WO1995014783A1 - Lipase gene and variant lipase - Google Patents

Lipase gene and variant lipase Download PDF

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Publication number
WO1995014783A1
WO1995014783A1 PCT/JP1994/001965 JP9401965W WO9514783A1 WO 1995014783 A1 WO1995014783 A1 WO 1995014783A1 JP 9401965 W JP9401965 W JP 9401965W WO 9514783 A1 WO9514783 A1 WO 9514783A1
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lipase
amino acid
ser
gene
leu
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PCT/JP1994/001965
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French (fr)
Japanese (ja)
Inventor
Tadashi Yoneda
Yoshiaki Miyota
Kei Ohno
Junji Sasuga
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Showa Denko K.K.
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Publication of WO1995014783A1 publication Critical patent/WO1995014783A1/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

Definitions

  • the present invention relates to a gene encoding a novel lipase useful in the fields of detergents, food processing, papermaking industry and the like, a nucleotide sequence thereof, and a mutation of a gene that changes the physical or chemical properties of the lipase and a mutant gene thereof It relates to the encoded variant lipase.
  • Lipase a lipolytic enzyme
  • a lipolytic enzyme is an enzyme for food processing for flavor formation in dairy products, a medical enzyme as a digestive agent, a diagnostic enzyme for measuring blood lipids, and the hydrolysis and modification of fats and oils. O It is widely used as an industrial enzyme for decomposing and removing lipid stains, especially as a component of detergent compositions.
  • Known lipase-producing microorganisms include the genus Eudomonas, the genus Alcaligenes, the genus Mucor, the genus Candida, and the genus Humicola. Among these, several have obtained lipase genes. Among them, a large number of lipase genes of microorganisms belonging to the genus Pseudomonas have been obtained.
  • Lipases secreted and produced by bacteria of the genus Pseudomonas are used in industrial fields such as detergents, food processing, and paper manufacturing. Lipases used in these fields need to be stable to operating conditions such as temperature, pH, oxygen, solvent, and pressure.
  • lipase as an adjuvant to be added to detergents to enhance its cleaning effect has high pH, temperature, oxygen, oxidizing agents and other additives in detergents in the alkaline region. Stability is required.
  • lipase LP novel lipase having excellent properties used in industrial fields such as detergents and having the amino acid sequence described in SEQ ID NO: 1, and has previously filed a patent.
  • An application has been filed (JP-A-6-38746). Purpose of the invention
  • An object of the present invention is to change the gene encoding the lipase LP, its nucleotide sequence, and the physical or chemical properties of the lipase LP to those more suitable for industrial use and the like.
  • An object of the present invention is to provide a mutant lipase and a gene whose nucleotide sequence has been changed so as to encode the mutant lipase. Disclosure of the invention
  • the present inventors are one of the bacteria belonging to the genus Pseudomonas which produces lipase LP. Obtained lipase gene from Pseudomonas mendocina SD702 deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology did. The Pseudomonas mendocina SD702 strain was deposited on May 1, 1992, at the depositary institution as described above, and has a deposit number of FERMP-129244.
  • the nucleotide sequence is changed so as to encode a mutant lipase in which one or more amino acids in the amino acid sequence of lipase LP have been substituted with another amino acid.
  • the modified gene was obtained.
  • various lipases were obtained by a usual separation method. Among these lipases, it was confirmed that the physical or chemical properties of lipase LP changed, and that mutant lipase more useful in industrial fields such as detergent applications could be obtained.
  • the present invention has been completed.
  • FIG. 1 shows a restriction map of plasmid pSDL23. Arrows in the figure indicate the positions of lipase structural genes. Detailed description of the invention
  • the amino acid substitution and the amino acid substituted mutant lipase are represented as follows. That is, the name of the original amino acid residue to be replaced by the mutation (three-letter notation), the position of substitution in the amino acid sequence, and the name of the amino acid residue after the substitution (three-letter notation). It is indicated by notation.
