WO2010074209A1 - Lipase mutante et utilisation associée - Google Patents

Lipase mutante et utilisation associée 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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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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    • 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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  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention a pour objet une lipase mutante produite par l'introduction d'une mutation dans une lipase produite par un micro-organisme appartenant au genre Cryptococcus, Gibberella, Ustilago ou analogue, de sorte à augmenter la stabilité de l'enzyme. Un acide L-aminé tel que la L-lysine, la L-thréonine ou le L-tryptophane peut être produit grâce à un processus de fermentation utilisant, en tant que source de carbone, du glycérol ou analogue qui est produit par la mise en réaction de la lipase mutante avec un produit d'huile et de graisse.
PCT/JP2009/071553 2008-12-26 2009-12-25 Lipase mutante et utilisation associée WO2010074209A1 (fr)

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JP2008-331771 2008-12-26
JP2008331771A JP5574400B2 (ja) 2008-12-26 2008-12-26 変異型リパーゼとその応用

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WO2010074209A1 true WO2010074209A1 (fr) 2010-07-01

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109161538A (zh) * 2018-09-29 2019-01-08 云南师范大学 一种热稳性提高的脂肪酶突变体及其应用
CN109182299A (zh) * 2018-10-30 2019-01-11 福建师范大学 耐受过氧化氢的枯草芽胞杆菌脂肪酶突变体及其制备方法
CN109929765A (zh) * 2019-03-20 2019-06-25 武汉大学 一株隐球酵母及其胞外多糖与应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101500073B1 (ko) * 2013-04-23 2015-03-06 씨제이제일제당 (주) L-아르기닌 생산능이 향상된 코리네박테리움속 미생물 및 이를 이용한 l-아르기닌의 생산 방법

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001252072A (ja) * 2000-03-13 2001-09-18 Tax Adm Agency 酵母由来の新規リパーゼｃｓ２
JP2004073123A (ja) * 2002-08-20 2004-03-11 National Research Inst Of Brewing リパーゼｃｓ２遺伝子
WO2006078039A1 (fr) * 2005-01-18 2006-07-27 Ajinomoto Co., Inc. Micro-organismes produisant un acide amine l et procede permettant de produire un acide amine l

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001252072A (ja) * 2000-03-13 2001-09-18 Tax Adm Agency 酵母由来の新規リパーゼｃｓ２
JP2004073123A (ja) * 2002-08-20 2004-03-11 National Research Inst Of Brewing リパーゼｃｓ２遺伝子
WO2006078039A1 (fr) * 2005-01-18 2006-07-27 Ajinomoto Co., Inc. Micro-organismes produisant un acide amine l et procede permettant de produire un acide amine l

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EGMOND MR. ET AL.: "Fusarium solani pisi cutinase", BIOCHIMIE, vol. 82, no. 11, 2000, pages 1015 - 1021 *
KAZUO MASAKI: "Shinki Koso ni yoru Seibunkaisei Plastic no Bunkai", POLYMER PREPRINTS, JAPAN, vol. 55, no. 2, 2006, pages 5670 - 5671 *
MASAKI K. ET AL.: "Cutinase-like enzyme from the yeast Cryptococcus sp. strain S-2 hydrolyzes polylactic acid and other biodegradable plastics", APPL. ENVIRON. MICROBIOL., vol. 71, no. 11, 2005, pages 7548 - 7550 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109161538A (zh) * 2018-09-29 2019-01-08 云南师范大学 一种热稳性提高的脂肪酶突变体及其应用
CN109161538B (zh) * 2018-09-29 2021-10-15 云南师范大学 一种热稳性提高的脂肪酶突变体及其应用
CN109182299A (zh) * 2018-10-30 2019-01-11 福建师范大学 耐受过氧化氢的枯草芽胞杆菌脂肪酶突变体及其制备方法
CN109182299B (zh) * 2018-10-30 2021-12-31 福建师范大学 耐受过氧化氢的枯草芽胞杆菌脂肪酶突变体及其制备方法
CN109929765A (zh) * 2019-03-20 2019-06-25 武汉大学 一株隐球酵母及其胞外多糖与应用
CN109929765B (zh) * 2019-03-20 2020-10-13 武汉大学 一株隐球酵母及其胞外多糖与应用

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