸に置換される変異
（Ｂ）５０位のチロシンがアスパラギンに置換される変異
（Ｃ）９５位のアラニンがアスパラギン酸に置換される変異
（Ｄ）１２６位のリジンがイソロイシンに置換される変異
（Ｅ）１３１位のシステインがセリン、トレオニン、イソロイシン、及びロイシンから選択されるアミノ酸残基に置換される変異
（Ｆ）１３３位のバリンがリジンに置換される変異
（Ｇ）１５３位のグリシンがプロリンに置換される変異
（Ｈ）１６４位のロイシンがグルタミンに置換される変異
（Ｉ）７位のアラニンがプロリンに置換される変異
（Ｊ）４２位のアラニンがグルタミンに置換される変異
（Ｋ）８３位のバリンがグルタミンに置換される変異
（Ｌ）８８位のグルタミンがアスパラギンに置換される変異
（Ｍ）１５４位のセリンがイソロイシンに置換される変異
（Ｎ）１７７位のアラニンがプロリンに置換される変異
（Ｏ）２０３位のアラニンがロイシンに置換される変異
（Ｐ）２０６位のリジンがグルタミンに置換される変異
（Ｑ）２０９位のグリシンがプロリンに置換される変異
（３）前記（ａ）～（ｑ）から選択される変異が、前記（ｄ）、（ｅ）、（ｆ）又は（ｏ）の変異である、前記変異型リパーゼ。
（４）前記（ｄ）、（ｅ）、（ｆ）又は（ｏ）の変異が、前記（Ｄ）、（Ｅ）、（Ｆ）又は（Ｏ）の変異である、前記変異型リパーゼ。
（５）前記変異を有しないリパーゼが、下記（Ｉ）又は（ＩＩ）のタンパク質である、前記変異型リパーゼ。
（Ｉ）配列番号２、４、又は６に記載のアミノ酸配列を有するタンパク質。
（ＩＩ）配列表の配列番号２、４、又は６に記載のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入、付加、又は逆位を含むアミノ酸配列からなり、かつ、リパーゼ活性を有するタンパク質。
（６）前記変異型リパーゼをコードするＤＮＡ。
（７）下記（ｉ）又は（ｉｉ）の塩基配列を有し、かつ、前記アミノ酸配列における変異に相当する変異を有する前記ＤＮＡ。
（ｉ）配列番号１、３、又は５に記載の塩基配列。
（ｉｉ）配列表の配列番号１、３、又は５に記載の塩基配列又は該配列から調製され得るプローブと、ストリンジェントな条件下でハイブリダイズする配列。
（８）前記ＤＮＡを含む形質転換微生物。
（９）前記形質転換微生物がエシェリヒア・コリである前記微生物。
（１０）前記形質転換微生物を培地中で培養し、培地中および／または微生物中に前記変異型リパーゼを蓄積させることを特徴とする、変異型リパーゼの製造方法。
（１１）前記変異型リパーゼを油脂に作用させて、グリセロールを生成させることを特徴とする、グリセロールの製造方法。
（１２）前記変異型リパーゼを油脂に作用させて、グリセロールを生成させ、生成したグリセロールを炭素源として添加した培地で、グリセロール資化能を有し、かつ、目的物質を産生する微生物を培養し、目的物質を培養物中から採取する、目的物質の製造方法。
（１３）前記変異型リパーゼを油脂に作用させて、グリセロール及び脂肪酸を生成させ、生成したグリセロール及び脂肪酸を炭素源として添加した培地で、グリセロール及び脂肪酸の資化能を有し、かつ、目的物質を産生する微生物を培養し、目的物質を培養物中から採取する、目的物質の製造方法。
（１４）前記微生物が、コリネ型細菌、及び腸内細菌科に属する微生物から選択される細菌である前記方法。
（１５）前記目的物質がＬ－アミノ酸である、前記方法。
（１６）前記Ｌ－アミノ酸が、Ｌ－リジン、Ｌ－スレオニン、及びＬ－トリプトファンから選択される前記方法。 (1) having the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence including 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 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 the above (a) to (q) are the above (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.
配列番号２、４及び６のアミノ酸配列（成熟タンパク質部分）のアラインメントを示す図。「Ｘ」は、３種のリパーゼで共通していないことを示す。「-」は、その位置にアミノ酸が存在しないことを示す。四角形で囲んだアミノ酸は、変異点を示す。アスタリスク（*）で示したアミノ酸は、配列番号２に示すCryptococcus sp.S-2のリパーゼに由来する変異型リパーゼの持つ他の変異点を示す。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. 60℃、20時間で加熱処理前後における、野生型リパーゼと各変異型リパーゼの残存エステラーゼ活性を示す図。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. 加熱処理前の、野生型リパーゼと各変異型リパーゼのエステラーゼ活性を示す図。The figure which shows the esterase activity of a wild-type lipase and each variant lipase before heat processing.
　以下、本発明を詳細に説明する。
　なお、以下に挙げる種々の遺伝子工学的な技法については、Molecular Cloning, 2nd edition, Cold Spring Harbor press (1989)、細胞工学ハンドブック、黒木登志夫ら編、羊土社（1992）、新遺伝子工学ハンドブック改訂第３版、村松ら編、羊土社（1999）など、多くの標準的な実験マニュアルがあり、これらの文献を参考にすることにより当業者であれば実施可能である。 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.
　本明細書におけるアミノ酸、ペプチド、核酸、塩基配列などの略号による表示は、IUPAC（International Union of Pure and Applied Chemistry）またはIUBMB（International Union of Biochemistry and Molecular Biology）による規定、「塩基配列又はアミノ酸配列を含む明細書等の作成のためのガイドライン」（日本国特許庁編）、「Standard for the presentation of nucleotide and amino acid sequence listings in patent applications」（WIPO Standard ST.25）、および当該分野における慣用記号に従うものとする。 The abbreviations such as amino acids, peptides, nucleic acids, and base sequences in this specification are defined by IUPAC (International Union of Pure Pure and Applied Chemistry) or IUBMB (International Union of Biochemistry and Molecular Molecular Biology), “base sequence or amino acid sequence. In accordance with “Guidelines for Preparation of Including Descriptions” (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 Shall.
１．本発明のリパーゼ
　本発明のリパーゼは、クリプトコッカス属由来のリパーゼ、例えCryptococcus sp.S-2が産生するリパーゼ、及びそれと一次構造が類似するリパーゼであって、さらに特定の変異を有する変異型リパーゼである。以下、本発明の変異型のリパーゼと区別して、本発明の変異型リパーゼが持つ変異を有しないリパーゼを、野生型のリパーゼと呼ぶことがある。尚、前記特定の変異以外の変異を有するものであって、特定の変異を有しないリパーゼは、本発明にいう野生型リパーゼである。 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.
　本発明においてリパーゼとは、油脂分解性を有し、油脂に作用して、グリセロールと脂肪酸に加水分解する酵素であり、トリアシルグリセロール　リパーゼ（triacylglycerol lipase）、トリアシルグリセリド　リパーゼ（triacylglyceride lipase）とも呼ばれる。油脂は、脂肪酸とグリセロールのエステルであり、トリグリセリドとも呼ばれる。また、リパーゼは、メタノールなどのアルコール存在下では、油脂とアルコールのエステル交換反応を触媒し、脂肪酸エステルを生成する（Jaeger, K. E. and Eggert, T. 2002. Curr. Opin. Biotechnol. 13: 390-397）。ここで、油脂の分解活性とは、前記のようにトリグリセリドに作用して、グリセロールと脂肪酸に加水分解する活性及び、エステル交換反応によって脂肪酸エステルとグリセロールを生成する活性を含む。リパーゼの基質となる油脂を構成する脂肪酸の長さは特に制限されず、その長さに応じて、分解により生成する脂肪酸又は脂肪酸エステルの長さは異なる。 In the present invention, a lipase is an enzyme that has oil and fat degradability and acts on fats and oils to hydrolyze into glycerol and fatty acids. It 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 esters (Jaeger, K. E. 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.
　本発明の変異型リパーゼは、野生型リパーゼに、前記特定の変異を導入することにより、得ることができる。本発明における野生型リパーゼは、これら上記の反応を触媒するリパーゼであって、前記特定の変異を導入することによって、酵素活性が上昇するものであれば、どのような種由来のリパーゼも用いることが可能である。野生型リパーゼとしてより具体的には、配列番号７に示すアミノ酸配列を有するリパーゼが挙げられる。また、野生型リパーゼは、配列番号７において、１若しくは数個のアミノ酸の置換、欠失、挿入、付加、又は逆位を含むアミノ酸配列を有するリパーゼであってもよい。 The mutant lipase of the present invention can be obtained by introducing the specific mutation into 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 may be a lipase having an amino acid sequence including substitution, deletion, insertion, addition, or inversion of one or several amino acids in SEQ ID NO: 7.
　上記のような野生型リパーゼとしては、特に酵母であるクリプトコッカス属あるいはその近縁種や、Gibberella属、またはUstilago属のリパーゼが好ましい。
例えばクリプトコッカス属としては、以下の微生物が挙げられる。
Cryptococcus neoformans JEC21
Cryptococcus neoformans B-3501A
Cryptococcus sp.S-2 (FERM P-15155) （特許3507860号公報）
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 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
　例えばクリプトコッカス属、特にCryptococcus sp.S-2が産生するリパーゼは以下の理化学的性質を有する。 For example, lipases produced by Cryptococcus spp., In particular Cryptococcus sp. S-2, have the following physicochemical properties.
（１）作用
　油脂分解性を有し、トリグリセリドに作用して、グリセロールと脂肪酸に加水分解する。 (1) Action It has fat and oil degradability, acts on triglycerides, and hydrolyzes into glycerol and fatty acids.
（２）基質特異性
　トリブチリン、トリカプリリン、トリパルミチン、トリオレインをよく分解する。トリアセチン、トリカプリン、トリラウリンは中程度に分解する。トリミリスチン、トリステアリンに対する分解力は弱い。 (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）位置特異性
　トリオレインに作用せしめると、オレイン酸と少量の1,2(2,3)-ジオレインが生成し、1,3-ジオレインとモノオレインは検出されない。 (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）至適pH及び安定pH範囲
至適pH：7.0
安定pH範囲：５～９ (4) Optimal pH and stable pH range Optimal pH: 7.0
Stable pH range: 5-9
（５）反応最適温度及び温度による失活の条件
反応最適温度：37℃
温度による失活の条件：温度上昇による活性の失活は緩やかであり、60℃、30分の熱処理においても活性を維持するが、60℃、20時間の熱処理により、活性は20%以下に低下する。 (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 decreases to 20% or less by heat treatment at 60 ° C for 20 hours. To do.
（６）有機溶媒に対する安定性、活性化
有機溶媒に安定であり、更にジメチルスルホキシド、ジエチルエーテルによってエステル交換活性が上昇する。 (6) Stability to organic solvent, stable to activated organic solvent, and further increased transesterification activity by dimethyl sulfoxide and diethyl ether.
（７）分子質量
21kDa（Expasy Compute pI/Mw） (7) Molecular mass
21kDa (Expasy Compute pI / Mw)
　このような性質を有するクリプトコッカス属のリパーゼあるいはそれと構造が類似するリパーゼは、特許3507860号に記載の方法と同様の方法で取得することが出来るし、下述するリパーゼをコードする遺伝子配列の相同性を利用しても取得することが出来る。 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.
　クリプトコッカス属由来のリパーゼをコードする遺伝子として、Cryptococcus sp.S-2 (FERM P-15155)のリパーゼ遺伝子CS2遺伝子が知られている（特開2004-73123号公報）。本CS2遺伝子の塩基配列を配列番号１に、本CS2遺伝子がコードするリパーゼの前駆体のアミノ酸配列を配列番号２に示す。配列番号２に示すアミノ酸配列において、-30～-1位はシグナルペプチド、1～250位は成熟タンパク質に相当すると予想される。Cryptococcus sp.S-2は、1995年9月5日付けで工業技術院生命工学工業技術研究所（現独立行政法人産業技術総合研究所 特許生物寄託センター）（〒305-5466 日本国茨城県つくば市東１丁目１番地１　中央第６）にFERM P-15155の受託番号で寄託され、2008年4月25日にブダペスト条約に基づく国際寄託に移管され、受託番号FERM BP-10961が付与されている。 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 predicted 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 in the center, 1-chome, 1-chome, 1st, 6th), transferred to an international deposit based on the Budapest Treaty on April 25, 2008, and given the accession number FERM BP-10961 .