  • a mutant in which the glycine at position 18 has been replaced with alanine is denoted Gly18Ala.
  • a lipase-encoding gene can be isolated from chromosomal DNA by a known method such as colony hybridization method or formation of a clear zone on a plate medium.
  • a chromosomal DNA library is prepared.
  • a homologous oligonucleotide probe is prepared, and colony hybridization is carried out using the probe to obtain a gene encoding lipase. Can be isolated.
  • a chromosomal DNA library is prepared using a lipase-producing bacterium and cultured on agar containing a lipase substrate. Bacteria that have a chromosomal DNA fragment containing the lipase gene form a clear zone around the colony where the lipase is degraded by the substrate, and use that to encode the lipase. Can be isolated.
  • the amino acid sequence of lipase LP represented by SEQ ID NO: 1
  • Nucleotide sequence has been modified to encode lipase.
  • a gene containing a nucleotide sequence encoding lipase LP may be substituted with a nucleotide sequence that causes amino acid substitution at a target site in the amino acid sequence of lipase LP. Any method can be used as long as it can cause mutation.
  • Site-directed mutagenesis refers to a method in which a wild-type enzyme gene is transformed into type II and a synthetic oligonucleotide having a base substitution at the target site of mutation is used as a mutagen (. J. Zoller, M. Smith, "Method in Enzymology”). vol.100, R. Wu, L. Grossmann, K. Moldave ed., P468, Academic Press (1983)) and the Kunkel method (Kunkel, TA (1985) Proc. Natl. Acad. USA., 82, 488) and improved methods such as the gapped duplex method (Karmer, W.
  • a random mutation method in which amino acid substitution is performed without particularly specifying the target site of the mutation.
  • Several methods are known for this. For example, there is a method of introducing a plasmid into which a lipase gene has been incorporated into a mutagenized strain of Escherichia coli. For example, a method of performing a polymerase chain reaction (PCR) reaction in the presence of magnesium ions, manganese ions, etc.
  • PCR polymerase chain reaction
  • a mutagen such as nitrous acid, formic acid, hydrazine or the like
  • Myers, RM, Lerman, L.S., Maniatis, T. (1985) Science 229, 242-247 a mutagen
  • the method of causing mutation is not limited to these, and any other method may be used.
  • a method for obtaining the gene according to the present invention from a strain in which a desired mutation has occurred due to a natural or artificial mutation such as ultraviolet light or the like can be considered.
  • a lipase gene obtained by incorporating a lipase gene into an Escherichia coli vector such as a pUC system can be used.
  • the mutant lipase gene obtained as described above is introduced into a host bacterium.
  • a host bacterium For example, when a genus Pseudomonas is used as a host, a method for stably maintaining the gene outside the chromosome by using a broad host range plasmid such as RSF11010, or a method for replicating a gene that cannot be replicated in the host bacterium. And integrate it into the chromosome.
  • mutant lipase is secreted into the culture medium. Separation and purification of the mutant lipase from the culture can be performed according to a conventional method. For example, ammonium sulfate is added to the culture solution, the mutant lipase is roughly fractionated, ammonium sulfate is removed by dialysis, and then fractionated on a CM cellulose column until it becomes a single band by SDS polyacrylamide gel electrophoresis. Purify the mutant lipase.
  • the method for producing and purifying the mutant lipase is not limited to the above-mentioned method, but may be other methods.
  • the mutant lipase of the present invention can have a stable structure, enhanced Z or function, and improved properties such as thermostability and specific activity by changing its physical or chemical properties.
  • Structural stabilization involves changing amino acid residues in a direction that enhances hydrophobic interactions inside the structure, that reduces the positive charge of the surface layer, that stabilizes the secondary structure, and that stabilizes the loop structure. Is achieved by
  • Preferred positions of the amino acid sequence for stabilizing the structure are: 18, 31, 60, 79, 93, 104, 125, 145, 155, 164, 183, 20 3, 2 16, 2 23, 242, 246, 267, and 292.