　また、本発明においてリパーゼは、上記配列番号１又は２の配列との相同性に基づき、クリプトコッカス属あるいは近縁種の酵母、その他の微生物からクローニングされるリパーゼ遺伝子のホモログであってもよい。アミノ酸配列および塩基配列の相同性は、例えばKarlin および AltschulによるアルゴリズムBLAST(Pro. Natl. Acad. Sci. USA, 90, 5873(1993))やPearsonによるFASTA(MethodsEnzymol., 183, 63 (1990))を用いて決定することができる。このアルゴリズムBLASTに基づいて、BLASTNやBLASTXとよばれるプログラムが開発されている(http://www.ncbi.nlm.nih.gov参照)。また、アミノ酸配列および塩基配列の相同性は、CLUSTALW（Thompson J.D., Higgins D.G. and Gibson T.J. Nucleic Acids Res. 1994, 22:4673-4680）によっても計算することができる。例えば、Cryptococcus sp.S-2のCS2遺伝子と相同性の高い遺伝子として、Gibberella zeae （ジベレラ・ゼアエ）PH-1の遺伝子（Genbank Accession No. gi|42549908|gb|EAA72751.1|[42549908] 、塩基配列：配列番号３、アミノ酸配列：配列番号４）、Ustilago maydis（ウスチラゴ・マイジス）521由来の遺伝子（Genbank Accession No.gi|46096123|gb| EAK81356.1|[46096123]、塩基配列：配列番号５、アミノ酸配列：配列番号６）を利用することが出来る。 In the present invention, the lipase may be a homologue of a lipase gene cloned from a yeast of the genus Cryptococcus or a related species or other microorganisms based on the 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 of 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, Gibberella zeae PH PH-1 gene (Genbank Accession No. gi | 42549908 | gb | EAA72751.1 | [42549908], which is highly homologous to the CS2 gene of Cryptococcus sp. S-2 Nucleotide sequence: SEQ ID NO: 3, amino acid sequence: SEQ ID NO: 4), gene derived from Ustilago maydis 521 (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.
　なお、配列番号４においてシグナルペプチドは-19～-1位、成熟型タンパク質は1～204位に相当し、配列番号６においてシグナルペプチドは-20～-1位、成熟型タンパク質は1～204位に相当すると予想される。
　尚、上記各リパーゼにおいて、シグナルペプチド及び成熟タンパク質の位置は、SignalP（http://www.cbs.dtu.dk/services/SignalP/）を用いて予測した。 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/).
　リパーゼ遺伝子のホモログとは、他の微生物由来または天然もしくは人工の変異型遺伝子で、クリプトコッカスのCS2遺伝子と構造が高い類似性を示し、リパーゼ活性を示す遺伝子を意味する。リパーゼ遺伝子のホモログは、配列番号２の1～205位のアミノ酸配列全体に対して、８０％以上、好ましくは９０％以上、より好ましくは９５％、特に好ましくは９８％以上の相同性を有し、かつ、リパーゼの機能を有するタンパク質をコードするものを意味する。なお、リパーゼの機能を有することは、これらの遺伝子を宿主細胞で発現させ、油脂分解能を確認することによって確かめることが出来る。
　尚、本明細書において、「相同性」（homology）」は、「同一性」（identity）を指すことがある。 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”.
　また本発明に使用できるリパーゼは、配列番号１、３、又は５の塩基配列に相補的な塩基配列又は該配列から調製され得るプローブとストリンジェントな条件下でハイブリダイズするＤＮＡであって、リパーゼとしての機能を有するタンパク質をコードするDNAであってもよい。ここで、「ストリンジェントな条件」とは、いわゆる特異的なハイブリッドが形成され、非特異的なハイブリッドが形成されない条件をいう。この条件を明確に数値化することは困難であるが、一例を示せば、相同性が高いＤＮＡ同士、例えば８０％、好ましくは９０％以上、より好ましくは９５％、特に好ましくは９８％以上の相同性を有するＤＮＡ同士がハイブリダイズし、それより相同性が低いＤＮＡ同士がハイブリダイズしない条件、あるいは通常のサザンハイブリダイゼーションの洗いの条件である６０℃、１×ＳＳＣ，０．１％ＳＤＳ、好ましくは、０．１×ＳＳＣ、０．１％ＳＤＳさらに好ましくは、６８℃、０．１×ＳＳＣ、０．１％ＳＤＳに相当する塩濃度、温度で、１回より好ましくは２～３回洗浄する条件が挙げられる。 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.
　前記配列番号２、４及び６のアミノ酸配列（成熟タンパク質部分）のアラインメントを、図１に示す。これらの配列の下に、共通配列を示した。共通配列中「Ｘ」で示したアミノ酸は、３種のリパーゼで共通していないことを示す。「-」は、その位置にアミノ酸が存在しないことを示す。また、四角形で囲んだアミノ酸は、本発明の変異型リパーゼが持つ変異点を示す。さらに、アスタリスク（*）で示したアミノ酸は、配列番号２に示すCryptococcus sp.S-2のリパーゼに由来する変異型リパーゼの持つ他の変異点を示す。 The 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 a mutant lipase derived from the lipase of Cryptococcus sp. S-2 shown in SEQ ID NO: 2.
　上記のように、３種のリパーゼは高い相同性を有している。また、後述するように、Cryptococcus sp.S-2のリパーゼにおいて、リパーゼ活性が上昇する１７箇所の変異のうち８箇所は、３種のリパーゼの間でアミノ酸が共通している。したがって、本発明において野生型リパーゼは、配列番号７に示すアミノ酸配列を有するタンパク質として特定することができる。
　尚、各リパーゼ成熟タンパク質間の相同性を表すSCOREをCLUSTALWversion1.83により決定したところ、配列番号２と配列番号４では64、配列番号２と配列番号６では58、配列番号４と配列番号６では59であった。 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．本発明の変異型リパーゼ
　本発明の変異型リパーゼとは、上記のような野生型リパーゼに変異を導入することにより、野生型リパーゼよりさらに安定性が高くなったリパーゼを意味する。安定性とは、熱安定性を意味する。変異型リパーゼは、60℃で20時間処理した後の残存活性が、好ましくは20%以上、より好ましくは30%以上であることが望ましい。 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.
　変異導入の方法としては、野生型リパーゼ遺伝子のコード領域の配列をヒドロキシルアミン等でインビトロ処理する方法、および該遺伝子を保持する微生物、例えばリパーゼ遺伝子を導入したエシェリヒア・コリを、紫外線またはN-メチル-N'-ニトロ-N-ニトロソグアニジン(NTG)もしくはエチルメタンスルフォネート（EMS）等の通常変異処理に用いられている変異剤によって処理する方法、エラープローンPCR（Cadwell,R.C. PCR Meth. Appl. 2, 28(1992)）、DNA shuffling（Stemmer,W.P. Nature 370, 389(1994)）、StEP-PCR（Zhao,H. Nature Biotechnol. 16, 258(1998)）等によって、遺伝子組換えにより人工的にリパーゼに変異を導入する方法が挙げられる。これらの変異型リパーゼ遺伝子のリパーゼ活性は、例えば、ｐ-ニトロフェニルブチル酸またはｐ-ニトロフェニルパルミチン酸を基質とした分光光度法によりエステラーゼ活性を測定することによって確認することが出来る。 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), etc., 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.
　また、変異型リパーゼの活性は、変異型リパーゼ、油脂、アルコールを混合、インキュベートした後に、原料である油脂含量の低下、もしくは脂肪酸エステルまたはグリセロールの生成を測定することによっても評価することができる。あるいは、変異型リパーゼ、油脂、水を混合、インキュベートした後に、原料である油脂含量の低下、もしくは脂肪酸またはグリセロールの生成を測定してもよい。 The activity of the mutant lipase can also be evaluated by measuring the decrease in the content of fat or oil, 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.
　リパーゼの安定性を上昇させる変異として具体的には、以下の（ａ）から（ｑ）のような変異を挙げることができる。尚、いずれの位置も、配列番号７における位置である。 Specific examples of mutations that increase the stability of lipase include the following mutations (a) to (q). Each position is the position in SEQ ID NO: 7.
（ａ）３２位のGlyが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Aspに置換されることが最も好ましい。 (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）５０位のTyrが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Asnに置換されることが最も好ましい。 (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）９５位のAlaが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Aspに置換されることが最も好ましい。 (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）１２６位のLysが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Ileに置換されることが最も好ましい。 (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）１３１位のCysが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Ser、Thr、Leu、又はIleに置換されることが最も好ましい。 (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）１３３位のValが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Lysに置換されることが最も好ましい。 (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）１５３位のGlyが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Proに置換されることが最も好ましい。 (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）１６４位のLeuが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Glnに置換されることが最も好ましい。 (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）７位のＸがAlaであって、このAlaが他のアミノ酸残基に置換される変異
　ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Proに置換されることが最も好ましい。 (I) 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, and His may be any amino acid. Of these, substitution to Pro is most preferable.
（ｊ）４２位のＸがAlaであって、このAlaが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Glnに置換されることが最も好ましい。 (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）８３位のＸがValであって、このValが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Glnに置換されることが最も好ましい。 (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）８８位のＸがGlnであって、このGlnが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Asnに置換されることが最も好ましい。 (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）１５４位のＸがSerであって、このSerが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Ileに置換されることが最も好ましい。 (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）１７７位のＸがAlaであって、このAlaが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Proに置換されることが最も好ましい。 (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）２０３位のＸがAlaであって、このAlaが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Leuに置換されることが最も好ましい。 (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）２０６位のＸがLysであって、このLysが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Gly, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Glnに置換されることが最も好ましい。 (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）２０９位のＸがGlyであって、このGlyが他のアミノ酸残基に置換される変異
ここで他のアミノ酸残基とは天然型アミノ酸であればいずれのアミノ酸でもよく、Lys, Glu, Thr, Val, Leu, Ile, Ser, Asp, Asn, Gln, Arg, Cys, Met, Phe, Trp, Tyr, Ala, Pro, Hisから選択されるアミノ酸であればいずれでもよい。なかでも、特に、Proに置換されることが最も好ましい。 (Q) A 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, 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.
　上記変異の中では、安定性の点からは、（ｄ）、（ｅ）、（ｆ）、及び（ｏ）が好ましい。また、変異型リパーゼの比活性（タンパク質量当りの酵素活性）の点からは、（ｋ）、（ｅ）及び（ｐ）が好ましい。 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.
　上記（ａ）～（ｑ）の７位、３２位、４２位、５０位、８３位、８８位、９５位、１２６位、１３１位、１３３位、１５３位、１５４位、１６４位、１７７位、２０３位、２０６位、２０９位は、それぞれ、配列番号２のアミノ酸配列においては、１位、２６位、３６位、４４位、７７位、８２位、８９位、１２０位、１２５位、１２７位、１４７位、１４８位、１５８位、１７１位、１９７位、２００位、２０３位に相当する。配列番号４及び６における変異の位置は、図１に示すアラインメントにおいて、（ａ）～（ｑ）に記載した位置に相当する。 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 the mutations in SEQ ID NOs: 4 and 6 correspond to the positions described in (a) to (q) in the alignment shown in FIG.
　本明細書において、変異型リパーゼが持つ変異は、表１に示すようにアミノ酸残基の略号および配列表に示すアミノ酸配列中の部位より特定する。例えば、配列番号２における変異に関し、変異型１の「Ｃ１２５Ｉ」とは、配列番号２の配列の第１２５位のアミノ酸残基システインをイソロイシンに置換したことを示す。すなわち、変異型の表記は、野生型（配列番号２で特定されるアミノ酸）のアミノ酸残基の種類；配列番号２に記載のアミノ酸配列におけるアミノ酸残基の位置；変異導入後のアミノ酸残基の種類を示す。他の変異型の表記も同様である。 In the present 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.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　本発明の変異型リパーゼは、優れた安定性を有する。すなわち、野生型のリパーゼに比べ、熱に対する耐性が高い。より具体的には、本発明の変異型リパーゼはそれぞれ、温度安定性などの特性について、野生型タンパク質よりも性能が向上している。 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.
　本発明の変異型リパーゼは、安定性に加えて、他の酵素学的性質が改善される変異を有していてもよい。そのような変異としては、例えば、比活性、ｐＨ特性、基質特異性等が挙げられる。上記変異型リパーゼが持つ変異の中には、リパーゼの比活性を低下させる変異も存在する。変異型リパーゼがそのような変異を有する場合、さらに比活性を高める変異を導入することによって、安定性及び比活性を両立させることができる。 The mutant lipase of the present invention may have a mutation that improves other enzymatic properties in addition to the stability. 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.
　本発明者らは、リパーゼの比活性を高める変異として、下記の変異を見出している。これらの変異は、配列番号７のアミノ酸配列、又は配列番号７において１若しくは数個のアミノ酸の置換、欠失、挿入、付加、又は逆位を含むアミノ酸配列を有する野生型リパーゼにおける変異である。 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）２２位のグリシンが他のアミノ酸残基に置換される変異
（ii）５３位のバリンが他のアミノ酸残基に置換される変異
（iii）５８位のフェニルアラニンが他のアミノ酸残基に置換される変異
（iv）１３１位のシステインが他のアミノ酸残基に置換される変異
（v）１６０位のバリンが他のアミノ酸残基に置換される変異
（vi）１７３位のバリンが他のアミノ酸残基に置換される変異
（vii）１８７位のロイシンが他のアミノ酸残基に置換される変異
（viii）１８０位のＸがフェニルアラニンであって、このフェニルアラニンがイソロイシンまたはバリンに置換する変異
（ix）１８５位のＸがグルタミンであって、このグルタミンがグルタミン酸またはロイシンに置換する変異 (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
　上記（i）～（vii）の変異としてより具体的には、下記変異が挙げられる。
（Ｉ）２２位のグリシンがアラニンに置換される変異
（II）５３位のバリンがロイシン又はアルギニンに置換される変異
（III）５８位のフェニルアラニンがイソロイシン、ロイシン、バリン、及びトリプトファンから選択されるアミノ酸残基に置換される変異
（IV）１３１位のシステインがアラニンに置換される変異
（V）１６０位のバリンがイソロイシンに置換される変異
（IV）１７３位のバリンがイソロイシンに置換される変異
（VII）１８７位のロイシンがフェニルアラニンに置換される変異
　上記（I）～（VII）、又は（viii）もしくは（ix）の変異を有する変異型リパーゼの比活性が野生型リパーゼより高いことは、後述の実施例１～５と同様の方法により確認されている。 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 187 leucine 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. This is confirmed by the same method as in Examples 1 to 5 described later.
２．本発明の変異型リパーゼの製造方法
　本発明の変異型リパーゼの製造方法、並びにこれに用いられる組換え体および形質転換体の作製方法について説明する。
　上記変異型リパーゼは、野生型リパーゼをコードするＤＮＡに上記変異を導入し、これを適当なベクターにクローニングした後、適切な宿主に導入し、発現させることによって変異型リパーゼを製造することができる。 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. .
　変異型リパーゼを設計、作製するに当っては、配列番号７のアミノ酸配列をコードするＤＮＡ、例えば配列番号２、４、６のアミノ酸配列をコードする遺伝子のうち、上記変異に対応するコドンを部位特異的変異法により改変し、得られた改変遺伝子を適当なベクターに組込んだ後宿主に導入し、形質転換体を培養すればよい。目的とする変異型リパーゼは形質転換体の培養菌体より回収することができる。得られた変異型リパーゼは、野生型リパーゼに比べて高い安定性を有することを確認することが好ましい。 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.
　ＤＮＡの目的部位に目的の変異を起こす部位特異的変異法としては、実施例に示したＰＣＲを用いる方法〔R．ヒグチ、DNA操作へのPCRの使用、イン PCR テクノロジー（Higuchi, R., Using PCR to engineer DNA, in PCR technology）、H．A．エーリッヒ（Erlich,H. A.編、ストックトンプレス（Stockton press））、第61頁、(1989)）〕;〔P．カーター、メソッズ インエンザイモロジー（Carter, P., Meth. In Enzymol）、第154巻、第382頁(1987)〕以外に、ファージを用いる方法（W．クレーマー、及びH．J．フリッツ（Kramer, W. and Frits, H. J.)、メソッズ イン エンザイモロジー、第154巻、第350頁 (1987)； T. Aクンケルほか（Kunkel，T．A．et，al）メソッズ イン エンザイモロジー、第154巻、第367頁 (1987)、などがある。 As a site-specific mutagenesis method for causing a target mutation in a target site of DNA, a method using PCR shown in Examples [R. Higuchi, use of PCR for DNA manipulation, in-PCR technology (Higuchi, R., Using PCR to engineer DNA, in PCR technology), H. A. Erich (Erlich, H. A., Stockton press), p. 61, (1989))]; In addition to Carter, Method In Enzymology (Carter, P., Meth. In Enzymol), vol. 154, p. 382 (1987)], a method using a phage (W. Kramer, and H. J. Fritz (Kramer , W and Frits, H. J.), Methods Enzymology, Volume 154, 350 (1987); T. A Kunkel et al. (Kunkel, TA et al) Methodology In Enzymology 154, p. 367 (1987).
　なお、変異型リパーゼをコードする遺伝子を組込むベクターとしては、宿主において複製可能なものであれば特に制限はない。宿主としてエシェリヒア・コリを用いる場合には当該細菌で自律複製できるプラスミドを挙げることができる。例えば、pUC19、pET、pGEMEX等が使用可能である。また、好ましい宿主としては、エシェリヒア・コリ菌株が挙げられるが、これ以外にも、構築した組換えＤＮＡの複製起点と変異型リパーゼ遺伝子が機能し、組換えＤＮＡが複製可能でかつ変異型リパーゼ遺伝子の発現が可能な微生物ならば、すべて宿主として利用できる。宿主としては、例えばエシェリヒア・コリ（Escherichia coli）等のエシェリヒア属細菌、エンペドバクター属細菌、スフィンゴバクテリウム属細菌、フラボバクテリウム属細菌、及びバチルス・ズブチリス（Bacillus subtilis）をはじめとする種々の原核細胞、サッカロマイセス・セレビシエ（Saccharomyces cerevisiae）、ピヒア・スティピティス（Pichia stipitis）、アスペルギルス・オリゼ（Aspergillus oryzae）をはじめとする種々の真核細胞を用いることができる。 The vector into which the gene encoding the mutant lipase is incorporated is not particularly limited as long as it can replicate 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 bacteria including Escherichia coli such as Escherichia coli, Empedobacter bacteria, Sphingobacteria bacteria, Flavobacterium bacteria, and Bacillus subtilis. Various eukaryotic cells including prokaryotic cells, Saccharomyces cerevisiae, Pichia stipitis, Aspergillus oryzae can be used.
　変異型リパーゼをコードする遺伝子を含むＤＮＡ断片とベクターとを連結させてなる組換えＤＮＡを宿主に導入する方法としては特に制限はなく、通常の方法により行うことができる。宿主としてエシェリヒア・コリを用いる場合は、塩化カルシウム法〔ジャーナル オブ モレキュラー バイオロジー(J.Mol. Biol.)、第53巻、第159頁 (1970)〕、ハナハン（Hanahan）法〔ジャーナル オブ モレキュラー バイオロジー、第166巻、第557頁 (1983)〕、SEM法〔ジーン（Gene)、第96巻、第23頁(1990)〕、チャング（Chung）らの方法〔プロシーディングズ オブ ザ ナショナル アカデミー オブ サイエンシーズ オブ ザ ＵＳＡ、第86巻、第2172頁(1989)〕などの方法を用いることができる。 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 the host, the calcium chloride method (Journal of Molecular Biology (J. Mol. Biol.), Vol. 53, pp. 159 (1970)), Hanahan method (Journal of Molecular Biology) 166, 557 (1983)], SEM (Gene, 96, 23 (1990)), Chung et al. [Proceedings of the National Academy Sciences of the USA, Vol. 86, p. 2172 (1989)] can be used.
　変異型リパーゼをコードする遺伝子を発現させるためのプロモーターは、野生型遺伝子固有のプロモーターが宿主で機能する場合は、固有のプロモーターを使用することができるが、他のプロモーターを使用してもよい。また、固有のプロモーターが宿主で機能しない場合は、その宿主で機能するプロモーターを使用する。例えば、エシェリヒア・コリで機能するプロモーターとしては、lacプロモーター、trpプロモーター、trcプロモーター、tacプロモーター、ラムダファージのPRプロモーター、PLプロモーター、tetプロモーター等が挙げられる。 As a promoter for expressing a gene encoding a mutant lipase, a unique promoter can be used if 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.
　また、変異型リパーゼは、シグナルペプチドを含む前駆体タンパク質として発現させてもよく、成熟タンパク質を直接発現させてもよい。さらに、宿主に適したシグナルペプチドに変異型リパーゼの前駆体タンパク質又は成熟タンパク質を連結したものを発現させてもよい。例えば、宿主がエシェリヒア・コリの場合は、pelB、ompT等の遺伝子のシグナルペプチドが挙げられる。 Further, the mutant lipase may be expressed as a precursor protein containing a signal peptide, or the mature protein may be directly expressed. 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.
　上記のようにして得られる変異型リパーゼをコードする遺伝子を含む組換えＤＮＡを導入した形質転換体は、炭素源、窒素源、無機イオン、更に必要ならば有機栄養源を含む適当な培地で培養することにより変異型リパーゼを発現させることができる。 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．変異型リパーゼの利用
　本発明の変異型リパーゼと油脂と水を反応させることによって、脂肪酸とグリセロールが生成する（加水分解反応）。また、変異型リパーゼ、油脂、アルコールを反応させることによって、脂肪酸エステルとグリセロールが生成する（エステル交換反応）。本発明の変異型リパーゼは、野生型リパーゼに比べて安定性が高いので、油脂を加水分解して得られるグリセロール、脂肪酸を効率よく生成させることができる。
　油脂のエステル交換反応及び加水分解反応は工業的にも重要な反応であり、多くの報告がある（Fukuda, H., Kondo, A., and Noda, H. 2001, J. Biosci. Bioeng. 92, 405-416; Selmi, B. and Thomas, D. 1998. J. Am. Oil Chem. Soc. 75. 691-695; Mittelbach, M. 1990. J. Am. Oil Chem. Soc. 67. 168-170; Rooney, D. and Weartherley, L. R. 2000. Proc. Biochem. 36. 947-953; Noor, I. M. et al. 2003. Proc. Biochem. 39. 13-20）。当業者であれば通常用いられる条件で反応を行うことができる。
　また、本発明の変異型リパーゼの反応最適温度は37℃であり、この温度近辺で反応を行うことが好ましいが、野生型リパーゼよりも安定性が高いので、より高い温度、例えば37～60℃、好ましくは37～50℃で反応を行うこともできる。反応時間は、通常５～９６時間、好ましくは５～１２０時間が挙げられる。 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.
The transesterification and hydrolysis of fats and oils are industrially important reactions, and there are many reports (Fukuda, H., Kondo, A., and Noda, H. 2001, J. Biosci. Bioeng. 92 , 405-416; Selmi, B. and Thomas, D. 1998. J. Am. Oil Chem. Soc. 75. 691-695; Mittelbach, M. 1990. J. Am. Oil Chem. Soc. 67. 168- 170; Rooney, D. and Weartherley, LR 2000. Proc. Biochem. 36. 947-953; Noor, IM et al. 2003. Proc. Biochem. 39. 13-20). A person skilled in the art can carry out the reaction under conditions usually used.
Further, the optimum reaction temperature of the mutant lipase of the present invention is 37 ° C., and it is preferable to carry out the reaction at around this temperature. The reaction can also be preferably carried out at 37 to 50 ° C. The reaction time is usually 5 to 96 hours, preferably 5 to 120 hours.
　リパーゼを作用させる油脂としては、動物（魚類を含む）及び植物由来の油脂の１種または２種以上がすべて使用可能であり、例えば、パーム油、オリーブ油、菜種油、大豆油、米糠油、クルミ油、ゴマ油、ツバキ油、ピーナッツ油等の植物油、バター、豚脂、牛脂、羊脂、鳥脂、鶏油等の動物油、クジラ油、イワシ油、ニシン油、タラ肝油等の魚油などが挙げられるが、これらに限定されるものではない。また、油脂は固形油脂でもよい。さらに、油脂原料は、純粋な油脂であってもよいし、油脂以外の物質を含む混合物であってもよい。例えば、油脂が植物由来のものである場合は、油脂を含む植物抽出物又はその分画物が挙げられる。 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.
　アルコールとしては、メタノール等の低級アルコールのほか各種のアルコールが使用可能であり、例えば、メチルアルコール、エチルアルコール、プロピルアルコール、イソプロピルアルコール、ブチルアルコール、イソブチルアルコール、Ｓ－ブチルアルコール、ｔ－ブチルアルコール、アルミアルコール、ヘキシルアルコール等脂肪族アルコール；アリルアルコール、プロパルギルアルコール等の不飽和脂肪族アルコール；シクロヘキサノール、シクロペンタノール等の脂環式アルコール；ベンジルアルコール、シンナミルアルコール等の芳香族アルコール；その他の各種アルコールなどが挙げられるが、これらに限定されるものではない。 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.