  • substitutions include: Glyl8Ala; Asn31Asp; A1a60Va1; Gly79Pro; Thr93Val; le; Glyl25Gln; Glyl45Ser; Glyl55Ser; Alal64Ser; Phel83Tyr; Arg203Ser; Val216 Phe; Leu223Phe; Arg242Thr; Arg246His; Leu267Phe, Gly292Ser where at least one of the substitutions is made Is mentioned.
  • the enhancement of the function is to increase the positive charge near the active center, to reduce the height of the amino acid residue, to prevent modification of the loop structure, and to reduce the interaction of the loop structure. Achieved by changing the acid residue.
  • Preferred amino acid sequence positions for enhanced function are: 21, 25, 43, 75, 107, 142, 148, 156, 159, 198, 2110, 22 2, 2 24, 2 47, 2 57, 2 66, and 2 76.
  • substitutions include: Met21Leu; Asp25Leu; Asp25Arg; Asp43Asn; Ile75Leu; Val107Ala ; I lel 42 Leu; T hrl 48 Ala; Serl 56 Gly; Thrl 59 Gly; Serl 98 Lys; Thr 2 10 Ser; Leu 22 2 A la Met224Leu; Met247Leu; Met257Leu; Thr2666Val; Asp2776Ser substitution; At least one of them has been done.
  • substitution position is as follows.
  • amino acid sequence of the lipase having homology with the amino acid sequence described in SEQ ID NO: 1 is juxtaposed, while providing a deletion position as necessary so that the homology of the two amino acid sequences is maximized.
  • Pseudomonas mendocina SD702 strain was inoculated into 3 Oml of L medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride) and incubated at 37. After culturing for 1 ⁇ , the supernatant was removed by centrifugation to obtain bacterial cells. This was suspended in 5 ml of a 50 mM Tris-HCl buffer (pH 8) containing 0.4 M sodium chloride and 10 mM EDTA. To this, 2.5 mg of lysozyme and 0.25 mg of RNase A were added, and the cells were lysed by gently shaking at 37 ° C for 30 minutes. Thereafter, the mixture was heated at 60 ° C. for 10 minutes to completely solubilize.
  • L medium 1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride
  • the N-terminal amino acid sequence of the purified lipase was analyzed using an amino acid sequencer, and the result shown in SEQ ID NO: 3 was obtained. Based on this, an oligonucleotide probe represented by SEQ ID NO: 4 was synthesized using a DNA synthesizer. Thereto, ⁇ - 32 ⁇ - A ⁇ ⁇ and T 4 Porinuku Reochi Dokinaze was added, reacted at at 37, was radiolabeled.
  • n XSSC means sodium chloride / sodium citrate solution with a concentration of n times that of 1 XSSC.
  • the bacterial residue was washed in.
  • SDS sodium dodecyl sulfate
  • 5X Denhardts solution 0.1% icoico11, 0.1% polyvinylpyrrolidone, 0.1% BSA
  • One shot was performed at 37 ° C for 1 hour.
  • the above-mentioned radiolabeled oligonucleotide probe was added to the same solution, and the filter was subjected to hybridization for 1 hour at 37 ° C. Thereafter, the filter was washed with 4 XSSC for 10 minutes, 2 XSSC twice for 20 minutes, and 1
  • This fragment was recovered, ligated to the EcoRI site of plasmid PUC119, and transformed into E. coli JMl01. This plasmid was recovered, digested with the restriction enzyme SacI, and subjected to Southern hybridization in the same manner. As a result, a 2.3 kbp fragment was obtained.
  • FIG. 1 shows a restriction map of plasmid PSDL23.
  • the Sanger dideoxy method (Sanger, F., Nicklen, S., Coulson, AR (1977) Pro Natl. Acad. Sci. USA., 74, 5463) Determined the nucleotide sequence of the lipase gene DNA. That is, the nucleotide sequence was analyzed by a DNA sequencer using ADI's didoxytermine overnight-sequencing kit. As a result, a nucleotide sequence encoding lipase such as SEQ ID NO: 2 was obtained. The amino acid sequence deduced from this sequence is shown in SEQ ID NO: 1.