　生成されるグリセロールや脂肪酸は、腸内細菌科に属する細菌を培養するための培地の炭素源として使用することができる。油脂のリパーゼとの反応生成物には、細菌の生育を大きく損なう不純物が含まれておらず、生成される脂肪酸やグリセロールを精製せずに、培養に利用することが可能である。また、油脂の加水分解反応は、油脂と水から、脂肪酸とグリセロールを生成する反応であり、室温付近の温度においては、グリセロールが溶解した水相である下層と、脂肪酸を含む油相である上層に分離しているのが、一般的である。水相に生じるグリセロール、及び、油相に生じる脂肪酸のいずれもが発酵原料として利用可能である。 Glycerol and fatty acids produced 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.
　油脂の加水分解物としてグリセロール及び脂肪酸を含むものを用いる場合は、油脂の加水分解物を乳化処理することが好ましい。乳化処理としては、乳化促進剤添加、攪拌、ホモジナイズ、超音波処理等が挙げられる。乳化処理によって、細菌がグリセロール及び脂肪酸を資化しやすくなり、Ｌ－アミノ酸発酵がより有効になると考えられる。乳化処理は、Ｌ－アミノ酸生産能を有する細菌が、脂肪酸とグリセロールの混合物を資化しやすくする処理であれば、どのようなものでも構わない。例えば、乳化方法として、乳化促進剤や界面活性剤を加える等が考えられる。ここで乳化促進剤としては、リン脂質やステロールが挙げられる。また界面活性剤としては、非イオン界面活性剤では、ポリ（オキシエチレン）ソルビタンモノオレイン酸エステル（Tween 80）などのポリオキシエチレンソルビタン脂肪酸エステル、n-オクチルβ-D-グルコシドなどのアルキルグルコシド、ショ糖ステアリン酸エステルなどのショ糖脂肪酸エステル、ポリグリセリンステアリン酸エステルなどのポリグリセリン脂肪酸エステル等が挙げられる。両性イオン界面活性剤としては、アルキルベタインであるN,N-ジメチル-N-ドデシルグリシンベタインなどが挙げられる。これ以外にも、トライトンX-100（Triton X-100）、ポリオキシエチレン(20)セチルエーテル（Brij-58）やノニルフェノールエトキシレート（Tergitol NP-40）等の一般的に生物学の分野で用いられる界面活性剤が利用可能である。 When using oil and fat hydrolyzate containing glycerol and fatty acid, it is preferable to emulsify the oil and fat hydrolyzate. Examples of the emulsification treatment include emulsification accelerator addition, stirring, homogenization, ultrasonic treatment and the like. It is considered 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 L-amino acid-producing ability make it easy to 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. In addition, as the surfactant, in the nonionic surfactant, polyoxyethylene sorbitan fatty acid ester such as poly (oxyethylene) sorbitan monooleate (Tween チ ル 80), alkyl glucoside 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. In addition, Triton X-100 (Triton X-100), polyoxyethylene (20) cetyl ether (Brij-58) and nonylphenol ethoxylate (Tergitol NP-40) are generally used in the field of biology. Available surfactants are available.
　さらに、脂肪酸のような難溶解性物質の乳化や均一化を促進するための操作も有効である。この操作は、脂肪酸とグリセロールの混合物の乳化や均一化を促進する操作であれば、どのような操作でも構わない。具体的には、攪拌処理、ホモジナイザー処理、ホモミキサー処理、超音波処理、高圧処理、高温処理などが挙げられるが、攪拌処理、ホモジナイザー処理、超音波処理およびこれらの組合せがより好ましい。 Furthermore, operations for promoting emulsification and homogenization of hardly soluble substances such as fatty acids are 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.
　上記乳化促進剤による処理と、攪拌処理、ホモジナイザー処理及び／または超音波処理を組み合わせることが特に好ましく、これらの処理は、脂肪酸がより安定なアルカリ条件下で行われることが望ましい。アルカリ条件としては、pH9以上が好ましく、pH10以上がより好ましい。 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 fatty acids are more stable. The alkaline condition is preferably pH 9 or higher, more preferably pH 10 or higher.
　グリセロール又は脂肪酸を炭素源とした発酵生産により出来る目的物質としては、Ｌ－アミノ酸（EP1715056、EP1715055、国際公開2007/013695号パンフレット）、有機酸（Dharmadi Y, Murarka A, Gonzalez R. 2006. Biotechnol Bioeng. 94:821-829.）、エタノール（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.）、水素（to T, Nakashimada Y, Senba K, Matsui T, Nishio N. 2005. J Biosci Bioeng. 100:260-255）等が挙げられる。尚、目的物質が塩を形成し得る場合は、その塩も目的物質に含まれる。 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). In addition, when the target substance can form a salt, the salt is also included in the target substance.
　Ｌ－アミノ酸は、Ｌ－アラニン、Ｌ－アルギニン、Ｌ－アスパラギン、Ｌ－アスパラギン酸、Ｌ－システイン、Ｌ－グルタミン酸、Ｌ－グルタミン、グリシン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－ロイシン、Ｌ－リジン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン及びＬ－バリン等を含む。特に、Ｌ－スレオニン、Ｌ－リジン、Ｌ－トリプトファン及びＬ－グルタミン酸が好ましい。 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.
　有機酸としては、コハク酸、クエン酸、フマル酸、リンゴ酸等を含むが、特にコハク酸が好ましい。
　エタノールとしては、エタノール、プロパノール、１，３－プロパンジオール等が挙げられる。
　発酵生産に使用できる微生物は、腸内細菌科に属する微生物、コリネ型細菌に属する微生物等が挙げられる。 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.
　腸内細菌科に属する微生物は、エシェリヒア、エンテロバクター、エルビニア、クレブシエラ、パントエア、フォトルハブドゥス、プロビデンシア、サルモネラ、セラチア、シゲラ、モルガネラ、イェルシニア等の属に属する細菌を含む。特に、NCBI（National Center for Biotechnology Information）のデータベース（http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=91347）で用いられている分類法により腸内細菌科に分類されている細菌が好ましい。 Microorganisms belonging to the family Enterobacteriaceae include bacteria belonging to genera such as Escherichia, Enterobacter, Erbinia, Klebsiella, Pantoea, Photorhabdus, Providencia, Salmonella, Serratia, Shigella, Morganella, and Yersinia. In particular, enterobacteria are identified by 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.
　エシェリヒア属に属する細菌とは、当該細菌が微生物学の専門家に知られている分類により、エシェリヒア属に分類されていることを意味する。本発明において使用されるエシェリヒア属に属する細菌の例としては、エシェリヒア・コリ（E.coli）が挙げられるが、これに限定されない。 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).
　本発明において使用することができるエシェリヒア属に属する細菌は、特に制限されないが、例えば、ナイトハルトらの著書（Neidhardt, F. C. Ed. 1996. Escherichia coli and Salmonella: Cellular and Molecular Biology/Second Edition pp. 2477-2483. Table 1. American Society for Microbiology Press, Washington, D.C.）に記述されている系統のものが含まれる。具体的には、プロトタイプの野生株Ｋ１２株由来のエシェリヒア・コリ W3110（ATCC 27325）、エシェリヒア・コリ MG1655（ATCC 47076）等が挙げられる。
　これらの菌株は、例えばアメリカン・タイプ・カルチャー・コレクション（住所　P.O. Box 1549 Manassas, VA 20108, United States of America）より分譲を受けることが出来る。すなわち各菌株に対応する登録番号が付与されており、この登録番号を利用して分譲を受けることが出来る。各菌株に対応する登録番号は、アメリカン・タイプ・カルチャー・コレクションのカタログに記載されている。 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 can be sold, for example, from 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.
　パントエア属に属する細菌とは、当該細菌が微生物学の専門家に知られている分類により、パントエア属に分類されていることを意味する。エンテロバクター・アグロメランスのある種のものは、最近、16S rRNAの塩基配列分析等に基づき、パントエア・アグロメランス、パントエア・アナナティス、パントエア・ステワルティイその他に再分類された（Int. J. Syst. Bacteriol., 43, 162173 (1993)）。本発明において、パントエア属に属する細菌には、このようにパントエア属に再分類された細菌も含まれる。 The bacterium belonging to the genus Pantoea means that the bacterium is classified into the genus Pantoea according to the classification known to microbiologists. Certain types of Enterobacter agglomerans have recently been reclassified as Pantoea agglomerans, Pantoea ananatis, Pantoea stewarty and others (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.
　コリネ型細菌としては、バージーズ・マニュアル・オブ・デターミネイティブ・バクテリオロジー（Bergey's Manual of Determinative Bacteriology）第8版599頁（1974）に定義されている一群の微生物であり、好気性，グラム陽性，非抗酸性，胞子形成能を有しない桿菌に分類される微生物が利用できる。なお、コリネ型細菌は、従来ブレビバクテリウム属に分類されていたが現在はコリネバクテリウム属細菌として統合された細菌（Int.J. Syst. Bacteriol., 41, 255 (1991)）、及びコリネバクテリウム属と非常に近縁なブレビバクテリウム属細菌及びミクロバテリウム属細菌を含む。 Coryneform bacteria are a group of microorganisms defined in the Bergey's （Manual 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.
　このようなコリネ型細菌の例として以下のものが挙げられる。
　コリネバクテリウム・アセトアシドフィラム
　コリネバクテリウム・アセトグルタミカム
　コリネバクテリウム・アルカノリティカム
　コリネバクテリウム・カルナエ
　コリネバクテリウム・グルタミカム
　コリネバクテリウム・リリウム
　コリネバクテリウム・メラセコーラ
　コリネバクテリウム・サーモアミノゲネス（コリネバクテリウム・エフィシェンス）
　コリネバクテリウム・ハーキュリス
　ブレビバクテリウム・ディバリカタム
　ブレビバクテリウム・フラバム
　ブレビバクテリウム・インマリオフィラム
　ブレビバクテリウム・ラクトファーメンタム
　ブレビバクテリウム・ロゼウム
　ブレビバクテリウム・サッカロリティカム
　ブレビバクテリウム・チオゲニタリス
　コリネバクテリウム・アンモニアゲネス
　ブレビバクテリウム・アルバム
　ブレビバクテリウム・セリヌム
　ミクロバクテリウム・アンモニアフィラム 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
　具体的には、下記のような菌株を例示することができる。
　コリネバクテリウム・アセトアシドフィラム ATCC13870
　コリネバクテリウム・アセトグルタミカム ATCC15806
　コリネバクテリウム・アルカノリティカム ATCC21511
　コリネバクテリウム・カルナエ ATCC15991
　コリネバクテリウム・グルタミカム ATCC13020, ATCC13032, ATCC13060
　コリネバクテリウム・リリウム ATCC15990
　コリネバクテリウム・メラセコーラ ATCC17965
　コリネバクテリウム・エッフィシエンス　AJ12340(FERM BP-1539)
　コリネバクテリウム・ハーキュリス ATCC13868
　ブレビバクテリウム・ディバリカタム ATCC14020
　ブレビバクテリウム・フラバム ATCC13826, ATCC14067, AJ12418(FERM BP-2205)
　ブレビバクテリウム・インマリオフィラム ATCC14068
　ブレビバクテリウム・ラクトファーメンタム ATCC13869（コリネバクテリウム・グルタミカムATCC13869）
　ブレビバクテリウム・ロゼウム ATCC13825
　ブレビバクテリウム・サッカロリティカム ATCC14066
　ブレビバクテリウム・チオゲニタリス ATCC19240
　コリネバクテリウム・アンモニアゲネス ATCC6871、ATCC6872
　ブレビバクテリウム・アルバム ATCC15111
　ブレビバクテリウム・セリヌム ATCC15112
　ミクロバクテリウム・アンモニアフィラム ATCC15354 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
　これらを入手するには、例えばアメリカン・タイプ・カルチャー・コレクションより分譲を受けることができる（住所　P.O. Box 1549, Manassas, VA 2010812301　Parklawn Drive, Rockville, Maryland 20852, United States of America）。すなわち、菌株毎に対応する登録番号が付与されており、この登録番号を利用して分譲を受けることができる。また、AJ12340株は、1987年10月27日付けで通商産業省工業技術院生命工学工業技術研究所（現独立行政法人産業技術総合研究所 特許生物寄託センター）（〒305-5466 日本国茨城県つくば市東１丁目１番地１　中央第６）にFERM　BP-1539の受託番号でブダペスト条約に基づいて寄託されている。また、AJ12418株は、1989年１月５日付けで通商産業省工業技術院生命工学工業技術研究所にFERM　BP-2205の受託番号でブダペスト条約に基づいて寄託されている。 To obtain these, you can obtain sales from the American Type Culture Collection (address: P.O. 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 Technology, Ministry of International Trade and Industry (currently National Institute of Advanced Industrial Science and Technology (AIST)) It has been deposited in accordance with the Budapest Treaty under the accession number of FERM BP-1539 at Tsukuba City Higashi 1-chome 1 1 Central 6). In addition, AJ12418 stock was deposited on January 5, 1989 to the Ministry of International Trade and Industry, Institute of Industrial Science, Biotechnology Institute of Technology, under the accession number of FERM BP-2205 under the Budapest Treaty.
　本発明に用いる細菌は、グリセロールの資化性を高めるために、glpR遺伝子（EP1715056）の発現が弱化されているか、glpA、glpB、glpC、glpD、glpE、glpF、glpG、glpK、glpQ、glpT、glpX、tpiA、gldA、dhK、dhaL、dhaM、dhaR、fsa及びtalC遺伝子等のグリセロール代謝遺伝子（EP1715055A、国際公開2007/013695号パンフレット）の発現が強化されていてもよい。 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.
　目的物質を産生する微生物は、グリセロール又は脂肪酸を炭素源として含む培地で培養したときに目的物質を産生するものであれば特に制限されない。例えば、Ｌ－アミノ酸を生産する微生物としては、特開2005-261433号公報（米国特許出願公開第20050214911A1号）に記載の微生物及び菌株が挙げられる。尚、Ｌ－アミノ酸生産能を付与又は増強する方法も、これらの公報に開示されている。 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. For example, microorganisms that produce L-amino acids include microorganisms and strains described in JP-A-2005-261433 (US Patent Application Publication No. 20050214911A1). Incidentally, methods for imparting or enhancing L-amino acid producing ability are also disclosed in these publications.
　Ｌ－アミノ酸生産菌の一例として、Ｌ－リジン生産菌、Ｌ－スレオニン生産菌、Ｌ－トリプトファン生産菌について以下に例示する。
　エシェリヒア属に属するＬ－リジン生産菌の例としては、Ｌ－リジンアナログに耐性を有する変異株が挙げられる。Ｌ－リジンアナログはエシェリヒア属に属する細菌の生育を阻害するが、この阻害は、Ｌ－リジンが培地に共存するときには完全にまたは部分的に解除される。Ｌ－リジンアナログの例としては、オキサリジン、リジンヒドロキサメート、Ｓ－（２－アミノエチル）－Ｌ－システイン(AEC)、γ－メチルリジン、α－クロロカプロラクタムなどが挙げられるが、これらに限定されない。これらのリジンアナログに対して耐性を有する変異株は、エシェリヒア属に属する細菌を通常の人工変異処理に付すことによって得ることができる。Ｌ－リジンの生産に有用な細菌株の具体例としては、Escherichia coli AJ11442 (FERM BP-1543, NRRL B-12185; 米国特許第4,346,170号参照)及びEscherichia coli VL611が挙げられる。これらの微生物では、アスパルトキナーゼのＬ－リジンによるフィードバック阻害が解除されている。 As examples of L-amino acid-producing bacteria, L-lysine-producing bacteria , L-threonine-producing bacteria, and L-tryptophan-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.
　WC196株は、Escherichia coliのＬ－リジン生産菌として使用できる。この菌株は、E.coli K-12に由来するW3110株から取得された株で、352位のスレオニンをイソロイシンに置換することによりＬ－リジンによるフィードバック阻害が解除されたアスパルトキナーゼIIIをコードする変異型lysC遺伝子（米国特許第5,661,012号）でW3110株の染色体上の野生型lysC遺伝子を置き換えた後、AEC耐性を付与することにより育種された。同株は、Escherichia coli AJ13069と命名され、1994年12月6日、工業技術院生命工学工業技術研究所（現 独立行政法人 産業技術総合研究所 特許生物寄託センター、〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に受託番号FERM P-14690として寄託され、1995年9月29日にブダペスト条約に基づく国際寄託に移管され、受託番号FERM BP-5252が付与されている(米国特許第5,827,698号)。 WC196 strain can be used as an L-lysine-producing bacterium of Escherichia coli. This strain was obtained from the W3110 strain derived from E. coli K-12, and encodes aspartokinase III in which feedback inhibition by L-lysine was released by replacing threonine at position 352 with isoleucine. After replacing the wild-type lysC gene on the chromosome of W3110 strain with a mutant lysC gene (US Pat. No. 5,661,012), it was bred by conferring AEC resistance. The stock was named Escherichia coli AJ13069. On December 6, 1994, Biotechnology Institute of Industrial Technology (currently National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center, 305-8566 茨 Ibaraki, Japan Deposited as FERM P-14690 at Tsukuba City Higashi 1-chome 1-1 1 Chuo 6), transferred to international deposit based on the Budapest Treaty on September 29, 1995, and assigned FERM BP-5252 (US Pat. No. 5,827,698).
　Ｌ－リジン生産菌又はそれを誘導するための親株の例としては、Ｌ－リジン生合成系酵素の１種又は２種以上の活性が増強されている株も挙げられる。かかる酵素の例としては、ジヒドロジピコリン酸シンターゼ(dapA)、アスパルトキナーゼ(lysC)、ジヒドロジピコリン酸レダクターゼ(dapB)、ジアミノピメリン酸デカルボキシラーゼ(lysA)、ジアミノピメリン酸デヒドロゲナーゼ(ddh) (米国特許第6,040,160号)、フォスフォエノールピルビン酸カルボキシラーゼ(ppc)、アスパルテートセミアルデヒドデヒドロゲナーゼ遺伝子、ジアミノピメリン酸エピメラーゼ(dapF)、テトラヒドロジピコリン酸スクシニラーゼ(dapD)、スクシニルジアミノピメリン酸デアシラーゼ(dapE)及びアスパルターゼ(aspA) (EP 1253195 A)が挙げられるが、これらに限定されない。これらの酵素の中では、ジヒドロジピコリン酸レダクターゼ、ジアミノピメリン酸デカルボキシラーゼ、ジアミノピメリン酸デヒドロゲナーゼ、フォスフォエノールピルビン酸カルボキシラーゼ、アスパラギン酸アミノトランスフェラーゼ、ジアミノピメリン酸エピメラーゼ、アスパルテートセミアルデヒドデヒドロゲナーゼ、テトラヒドロジピコリン酸スクシニラーゼ、及び、スクシニルジアミノピメリン酸デアシラーゼが特に好ましい。また、親株は、エネルギー効率に関与する遺伝子(cyo) (EP 1170376 A)、ニコチンアミドヌクレオチドトランスヒドロゲナーゼをコードする遺伝子(pntAB) (米国特許第5,830,716号)、ybjE遺伝子(WO2005/073390)、または、これらの組み合わせの発現レベルが増大していてもよい。 Examples of L-lysine-producing bacteria or parent strains for inducing them 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. The parent strain is a gene involved in energy efficiency (cyo) (EP 1170376 A), a gene encoding nicotinamide nucleotide transhydrogenase (pntAB) (US Patent No. 5,830,716), ybjE gene (WO2005 / 073390), or The expression level of these combinations may be increased.
　Ｌ－リジン生産菌又はそれを誘導するための親株の例としては、Ｌ－リジンの生合成経路から分岐してＬ－リジン以外の化合物を生成する反応を触媒する酵素の活性が低下または欠損している株も挙げられる。Ｌ－リジンの生合成経路から分岐してＬ－リジン以外の化合物を生成する反応を触媒する酵素の例としては、ホモセリンデヒドロゲナーゼ、リジンデカルボキシラーゼ(米国特許第5,827,698号)、及び、リンゴ酸酵素(WO2005/010175)が挙げられる。 Examples of L-lysine-producing bacteria or parent strains for deriving the same include reduction or loss of the activity of enzymes that catalyze reactions that branch off from the L-lysine biosynthetic pathway to produce compounds 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).
　好ましいＬ－リジン生産菌として、エシェリヒア・コリWC196ΔcadAΔldc/pCABD2が挙げられる（WO2006/078039）。この菌株は、リジンデカルボキシラーゼをコードするcadA及びldcC遺伝子が破壊されたWC196株に、米国特許第6040160に記載されたプラスミドpCABD2が導入することにより得られた株である。pCABD2は、Ｌ－リジンによるフィードバック阻害が解除された変異を有するエシェリヒア・コリ由来のジヒドロジピコリン酸合成酵素（DDPS）をコードする変異型dapA遺伝子と、Ｌ－リジンによるフィードバック阻害が解除された変異を有するエシェリヒア・コリ由来のアスパルトキナーゼIIIをコードする変異型lysC遺伝子と、エシェリヒア・コリ由来のジヒドロジピコリン酸レダクターゼをコードするdapB遺伝子と、ブレビバクテリウム・ラクトファーメンタム由来ジアミノピメリン酸デヒドロゲナーゼをコードするddh遺伝子を含んでいる。WC196ΔcadAΔldcCは、AJ110692と命名され、2008年10月7日に工業技術院生命工学工業技術研究所（現 独立行政法人 産業技術総合研究所 特許生物寄託センター、〒305-8566 日本国茨城県つくば市東１丁目１番地１ 中央第６）に国際寄託され、受託番号FERM BP-11027が付与されている。 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 that is desensitized to feedback inhibition by L-lysine, and a mutation that is desensitized to feedback inhibition by L-lysine. 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. WC196ΔcadAΔldcC was named AJ110692, and on October 7, 2008, the Institute of Biotechnology, National Institute of Advanced Industrial Science and Technology (currently the National Institute of Advanced Industrial Science and Technology (AIST), Patent Biological Deposit Center, 305-8566, Tsukuba City East, Ibaraki Prefecture, Japan It is deposited internationally at 1st Street, 1st Floor, Central 6th), and is given the accession number FERM BP-11027.
Ｌ－スレオニン生産菌
　Ｌ－スレオニン生産菌又はそれを誘導するための親株の例としては、E. coli TDH-6/pVIC40 (VKPM B-3996) (米国特許第5,175,107号、米国特許第5,705,371号)、E. coli 472T23/pYN7 (ATCC 98081) (米国特許第5,631,157号)、E. coli NRRL-21593 (米国特許第5,939,307号)、E. coli FERM BP-3756 (米国特許第5,474,918号)、E. coli FERM BP-3519及びFERM BP-3520 (米国特許第5,376,538号)、E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978))、E. coli VL643及びVL2055 (EP 1149911 A)などのエシェリヒア属に属する株が挙げられるが、これらに限定されない。 Examples of L-threonine-producing bacteria L-threonine-producing bacteria or parent strains for inducing them include E. coli TDH-6 / pVIC40 (VKPM B-3996) (US Pat. No. 5,175,107, US Pat. No. 5,705,371) E. coli 472T23 / pYN7 (ATCC 98081) (U.S. Pat.No. 5,631,157), E. coli NRRL-21593 (U.S. Pat.No. 5,939,307), E. coli FERM BP-3756 (U.S. Pat.No. 5,474,918), E. coli. coli FERM BP-3519 and FERM BP-3520 (US Pat.No. 5,376,538), E. coli MG442 (Gusyatiner et al., Genetika (in Russian), 14, 947-956 (1978)), E. coli VL643 and VL2055 Examples include, but are not limited to, strains belonging to the genus Escherichia, such as (EP 1149911 A).
　TDH-6株はthrC遺伝子を欠損し、スクロース資化性であり、また、そのilvA遺伝子がリーキー(leaky)変異を有する。この株はまた、rhtA遺伝子に、高濃度のスレオニンまたはホモセリンに対する耐性を付与する変異を有する。B-3996株は、RSF1010由来ベクターに、変異thrA遺伝子を含むthrA*BCオペロンを挿入したプラスミドpVIC40を保持する。この変異thrA遺伝子は、スレオニンによるフィードバック阻害が実質的に解除されたアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードする。B-3996株は、1987年11月19日、オールユニオン・サイエンティフィック・センター・オブ・アンチビオティクス(Nagatinskaya Street 3-A, 117105 Moscow, Russia)に、受託番号RIA 1867で寄託されている。この株は、また、1987年4月7日、ルシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオルガニズムズ(VKPM)に、受託番号B-3996で寄託されている。 The TDH-6 strain lacks the thrC gene, is sucrose-utilizing, and the ilvA gene has a leaky mutation. This strain also has a mutation in the rhtA gene that confers resistance to high concentrations of threonine or homoserine. The B-3996 strain carries the plasmid pVIC40 in which the thrA * BC operon containing the mutated thrA gene is inserted into the RSF1010-derived vector. This mutant thrA gene encodes aspartokinase homoserine dehydrogenase I which is substantially desensitized to feedback inhibition by threonine. B-3996 was deposited on 19 November 1987 at the All Union Scientific Center of Antibiotics (Nagatinskaya Street 3-A, 117105 Moscow, Russia) under the deposit number RIA 1867. . This strain was also deposited with the Lucian National Collection of Industrial Microorganisms (VKPM) on April 7, 1987 under the deposit number B-3996.