  • the V a1 or Le u 223 Phe gene was constructed as follows. In order to convert amino acids in a partially specific manner, the oligonucleotides shown in SEQ ID NO: 5 and SEQ ID NO: 6 were chemically synthesized and phosphorylated at the 5 'end using T4 polynucleotide kinase before use. On the other hand, for the type I mutation, a plasmid pSDL23 (FIG. 1) was used. E.
  • Annealing buffer (2 OmM Tris-HCl buffer (pH 8), 10 mM magnesium chloride, 5 OmM sodium chloride, ImM DTT) was prepared by annealing the mutagenic oligonucleotide and type I DNA prepared as described above. The mixture was allowed to stand at 65 ° C for 15 minutes and then at 37 ° C for 15 minutes to allow the mutagenic oligonucleotide to anneal to the target site of the mutation.
  • the DN A mixture of 2.5 volumes of Extension buffer (50 mM T ris HC l, pH8.0, 6 OmM CHgCOONH ,. 5 mM M g C l 2, 5 m MD TT, I mM NAD, 0.5 m M dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP), and E. coli DNA ligase and T4 DNA polymerase.
  • Escherichia coli BMH71-18 mut S was transformed with the DNA, and ampicillin-resistant colonies were selected. Plasmid DNA was prepared from several of the above transformants, the nucleotide sequence was determined by the dideoxy method as in Example 5, and converted to codons corresponding to the target amino acid residues. It was confirmed.
  • the DNA fragment containing the mutant lipase gene portion was fractionated by agarose gel electrophoresis, and extracted and purified from agarose gel.
  • pM FY42 which is a broad host range vector, is completely digested with SacI, it is mixed with the DNA fragment containing the mutant lipase gene portion purified as described above, and mixed with T4 DNA ligase.
  • a ligation reaction was performed to transform Escherichia coli JMl01 strain, and a kanamycin-resistant colony was selected.
  • Plasmid DNA was extracted, purified, and analyzed from these transformants to obtain a plasmid in which a SacI DNA fragment containing a mutant lipase gene was inserted into the SacI cleavage site of PMFY42.
  • Example 7 Preparation of lipase
  • the lipase-deficient strain LD9 (Pseudomonas mendocina) SD702 was used.
  • a mutant strain obtained by reacting with N-methyl-N, mono-nitro-N-nitrosoguanidine, culturing on a plate medium containing a lipase substrate, and selecting a strain that does not form a clear zone) was obtained. Transformation was carried out by the kanamycin method, and kanamycin-resistant colonies were selected. That is, first, 9 strains of LD were grown in 5 ml of L medium until OD 0.7.
  • This transformant was shaken at 35 ° C for 14 hours in 30 Om1 of a lipase production medium containing 1% Tween 80 and adjusted to PH9 with sodium carbonate. After culturing, the mutant lipase was secreted and produced in the medium. The culture was centrifuged, and the supernatant was taken to prepare a crude enzyme solution. This was subjected to ammonium sulfate fractionation, and after removing ammonium sulfate by dialysis, treated with a CM cellulose column and purified by SDS polyacrylamide gel electrophoresis until a single band was obtained.
  • the number of molecules of the mutant lipase due to the site-specific mutation purified in Example 7 was determined.
  • Activity was measured at a constant absorbance of 280 nm and compared to wild-type lipase. The activity was measured by the following method.
  • p NPP p-nitrophenyl palmitate
  • isopropyl alcohol a concentration of 2 mgZm1.
  • This pNPP solution and 100 mM Bicine buffer (pH 8.0) are mixed at a ratio of 1:10 to prepare a substrate solution.