　E. coli VKPM B-5318 (EP 0593792B)も、Ｌ－スレオニン生産菌又はそれを誘導するための親株として使用できる。B-5318株は、イソロイシン非要求性であり、プラスミドpVIC40中のスレオニンオペロンの制御領域が、温度感受性ラムダファージC1リプレッサー及びPRプロモーターにより置換されている。VKPM B-5318は、1990年5月3日、ルシアン・ナショナル・コレクション・オブ・インダストリアル・マイクロオルガニズムズ(VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia)に、受託番号VKPM B-5318で寄託されている。 E. coli VKPM B-5318 (EP 0593792B) can also be used as an L-threonine producing bacterium or a parent strain for inducing it. The B-5318 strain is isoleucine non-required, and the control region of the threonine operon in the plasmid pVIC40 is replaced by a temperature sensitive lambda phage C1 repressor and a PR promoter. VKPM B-5318 was assigned to Lucian National Collection of Industrial Microorganisms (VKPM) (1 Dorozhny proezd., 1 Moscow 117545, Russia) on May 3, 1990 under the accession number VKPM B-5318. It has been deposited.
　好ましくは、本発明に用いる細菌は、さらに、下記の遺伝子の１種以上の発現が増大するように改変されたものである。
－スレオニンによるフィードバック阻害に耐性のアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードする変異thrA遺伝子
－ホモセリンキナーゼをコードするthrB遺伝子
－スレオニンシンターゼをコードするthrC遺伝子
－推定トランスメンブランタンパク質をコードするrhtA遺伝子
－アスパルテート－β－セミアルデヒドデヒドロゲナーゼをコードするasd遺伝子
－アスパルテートアミノトランスフェラーゼ（アスパルテートトランスアミナーゼ）をコードするaspC遺伝子 Preferably, the bacterium used in the present invention is further modified so that expression of one or more of the following genes is increased.
-A thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine-A thrB gene encoding homoserine kinase-A thrC gene encoding threonine synthase-A rhtA gene encoding a putative transmembrane protein-Aspartate- asd gene encoding β-semialdehyde dehydrogenase-aspC gene encoding aspartate aminotransferase (aspartate transaminase)
　E. coliのアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードするthrA遺伝子は明らかにされている(ヌクレオチド番号337～2799, GenBank accession NC_000913.2, gi: 49175990)。thrA遺伝子は、E. coli K-12の染色体において、thrL遺伝子とthrB遺伝子との間に位置する。E. coliのホモセリンキナーゼをコードするthrB遺伝子は明らかにされている(ヌクレオチド番号2801～3733, GenBank accession NC_000913.2, gi: 49175990)。thrB遺伝子は、E. coli K-12の染色体において、thrA遺伝子とthrC遺伝子との間に位置する。E. coliのスレオニンシンターゼをコードするthrC遺伝子は明らかにされている(ヌクレオチド番号3734～5020, GenBank accession NC_000913.2, gi: 49175990)。thrC遺伝子は、E. coli K-12の染色体において、thrB遺伝子とyaaXオープンリーディングフレームとの間に位置する。これら三つの遺伝子は、全て、単一のスレオニンオペロンとして機能する。スレオニンオペロンの発現を増大させるには、転写に影響するアテニュエーター領域を、好ましくは、オペロンから除去する(WO2005/049808, WO2003/097839)。 The thrA gene encoding aspartokinase homoserine dehydrogenase I of E. coli has been revealed (nucleotide numbers 337-2799, GenBank accession NC_000913.2, gi: 49175990). The thrA gene is located between the thrL gene and the thrB gene in the chromosome of E. coli K-12. The thrB gene encoding E. セ リ ン coli homoserine kinase has been elucidated (nucleotide numbers 2801 to 3733, GenBank accession NC_000913.2, gi: 99049175990). The thrB gene is located between the thrA gene and the thrC gene in the chromosome of E. coli K-12. The thrC gene encoding threonine synthase from E.coli has been elucidated (nucleotide numbers 3734 to 5020, GenBank accession NC_000913.2, gi: 49175990). The thrC gene is located between the thrB gene and the yaaX open reading frame in the chromosome of E. coli K-12. All three of these genes function as a single threonine operon. To increase the expression of the threonine operon, the attenuator region that affects transcription is preferably removed from the operon (WO2005 / 049808, WO2003 / 097839).
　スレオニンによるフィードバック阻害に耐性のアスパルトキナーゼホモセリンデヒドロゲナーゼＩをコードする変異thrA遺伝子、ならびに、thrB遺伝子及びthrC遺伝子は、スレオニン生産株E. coli VKPM B-3996に存在する周知のプラスミドpVIC40から一つのオペロンとして取得できる。プラスミドpVIC40の詳細は、米国特許第5,705,371号に記載されている。 The mutant thrA gene encoding aspartokinase homoserine dehydrogenase I resistant to feedback inhibition by threonine, and the thrB and thrC genes are one operon from the well-known plasmid pVIC40 present in the threonine producing strain E. coli VKPM B-3996. Can be obtained as Details of plasmid pVIC40 are described in US Pat. No. 5,705,371.
　rhtA遺伝子は、グルタミン輸送系の要素をコードするglnHPQ オペロンに近いE. coli染色体の18分に存在する。rhtA遺伝子は、ORF1 (ybiF遺伝子, ヌクレオチド番号764～1651, GenBank accession number AAA218541, gi:440181)と同一であり、pexB遺伝子とompX遺伝子との間に位置する。ORF1によりコードされるタンパク質を発現するユニットは、rhtA遺伝子と呼ばれている(rht: ホモセリン及びスレオニンに耐性)。また、rhtA23変異が、ATG開始コドンに対して-1位のG→A置換であることが判明している(ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A)。 The rhtA gene is present on the 18th minute of the E. 染色体 coli chromosome close to the glnHPQ operon, which encodes an element of the glutamine transport system. The rhtA gene is the same as ORF1 (ybiF gene, nucleotide numbers 764 to 1651, GenBank accession number AAA218541, gi: 440181), and is located between the pexB gene and the ompX gene. The unit that expresses the protein encoded by ORF1 is called rhtA gene (rht: resistant to homoserine and threonine). It has also been found that the rhtA23 mutation is a G → A substitution at position -1 relative to the ATG start codon (ABSTRACTS of the 17th International Congress of Biochemistry and Molecular Biology in conjugation with Annual Meeting of the American Society for Biochemistry and Molecular Biology, San Francisco, California August 24-29, 1997, abstract No. 457, EP 1013765 A).
　E. coliのasd遺伝子は既に明らかにされており(ヌクレオチド番号3572511～3571408, GenBank accession NC_000913.1, gi:16131307)、その遺伝子の塩基配列に基づいて作製されたプライマーを用いるPCRにより得ることができる(White, T. J., Arnheim, N., and Erlich, H. A. 1989. Trends Genet. 5: 185-189参照)。他の微生物のasd遺伝子も同様に得ることができる。 The E. coli asd gene has already been clarified (nucleotide numbers 3572511 to 3571408, GenBank accession NC_000913.1, gi: 16131307), and can be obtained by PCR using primers prepared based on the nucleotide sequence of the gene. (See White, T. J., Arnheim, N., and Erlich, H. A. 1989. Trends Genet. 5: 185-189). The asd gene of other microorganisms can be obtained similarly.
　また、E. coliのaspC遺伝子も既に明らかにされており(ヌクレオチド番号983742～984932, GenBank accession NC_000913.1, gi:16128895)、PCRにより得ることができる。他の微生物のaspC遺伝子も同様に得ることができる。

Ｌ－トリプトファン生産菌
　Ｌ－トリプトファン生産菌又はそれを誘導するための親株の例としては、変異trpS遺伝子によりコードされるトリプトファニル-tRNAシンテターゼが欠損したE. coli JP4735/pMU3028 (DSM10122)及びJP6015/pMU91 (DSM10123) (米国特許第5,756,345号)、セリンによるフィードバック阻害を受けないフォスフォグリセリレートデヒドロゲナーゼをコードするserAアレル及びトリプトファンによるフィードバック阻害を受けないアントラニレートシンターゼをコードするtrpEアレルを有するE. coli SV164 (pGH5) (米国特許第6,180,373号)、トリプトファナーゼが欠損したE. coli AGX17 (pGX44) (NRRL B-12263)及びAGX6(pGX50)aroP (NRRL B-12264) (米国特許第4,371,614号)、フォスフォエノールピルビン酸生産能が増大したE. coli AGX17/pGX50,pACKG4-pps (WO9708333, 米国特許第6,319,696号)などのエシェリヒア属に属する株が挙げられるが、これらに限定されない。yedA遺伝子またはyddG遺伝子にコードされるタンパク質の活性が増大したエシェリヒア属に属するＬ－トリプトファン生産菌も使用できる(米国特許出願公開2003/0148473 A1及び2003/0157667 A1)。 The aspC gene of E. coli has already been clarified (nucleotide numbers 983742 to 984932, GenBank accession NC — 000913.1, gi: 16128895), and can be obtained by PCR. The aspC gene of other microorganisms can be obtained similarly.

Examples of L-tryptophan-producing bacteria L-tryptophan-producing bacteria or parent strains for inducing them include E. coli JP4735 / pMU3028 (DSM10122) and JP6015 / pMU91 lacking the tryptophanyl-tRNA synthetase encoded by the mutant trpS gene (DSM10123) (U.S. Pat.No. 5,756,345), E. coli having a serA allele encoding phosphoglycerate dehydrogenase not subject to feedback inhibition by serine and a trpE allele encoding an anthranilate synthase not subject to feedback inhibition by tryptophan. SV164 (pGH5) (US Pat.No. 6,180,373), E. coli AGX17 (pGX44) (NRRL B-12263) and AGX6 (pGX50) aroP (NRRL B-12264) lacking tryptophanase (US Pat.No. 4,371,614) E. coli AGX17 / pGX50, pACKG4-pps (WO9708333, U.S. Pat.No. 6,319,696) with increased ability to produce phosphoenolpyruvate Strains include belonging to Erihia genus, but is not limited thereto. L-tryptophan-producing bacteria belonging to the genus Escherichia with increased activity of the protein encoded by the yedA gene or the yddG gene can also be used (US Patent Application Publications 2003/0148473 A1 and 2003/0157667 A1).
　Ｌ－トリプトファン生産菌又はそれを誘導するための親株の例としては、アントラニレートシンターゼ(trpE)、フォスフォグリセレートデヒドロゲナーゼ(serA)、及び、トリプトファンシンターゼ(trpAB)から選ばれる酵素の活性の一種以上が増大した株も挙げられる。アントラニレートシンターゼ及びフォスフォグリセレートデヒドロゲナーゼは共にＬ－トリプトファン及びＬ－セリンによるフィードバック阻害を受けるので、フィードバック阻害を解除する変異をこれらの酵素に導入してもよい。このような変異を有する株の具体例としては、脱感作型アントラニレートシンターゼを保持するE. coli SV164、及び、フィードバック阻害が解除されたフォスフォグリセレートデヒドロゲナーゼをコードする変異serA遺伝子を含むプラスミドpGH5 (WO 94/08031)をE. coli SV164に導入することにより得られた形質転換株が挙げられる。 Examples of L-tryptophan-producing bacteria or parent strains for inducing the same include anthranilate synthase (trpE), phosphoglycerate dehydrogenase (serA), and a kind of activity of an enzyme selected from tryptophan synthase (trpAB) Also included are strains with increased above. Since both anthranilate synthase and phosphoglycerate dehydrogenase are subject to feedback inhibition by L-tryptophan and L-serine, mutations that cancel the feedback inhibition may be introduced into these enzymes. Specific examples of strains having such mutations include E. coli SV164 carrying a desensitized anthranilate synthase and a mutant serA gene encoding phosphoglycerate dehydrogenase with desensitized feedback inhibition Examples include a transformant obtained by introducing the plasmid pGH5 (WO 94/08031) into E.coli SV164.
　Ｌ－トリプトファン生産菌又はそれを誘導するための親株の例としては、阻害解除型アントラニレートシンターゼをコードする遺伝子を含むトリプトファンオペロンが導入された株(特開昭57-71397号, 特開昭62-244382号, 米国特許第4,371,614号)も挙げられる。さらに、トリプトファンオペロン(trpBA)中のトリプトファンシンターゼをコードする遺伝子の発現を増大させることによりＬ－トリプトファン生産能を付与してもよい。トリプトファンシンターゼは、それぞれtrpA及びtrpB遺伝子によりコードされるα及びβサブユニットからなる。さらに、イソシトレートリアーゼ-マレートシンターゼオペロンの発現を増大させることによりＬ－トリプトファン生産能を改良してもよい(WO2005/103275)。
Examples of L-tryptophan-producing bacteria or parent strains for deriving the same include strains into which a tryptophan operon containing a gene encoding an inhibitory anthranilate synthase has been introduced (Japanese Patent Laid-Open Nos. 57-71397 and 57). 62-244382, US Pat. No. 4,371,614). Furthermore, L-tryptophan-producing ability may be imparted by increasing the expression of a gene encoding tryptophan synthase in the tryptophan operon (trpBA). Tryptophan synthase consists of α and β subunits encoded by trpA and trpB genes, respectively. Furthermore, L-tryptophan production ability may be improved by increasing the expression of the isocitrate triase-malate synthase operon (WO2005 / 103275).
　以下に実施例を挙げ、本発明をさらに詳しく説明する。本発明は、この実施例により何ら限定されない。 Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited in any way by this example.
〔実施例１〕リパーゼ遺伝子の大腸菌における発現
　Cryptococcus sp.S-2 (FERM BP-10961)の分泌シグナルを除いた成熟型リパーゼをコードする遺伝子（CS遺伝子）をpET-22b(+)ベクター（Novagen社製）のcloning/expression region の欄にあるMscI（pelB leader直後付近）とNotI（His・Tagの上流）で切断し、pelBleader直下に成熟型リパーゼの遺伝子を挿入した。
　CS遺伝子をpET-22b(+)ベクター（Novagen社製）のpelB leader直下へ挿入して、プラスミドpET-22b(+)_CLEを作製した。pelB遺伝子は、T7lacプロモーターによって制御され、イソプロピル-β-Ｄ-チオガラクトピラノシドで発現が誘導される。このpET-22b(+)_CLEを用いて、エシェリヒア・コリ OrigamiB(DE3)を形質転換し、100μg/ｍｌのアンピシリンを含むＬＢ寒天培地に植菌しアンピシリン耐性を指標として目的のプラスミドを有する株を選択した。なお、pET-22b(+)_CLEを保有するエシェリヒア・コリ OrigamiB(DE3)をpET-22b(+)_CLE/OrigamiB(DE3)株とも表す。 [Example 1] Expression of lipase gene in Escherichia coli A gene encoding a mature lipase (CS gene) excluding the secretion signal of Cryptococcus sp. S-2 (FERM BP-10961) is expressed in pET-22b (+) vector (Novagen Cleavage was performed with MscI (near pelB leader) and NotI (upstream of His • Tag) in the cloning / expression region column, and the mature lipase gene was inserted directly under pelBleader.
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)を、100μg/mlのアンピシリンを含むＬＢ培地にて25℃で18時間振盪培養した。この培養液を、100μg/mlのアンピシリンを含むＬＢ培地に適量加え、培養液の600nmにおける濁度（OD600）の値が1.0になるまで37℃で振盪培養した後、終濃度が0.5mMとなるようにイソプロピル-β-Ｄ-チオガラクトピラノシドを加え、25℃で18時間振盪培養した。得られた培養液中に含まれる菌体からの粗抽出液のエステラーゼ活性を実施例５の手順に従い測定し、粗抽出液がエステラーゼ活性を有することを確認した。また、pET-22b(+)_CLE/ OrigamiB(DE3)の代わりに、形質転換を行わないエシェリヒア・コリ OrigamiB(DE3)を用いて同様に培養し得られた菌体粗抽出液は、エステラーゼ活性を有しないことを確認した。 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.
〔実施例２〕変異型リパーゼの構築
　変異型リパーゼを構築するために、pET-22b(+)_CLEプラスミドを部位特異的突然変異誘発法の鋳型として使用した。突然変異誘発は、ＰＣＲ反応を利用する市販のポリメラーゼ（PrimeSTAR HS Polymerase, TaKaRa Bio,Inc..）を使用し、製造元のプロトコルに従い行った。各標的残基への部位特異的変異の導入には、各標的残基に対応するコドンを中央に含み、その前後に野生型リパーゼ配列に相当する15mer程度ずつを加えたプライマーを用いた。各プライマーの配列を表２に示す。 [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.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２中の各プライマーの配列中央部「XXX」は、導入したいアミノ酸残基の種類に応じて、対応するコドンの配列に置き換えて用いた。アミノ酸残基に対応する各コドンを、表３に示す。 The central part “XXX” of each primer in Table 2 was used by replacing it with the corresponding codon sequence according to the type of amino acid residue to be introduced. Table 3 shows each codon corresponding to an amino acid residue.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　変異導入ＰＣＲ後、DpnIで処理し鋳型の二本鎖ＤＮＡ（pET-22b(+)_CLEプラスミド）を消化したＰＣＲ産物を用いて、エシェリヒア・コリ JM109株を形質転換し、アンピシリン耐性を指標として、変異型リパーゼ遺伝子を含む目的のプラスミドを有する株を選択した。なお、変異型リパーゼ遺伝子を含む目的のプラスミドを包括的にpET-22b(+)_CLE_Mと命名した。形質転換株からpET-22b(+)_CLE_Mを単離し、エシェリヒア・コリOrigamiB(DE3)を形質転換した。pET-22b(+)_CLE_Mを保有するエシェリヒア・コリ OrigamiB(DE3)pET-22b(+)_CLE_M/OrigamiB(DE3)株とも表す。 After the mutation-introducing PCR, the PCR product obtained by treating with DpnI and digesting the template double-stranded DNA (pET-22b (+) _ CLE plasmid) was transformed into Escherichia coli JM109 strain, with 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 riOrigamiB (DE3) pET-22b (+) _ CLE_M / OrigamiB (DE3) strain carrying pET-22b (+) _ CLE_M.
　また、pET-22b(+)_CLE_Mについてその具体的な変異型を示す場合、pET-22b(+)_CLE_L181Fのように、「M」の部分を変異型の内容で置き換えて表示する場合がある。また、２種以上の変異を含む場合には変異型を「/」で区切り、各々の変異型を列挙する場合がある。例えば、pET-22b(+)_CLE_A50P/I125Sは、pET-22b(+)_CLEに搭載するリパーゼの遺伝子にA50PおよびC125Sの変異が導入されたものである。各プラスミドに目的の変異のみが導入されていることは、塩基配列決定により確認した。 In addition, when pET-22b (+) _ CLE_M indicates its specific mutation type, as in pET-22b (+) _ CLE_L181F, the “M” part may be replaced with the content of the mutation type and displayed. 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.
〔実施例３〕リパーゼの発現
　pET-22b(+)_CLE/OrigamiB(DE3)およびpET-22b(+)_CLE_M/OrigamiB(DE3)を、100μg/mlのアンピシリンを含むＬＢ培地にて37℃で16時間振盪培養した。この培養液を、100μg/mlのアンピシリンを含むＬＢ培地に適量加え、培養液の600nmにおける濁度（OD600）の値が1.0になるまで37℃で振盪培養した後、終濃度が0.5mMとなるようにイソプロピル-β-Ｄ-チオガラクトピラノシドを加え、25℃で18時間振盪培養した。 [Example 3] Expression of lipase pET-22b (+) _ CLE / OrigamiB (DE3) and pET-22b (+) _ CLE_M / OrigamiB (DE3) were added at 37 ° C. in LB medium containing 100 μg / ml ampicillin. Cultured with shaking for 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.
〔実施例４〕リパーゼ発現菌体からの粗抽出液の調製
　1.5mLチューブに実施例３で得られた培養液を1mL取り、14000gで1分間遠心分離した。上清を廃棄し、沈殿した菌体に400μlの非イオン界面活性剤を含む細胞溶解液(CelLytic B, Sigma)を加え、2分間ボルテックスした。室温で10分間インキュベーションした後、14000gで5分間遠心分離し、上清を粗抽出液として得た。リパーゼが粗抽出液に含まれていることは、SDS-PAGEで対応する分子量のバンドが存在することと、後に述べる方法で粗抽出液がエステラーゼ活性を有することから確認した。なお、エステラーゼ活性の測定を行う際、粗抽出液をPBS(-)あるいは100 mM 酢酸ナトリウム pH5.5で10倍に希釈して用いた。 [Example 4] Preparation of crude extract from lipase-expressing cells 1 mL of the culture solution obtained in Example 3 was placed 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.
〔実施例５〕リパーゼ発現菌体からの粗抽出液のエステラーゼ活性測定
　エステラーゼ活性は、ｐ-ニトロフェニルブチル酸を基質とした分光光度法により測定した。測定に用いる反応溶液として、体積比で4(w/w)% Triton X-100 52.6％、1M 酢酸バッファ（pH 5.5）10.5％、超純水36.9％からなる混合液に、終濃度が5.26mMとなるよう基質を溶かした溶液を調製した。反応液を95μLずつ96穴プレート(Nunc-Immuno Plate, 475094)に分注し、実施例４に記載した方法により得られる粗抽出液5μLを加えた。 [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.
　反応液を37℃、20分間インキュベートし、反応を停止させるために150μLずつアセトンを加えた。上記操作後、吸光度計（Micro plate reader）にて410nmの吸光度を測定した。
　100 mM 酢酸ナトリウム pH5.5で10倍希釈した粗抽出液を、60℃で20時間熱処理し、上記と同様にしてエステラーゼ活性を測定した。 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.
　変異型１から２０のリパーゼ粗抽出液の、熱処理前後におけるエステラーゼ活性の残存率を図２に示す。熱処理後、いずれの変異型リパーゼも、ｐ-ニトロフェニルブチル酸を基質として、野生型リパーゼよりも高い割合でエステラーゼ活性を保持した。中でも、変異型１１（C125S）は、６０℃で２０時間加熱処理を行っても高い活性保持率を示した。 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.
　60℃で20時間加熱処理した後の、野生型、変異型の残存エステラーゼ活性を図２に示す。野生型リパーゼより変異型リパーゼ、特に、Ｃ１２５Ｓが高い残存エステラーゼ活性を示した。 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.
　また、これらの変異型リパーゼの、野生型リパーゼに対する相対加水分解活性を図３に示す。 In addition, the relative hydrolytic activity of these mutant lipases with respect to wild-type lipase is shown in FIG.
　野生型リパーゼと変異型リパーゼが何れもエステル分解活性を有することから、以下に示す、野生型リパーゼで実施した油脂分解およびその分解物を用いたＬ－リジン発酵が、変異型リパーゼを用いた油脂分解物でも可能であると考えられる。 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 that fat and oil using mutant-type lipase It is thought that a decomposition product is also possible.
〔実施例６〕野生型リパーゼの精製
　実施例３で得られたpET-22b(+)_CLE/OrigamiB(DE3)の培養液から遠心機を用いて菌体を回収し、100mM リン酸バッファ（pH 7.0）で菌体を懸濁した後、再び菌体を回収した。回収した菌体を適量の100mM リン酸バッファ（pH 7.0）で懸濁した後、超音波破砕機（INSONATOR 201M、KUBOTA）にて180Wで25分間超音波破砕を行った。得られた液を15000rpmで20分間遠心し、上清を回収後、再び15000rpmで20分間遠心し上清を回収した後、得られた上清を0.22μmのフィルター（MILLEX-GV、MILLIPORE）で濾過した。その後、得られた濾過液を、ＦＰＬＣシステム（Akta explorer 10S、GE Healthcare Bio-Sciences）を用いた疎水相互作用クロマトグラフィーにて精製した。その具体的な手順を以下に示す。 [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.