  • Enzyme solution 201 in 500 ⁇ l substrate solution After reacting at room temperature for 1 to 10 minutes, add 1N hydrochloric acid (200 1) to stop the reaction, and measure the absorbance at 405 nm using a spectrophotometer.
  • Table 1 shows specific activity values when the activity of the wild-type enzyme is 100.
  • Example 9 Thermostability of mutant lipase
  • thermostability of the wild-type lipase and the mutants (Ala60Val and Leu223Phe) produced by the site-specific mutation prepared above were measured as follows.
  • the mutant lipase solution and the wild-type lipase solution having the same activity value were incubated in a 5 OmM borate buffer (pHIO) at 60 ° C for 10 minutes, and then the lipase activity was measured.
  • Table 1 shows the results when the activity of the wild-type enzyme before heat treatment was set to 100.
  • the gene of the present invention facilitates the production and modification of the lipase according to the present invention, that is, the wild-type lipase LP or its mutant lipase.
  • the modified lipase LP according to the present invention has a novel amino acid sequence, and its physical properties or chemical properties, such as an increase in heat stability or specific activity, are different from those of lipase LP depending on the field of use. It is possible to obtain a mutant lipase which has been changed so as to be compatible, and it is possible to provide a lipase useful in industrial fields such as detergents, food processing, and papermaking. Sequence Listing SEQ ID NO: 1
  • Organism name Pseudomonas mendocina; strain name: SD 7 0 2
  • Trp Tyr Gly lie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr
  • Sequence type nucleic acid
  • Organism name Pseudomonas mendocina Stock Name: SD 7 0 2
  • I9S J3S usy an nsq • iqi naq ⁇
  • Organism Shootmonas mendocina (Pseudomonas mendocina).
  • Sequence type nucleic acid
  • Sequence type nucleic acid
  • Sequence type nucleic acid

Abstract

A lipase gene isolated from a chromosome DNA of Pseudomonas mendocian SD702; a variant lipase gene obtained by the variation of the above gene; and a variant lipase coded for by the above variant gene and having physical or chemical properties changed thereby. The invention gene serves to facilitate production and modification of lipase LP or a variant lipase thereof. The modification of lipase LP permits production of a variant lipase that has a new amino acid sequence and is more suitable to industrial and other application than the lipase LP, thus providing a lipase useful in the fields of detergent, food processing, papermaking and so forth.

Description

明細: リパーゼ遺伝子及び変異体リパ―ゼ  Description: Lipase gene and mutant lipase
技術分野 Technical field
本発明は、 洗剤、 食品加工、 製紙工業等の分野で有用な新規リパーゼ をコードする遺伝子及びそのヌクレオチド配列、 さらにそのリパーゼの 物理的性質もしくは化学的性質を変化させる遺伝子の変異及びその変異 遺伝子によりコードされる変異体リパーゼに関する。 背景技術  The present invention relates to a gene encoding a novel lipase useful in the fields of detergents, food processing, papermaking industry and the like, a nucleotide sequence thereof, and a mutation of a gene that changes the physical or chemical properties of the lipase and a mutant gene thereof It relates to the encoded variant lipase. Background art
脂質分解酵素であるリパーゼは、 乳製品のフレーバー形成のための食 品加工用酵素、 消化剤としての医療用酵素、 血中脂質の測定のための診 断用酵素、 油脂の加水分解ゃ改質、 特に洗剤組成物の成分として脂質性 の汚垢を分解除去するための工業用酵素等として広範囲に使用されてい o  Lipase, a lipolytic enzyme, is an enzyme for food processing for flavor formation in dairy products, a medical enzyme as a digestive agent, a diagnostic enzyme for measuring blood lipids, and the hydrolysis and modification of fats and oils. O It is widely used as an industrial enzyme for decomposing and removing lipid stains, especially as a component of detergent compositions.