　カラムには疎水相互作用カラム（HiLoad 16/10 Phenyl Sepharose High Performance、GE Healthcare Bio-Sciences）を用い、溶出液としてＡ液（100ｍＭ リン酸バッファ（pH 7.0））およびＢ液（エチレングリコール 80％、100ｍＭ リン酸バッファ（pH 7.0） 20％）を用いた。Ａ液75％、Ｂ液25％の組成で溶出液を20.1ml流してカラムを平衡化した後、前記の濾過液をＦＰＬＣシステムへインジェクトし、さらにＡ液75％、Ｂ液25％の組成で溶出液を90.5ml流した。続けて、溶出液を265.5ml流す間に溶出液の組成がＡ液75％、Ｂ液25％からＡ液42％、Ｂ液58％へと直線的に変化するように溶出液を流した後、さらに溶出液を20.1ml流す間に溶出液の組成がＡ液42％、Ｂ液58％からＡ液0％、Ｂ液100％へと直線的に変化するように溶出液を流し、続けてＢ液100%の組成で溶出液を36.2ml流した。得られた各画分をＳＤＳ－ＰＡＧＥにて分析し、目的のリパーゼのみ存在が確認された画分を回収し、実施例５の手順に従いエステラーゼ活性を有することを確認した。 The column used was a hydrophobic interaction column (HiLoad 16/10 Phenyl Sepharose High Performance, GE Healthcare Bio-Sciences), and as eluents solution A (100 mM phosphate buffer (pH 7.0)) and solution B (ethylene glycol 80%, 100 mM phosphate buffer (pH 7.0) 20%) was used. 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.
〔実施例７〕オリーブ油の加水分解反応物の調製
　実施例６で得られた精製酵素液の溶媒を、分画分子量10,000Daの遠心式濃縮フィルターユニット（Amicon Ultra-15 10k、MILLIPORE）を用いて100mM NaClを含む10mM 酢酸バッファ（pH 5.5）に置換し、0.37mMの酵素溶液を調製した。この酵素溶液11ｍｌとオリーブ油（Sigma-Aldrich）120mlと超純水120mlを1L三角フラスコに張り込み、30℃、180rpmの条件にて168時間振盪した。 [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.
〔実施例８〕オリーブ油のエステル交換反応物の調製
　実施例７と同じ酵素溶液11mlとオリーブ油（Sigma-Aldrich）120mlとメタノール（純正化学株式会社、特級試薬）1.64mlを1L三角フラスコに張り込み、30℃、180rpmの条件にて10時間振盪した。その後、メタノール1.64mlを添加し同条件で24時間振盪後、さらにメタノール1.64mlを添加し同条件で134時間振盪した。 [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.
〔実施例９〕油脂分解物を炭素源に用いたＬ-リジン生産培養
　Ｌ－リジン生産菌として国際公開第2006/078039号パンフレット記載のエシェリヒア・コリWC196ΔcadAΔldc/pCABD2 (以降、「WC196LC/pCABD2」と記載)を用いた。WC196LC/pCABD2を、ストレプトマイシン硫酸塩20mg/Lを含有するＬＢ寒天培地（トリプトン10ｇ/L、酵母エキス5ｇ/L、NaCl 10ｇ/L、寒天15ｇ/L）にて37℃で24時間培養した。寒天培地上の細胞を掻き取り、本培養培地20mlを張り込んだ500ml容坂口フラスコに植菌し、37℃で48時間培養を行った。本培養培地には、野生型のリパーゼを用いて、実施例７のオリーブ油の加水分解反応物、もしくは実施例８のオリーブ油のエステル交換反応物、またはグリセロール（ナカライテスク社　試薬特級）を炭素源として用いた。
　前記オリーブ油の加水分解反応物及びエステル交換反応物中のグリセロールは、いずれも固定化酵素電極法バイオセンサ（BF-5、王子計測機器株式会社）により測定した。測定されたグリセロールの量が40g/Lとなるように、各炭素源を培地に加えた。本培養培地の組成を以下に示す。 [Example 9] 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.
［本培養培地組成］
炭素源                      40g/L
酵母エキス                  2g/L
FeSO4・7H2O                 10mg/L
MnSO4・4H2O                 10mg/L
KH2PO4                      1.0g/L
MgSO4・7H2O                 1g/L
(NH4)2SO4                   24g/L
ストレプトマイシン硫酸塩    20mg/L [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
　培養終了後、培地に残存するグリセロールの濃度を固定化酵素電極法バイオセンサにて、生育度を600nmにおける濁度（OD600）にて、それぞれ測定した。Ｌ－リジン量をバイオテックアナライザー（AS210、サクラ精機）にて測定した。各炭素源につきフラスコ２本ずつ培養を行った。その結果の平均値を表４に示す。 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.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　本発明により、安定性に優れた変異型リパーゼが提供される。本発明の変異型リパーゼは、油脂の分解物の製造に好適に使用することができる。本発明により得られる油脂の分解物はグリセロール及び脂肪酸を含み、アミノ酸や核酸等の発酵生産における原料として使用することができる。 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.

Claims (16)

  1. 　配列番号７のアミノ酸配列、又は配列番号７において１若しくは数個のアミノ酸の置換、欠失、挿入、付加、又は逆位を含むアミノ酸配列を有し、かつ、下記（ａ）～（ｑ）から選択される変異を含む、変異型リパーゼ。
    （ａ）３２位のグリシンが他のアミノ酸残基に置換される変異
    （ｂ）５０位のチロシンが他のアミノ酸残基に置換される変異
    （ｃ）９５位のアラニンが他のアミノ酸残基に置換される変異
    （ｄ）１２６位のリジンが他のアミノ酸残基に置換される変異
    （ｅ）１３１位のシステインが他のアミノ酸残基に置換される変異
    （ｆ）１３３位のバリンが他のアミノ酸残基に置換される変異
    （ｇ）１５３位のグリシンが他のアミノ酸残基に置換される変異
    （ｈ）１６４位のロイシンが他のアミノ酸残基に置換される変異
    （ｉ）７位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
    （ｊ）４２位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
    （ｋ）８３位のＸがバリンであって、このバリンが他のアミノ酸残基に置換される変異
    （ｌ）８８位のＸがグルタミンであって、このグルタミンが他のアミノ酸残基に置換される変異
    （ｍ）１５４位のＸがセリンであって、このセリンが他のアミノ酸残基に置換される変異
    （ｎ）１７７位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
    （ｏ）２０３位のＸがアラニンであって、このアラニンが他のアミノ酸残基に置換される変異
    （ｐ）２０６位のＸがリジンであって、このリジンが他のアミノ酸残基に置換される変異
    （ｑ）２０９位のＸがグリシンであって、このグリシンが他のアミノ酸残基に置換される変異     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
  2. 　前記（ａ）～（ｑ）の変異が、それぞれ下記（Ａ）～（Ｑ）の変異である、請求項１に記載の変異型リパーゼ。
    （Ａ）３２位のグリシンがアスパラギン酸に置換される変異
    （Ｂ）５０位のチロシンがアスパラギンに置換される変異
    （Ｃ）９５位のアラニンがアスパラギン酸に置換される変異
    （Ｄ）１２６位のリジンがイソロイシンに置換される変異
    （Ｅ）１３１位のシステインがセリン、トレオニン、イソロイシン、及びロイシンから選択されるアミノ酸残基に置換される変異
    （Ｆ）１３３位のバリンがリジンに置換される変異
    （Ｇ）１５３位のグリシンがプロリンに置換される変異
    （Ｈ）１６４位のロイシンがグルタミンに置換される変異
    （Ｉ）７位のアラニンがプロリンに置換される変異
    （Ｊ）４２位のアラニンがグルタミンに置換される変異
    （Ｋ）８３位のバリンがグルタミンに置換される変異
    （Ｌ）８８位のグルタミンがアスパラギンに置換される変異
    （Ｍ）１５４位のセリンがイソロイシンに置換される変異
    （Ｎ）１７７位のアラニンがプロリンに置換される変異
    （Ｏ）２０３位のアラニンがロイシンに置換される変異
    （Ｐ）２０６位のリジンがグルタミンに置換される変異
    （Ｑ）２０９位のグリシンがプロリンに置換される変異     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
  3. 　前記（ａ）～（ｑ）から選択される変異が、前記（ｄ）、（ｅ）、（ｆ）又は（ｏ）の変異である、請求項１に記載の変異型リパーゼ。 The mutant lipase according to claim 1, wherein the mutation selected from (a) to (q) is the mutation of (d), (e), (f), or (o).
  4. 　前記（ｄ）、（ｅ）、（ｆ）又は（ｏ）の変異が、前記（Ｄ）、（Ｅ）、（Ｆ）又は（Ｏ）の変異である、請求項３に記載の変異型リパーゼ。 The mutant lipase according to claim 3, wherein the mutation (d), (e), (f) or (o) is the mutation (D), (E), (F) or (O). .
  5. 　前記変異を有しないリパーゼが、下記（Ｉ）又は（ＩＩ）のタンパク質である、請求項１～５のいずれか一項に記載の変異型リパーゼ。
    （Ｉ）配列番号２、４、又は６に記載のアミノ酸配列を有するタンパク質。
    （ＩＩ）配列表の配列番号２、４、又は６に記載のアミノ酸配列において、１若しくは数個のアミノ酸の置換、欠失、挿入、付加、又は逆位を含むアミノ酸配列からなり、かつ、リパーゼ活性を有するタンパク質。     The mutant lipase according to any one of claims 1 to 5, wherein the lipase having no mutation is the following protein (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. 　請求項１～５のいずれか一項に記載の変異型リパーゼをコードするＤＮＡ。 DNA encoding the mutant lipase according to any one of claims 1 to 5.
  7. 　下記（ｉ）又は（ｉｉ）の塩基配列を有し、かつ、前記アミノ酸配列における変異に相当する変異を有する請求項６に記載のＤＮＡ。
    （ｉ）配列番号１、３、又は５に記載の塩基配列。
    （ｉｉ）配列表の配列番号１、３、又は５に記載の塩基配列又は該配列から調製され得るプローブと、ストリンジェントな条件下でハイブリダイズする配列。     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.
  8. 　請求項６又は７に記載のＤＮＡを含む形質転換微生物。 A transformed microorganism containing the DNA according to claim 6 or 7.
  9. 　前記形質転換微生物がエシェリヒア・コリである請求項６に記載の微生物。 The microorganism according to claim 6, wherein the transformed microorganism is Escherichia coli.
  10. 　請求項８または９に記載の形質転換微生物を培地中で培養し、培地中および／または微生物中に請求項１～５のいずれか一項に記載の変異型リパーゼを蓄積させることを特徴とする、変異型リパーゼの製造方法。 10. 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.
  11. 　請求項１～５に記載の変異型リパーゼを油脂に作用させて、グリセロールを生成させることを特徴とする、グリセロールの製造方法。 A method for producing glycerol, wherein the mutated lipase according to any one of claims 1 to 5 is allowed to act on fats and oils to produce glycerol.
  12. 　請求項１～５に記載の変異型リパーゼを油脂に作用させて、グリセロールを生成させ、生成したグリセロールを炭素源として添加した培地で、グリセロール資化能を有し、かつ、目的物質を産生する微生物を培養し、目的物質を培養物中から採取する、目的物質の製造方法。 A lipase produced by allowing the mutant lipase according to any one of claims 1 to 5 to act on oils and fats to produce glycerol, and having the produced glycerol 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.
  13. 　請求項１～５に記載の変異型リパーゼを油脂に作用させて、グリセロール及び脂肪酸を生成させ、生成したグリセロール及び脂肪酸を炭素源として添加した培地で、グリセロール及び脂肪酸の資化能を有し、かつ、目的物質を産生する微生物を培養し、目的物質を培養物中から採取する、目的物質の製造方法。 A medium in which the mutated lipase according to any one of claims 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.
  14. 　前記微生物が、コリネ型細菌、及び腸内細菌科に属する微生物から選択される細菌である請求項１３に記載の方法。 The method according to claim 13, wherein the microorganism is a bacterium selected from coryneform bacteria and microorganisms belonging to the family Enterobacteriaceae.
  15. 　前記目的物質がＬ－アミノ酸である、請求項１３又は１４に記載の方法。 The method according to claim 13 or 14, wherein the target substance is an L-amino acid.
  16. 　前記Ｌ－アミノ酸が、Ｌ－リジン、Ｌ－スレオニン、及びＬ－トリプトファンから選択される請求項１５に記載の方法。 The method according to claim 15, wherein the L-amino acid is selected from L-lysine, L-threonine, and L-tryptophan.
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CN109929765A (en) * 2019-03-20 2019-06-25 武汉大学 One plant of Cryptococcus and its exocellular polysaccharide and application
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