リパーゼを生産する微生物としては、 シユー ドモナス （ eudomonas) 属ヽ アルカリゲネス （Alcal igenes) 属ヽ ムコール （Mucor) 属、 キャン デイダ （Candida) 属、 フ ミコーラ （Humicola) 属などが知られている。 これらの中にはリパーゼ遺伝子が取得されているものがいくつかあるが、 中でもシュ一 ドモナス (Pseudomonas) 属に属する微生物のリパーゼ遺 伝子が数多く取得されている。 これまでに知られているものとしては、 シュ一 ドモナス · フラジ （Pseudomona fragi) (特開昭 62- 228279 、 特開平 2- 39890) 、 シユー ドモナス ' セパシァ （Pseudomonas cepacia) (特開平 3- 87187 、 特開平 3 - 47079) 、 シュ一 ドモナス · プチダ (Pseudomonas putida) ヽ シュー ドモナス · シユー ドアルカ リゲネス (Pseudomonas pseudoalcal igenes) (特開平 3 - 500845) 、 シユー ドモ ナス * ァェゾレギノサ (Pseudomonas aeruginosa (J. Gen. Microbio l . (1992) 138, 1325-1335) 、 シュ一 ドモナス s p . (Pseudomonas sp. ) 1 0 9 (J. Biol . Chem. (1991) 266, 18135-18140) 、 シユー ドモナス * ク、、ゾレマエ (Pseudomonas gluiae) (Appl. Envir. Microbiol. (1992) 58, 12, 3787-3791) がある。 Known lipase-producing microorganisms include the genus Eudomonas, the genus Alcaligenes, the genus Mucor, the genus Candida, and the genus Humicola. Among these, several have obtained lipase genes. Among them, a large number of lipase genes of microorganisms belonging to the genus Pseudomonas have been obtained. Known so far include Pseudomona fragi (JP-A-62-228279, JP-A-2-39890), Pseudomonas cepacia (JP-A-3-87187, JP-A-3-47079), Pseudomonas putida シ ュ ー Pseudomonas putuari lignes (Pseudomonas pseudoalcal igenes) (JP-A-3-500845), Pseudomonas aeruginosa (J. Gen. Microbiol. (1992) 138, 1325-1335), Pseudomonas sp. (Pseudomonas sp.) 1 0 9 (J. Biol. Chem. (1991) 266, 18135-18140), Pseudomonas gluiae (Appl. Envir. Microbiol. (1992) 58, 12, 3787-3791) .
シユー ドモナス (Pseudomonas) 属細菌が分泌生産する リパーゼは、 洗剤用、 食品加工用、 製紙工業用等の工業分野に用いられている。 これ らの分野で利用するリパーゼは、 温度、 P H、 酸素、 溶媒、 圧力などの 使用条件に対して安定であることが必要である。 特に、 洗剤に配合して その洗浄効果を高める 剤補助剤としてのリパーゼには、 アル力リ域で の p H、 温度、 酸素、 酸化剤をはじめとする洗剤中の添加物に対して高 い安定性が求められる。 この様な化学的に安定な特性を得る方法として、 酵素のァミノ酸配列を変えることが知られている。 その例としては、 特 開平 4-500608におけるプロテア一ゼ及び酸化剤に対する改良などがある。 本出願人は、 洗剤用等の工業分野に用いられる優れた特性を有し、 配 列番号 1に記載のァミノ酸配列を有する新規リパーゼ （以下リパーゼ L P と略記する。 ） を見出し、 先に特許出願を行っている （特開平 6-38746) 。 発明の目的  Lipases secreted and produced by bacteria of the genus Pseudomonas are used in industrial fields such as detergents, food processing, and paper manufacturing. Lipases used in these fields need to be stable to operating conditions such as temperature, pH, oxygen, solvent, and pressure. In particular, lipase as an adjuvant to be added to detergents to enhance its cleaning effect has high pH, temperature, oxygen, oxidizing agents and other additives in detergents in the alkaline region. Stability is required. As a method for obtaining such chemically stable properties, it is known to change the amino acid sequence of the enzyme. Examples include improvements to proteases and oxidizing agents in JP 4-500608. The present applicant has found a novel lipase (hereinafter abbreviated as lipase LP) having excellent properties used in industrial fields such as detergents and having the amino acid sequence described in SEQ ID NO: 1, and has previously filed a patent. An application has been filed (JP-A-6-38746). Purpose of the invention
本発明の目的は、 上記リパーゼ L Pをコ一 ドする遺伝子及びそのヌク レオチ ド配列、 及びリパーゼ L Pの物理的性質もしく は化学的性質をェ 業用等の用途により適したものに変化させた変異体リパ一ゼ、 及び該変 異体リパーゼをコ一ドするようにヌクレオチド配列を変更した遺伝子を 提供することにある。 発明の開示 An object of the present invention is to change the gene encoding the lipase LP, its nucleotide sequence, and the physical or chemical properties of the lipase LP to those more suitable for industrial use and the like. An object of the present invention is to provide a mutant lipase and a gene whose nucleotide sequence has been changed so as to encode the mutant lipase. Disclosure of the invention
本発明者等は、 上記の目的を達成するために、 まずリパーゼ L Pを生 産するシユ ー ドモナス属細菌の 1つであり、 本出願人により日本国通商 産業省工業技術院生命工学工業技術研究所（National Inst itute of Bioscience and Human-Technology, Agency of Industrial Science and Techno l ogy) に寄託さ れてい る シュ 一 ドモナス · メ ン ドシナ (Pseudomonas mendocina) S D 7 0 2株に由来する リパーゼ遺伝子を 取得 した。 なお、 シユ ー ドモ ナ ス · メ ン ド シ ナ （Ps eudomona s mendocina) S D 7 0 2株は、 1992年 5月 1日付で上記寄託機関に寄託 され F E R M P— 1 2 9 4 4なる受託番号が付与され、 1993年 5月 1 2日付で上記寄託機関における 「特許手続上の微生物の寄託の国際的承 認に関するブダぺスト条約」 （BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSE OF PATENT PPROCEDURE)に基づく国際寄託に移管され、 F E R M B P— 4 2 9 1なる受託番号が付与されている。  In order to achieve the above object, the present inventors are one of the bacteria belonging to the genus Pseudomonas which produces lipase LP. Obtained lipase gene from Pseudomonas mendocina SD702 deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology did. The Pseudomonas mendocina SD702 strain was deposited on May 1, 1992, at the depositary institution as described above, and has a deposit number of FERMP-129244. The Budapest Treaty on the International Recognition of the Deposit of the Microorganism for the Purchasing of the International Recognition of the Deposit of Microorganisms in Patent Procedures was granted on May 12, 1993, (PATENT PPROCEDURE) and have been assigned a deposit number of FERMBP—4291.
更に、 S D 7 0 2株由来遺伝子の特異的な変異により、 リパーゼ L P のァミノ酸配列の 1個以上のァミノ酸を他のァミノ酸に置換した変異体 リパーゼをコ一ドするようにヌクレオチド配列が変更された遺伝子を取 得した。 この遺伝子を宿主菌に導入し、 得られた形質転換体を培養した 後、 通常の分離方法により各種のリパーゼを得た。 これらのリパーゼの 中に、 リパーゼ L Pの物理的性質もしく は化学的性質が変化し、 洗剤用 途等の工業用分野でより有用な変異体リパ一ゼが得られるこ.とを確認し、 本発明を完成するに至った。  Furthermore, due to a specific mutation in the gene derived from the strain SD702, the nucleotide sequence is changed so as to encode a mutant lipase in which one or more amino acids in the amino acid sequence of lipase LP have been substituted with another amino acid. The modified gene was obtained. After introducing this gene into a host bacterium and culturing the resulting transformant, various lipases were obtained by a usual separation method. Among these lipases, it was confirmed that the physical or chemical properties of lipase LP changed, and that mutant lipase more useful in industrial fields such as detergent applications could be obtained. The present invention has been completed.
すなわち、 本発明は、  That is, the present invention
1 ) 配列番号 1に記載されたリパーゼをコ一ドする配列番号 2に記載 されたヌクレオチ ド配列の全部もしく は一部を含む遺伝子、 2) 配列番号 2に記載されたヌクレオチド配列の一部を含む前記 1記 載の遺伝子、 1) a gene that encodes the lipase described in SEQ ID NO: 1 and includes all or part of the nucleotide sequence described in SEQ ID NO: 2; 2) the gene of 1 above, which comprises a portion of the nucleotide sequence set forth in SEQ ID NO: 2;
3) 遺伝子が、 配列番号 2に記載されたヌクレオチ ド配列の全部を含 むリパーゼ遺伝子である前記 1記載の遺伝子、  3) the gene according to 1 above, wherein the gene is a lipase gene containing the entire nucleotide sequence of SEQ ID NO: 2;
4) 配列番号 1に記載されたアミノ酸配列のうち少なく とも一箇所の ァミノ酸残基が他のァミノ酸残基で置換された変異体リパーゼ、 4) a mutant lipase in which at least one amino acid residue in the amino acid sequence described in SEQ ID NO: 1 has been substituted with another amino acid residue;
5) 配列番号 1に記載されたァミノ酸配列もしく はそのァミノ酸配列 と少なく とも 5 0%以上の相同性を有するアミノ酸配列における次の位 置 ： 1 8、 2 1、 2 5、 3 1、 43、 60、 75、 79、 93、 104、 1 0 7、 1 25、 1 42、 1 45、 1 48、 1 55、 1 5 6、 1 5 9、 1 64、 1 8 3、 1 9 8、 2 0 3、 2 1 0、 2 1 6、 22 2、 2 2 3、 224、 242、 246、 24 7、 2 5 7、 2 6 6、 2 6 7、 2 7 6、 および 2 92のうちの少なく とも一箇所のァミノ酸残基が他のァミノ酸 残基に置換された変異体リパーゼ、 5) The amino acid sequence described in SEQ ID NO: 1 or the following position in the amino acid sequence having at least 50% homology with the amino acid sequence: 18, 21, 25, 31 , 43, 60, 75, 79, 93, 104, 107, 125, 142, 145, 148, 155, 156, 159, 164, 183, 198 , 203, 211, 216, 222, 223, 224, 242, 246, 247, 2557, 26, 26, 27, and 292 A mutant lipase in which at least one amino acid residue has been replaced with another amino acid residue;
6) 次の置換： G 1 y 1 8 A 1 a ； M e t 2 1 L e u ； A s p 2 5 L e u ； A s p 25 A r g ； A s n 3 1 A s p ； A s p 4 3 A s n ； A 1 a 6 0 V a 1 ； I l e 75 L e u ; G l y 7 9 P r o ; T h r 9 3 V a 1 ； V a 1 1 04 I l e ; V a l l 0 7 A l a ; G l y l 2 5 G l n ; I l e l 42 L e u ; G l y l 45 S e r ; T h r l 4 8 A l a ; G l y l 5 5 S e r ; S e r l 5 6 G l y ; T h r l 5 9 G l y ; A l a l 64 S e r ； P h e l 8 3 T y r ； S e r l 9 8 L y s ； A r g 2 0 3 S e r ； T h r 2 1 0 S e r ； V a l 2 1 6 P h e ； L e u 222 A 1 a ； L e u 22 3 P h e ； M e t 2 2 4 L e u ； A r g 2 42 T h r ； A r g 246 H i s ； M e t 24 7 L e u ； M e t 2 5 7 L e u ； T h r 2 6 6 V a 1 ； L e u 2 6 7 P h e ； A s p 2 7 6 S e r ； および G i y 2 9 2 S e rのうちの少なく とも 1つを含む前記 5記載の変異体リ 八0—ゼヽ 6) The following substitutions: G1y18A1a; Met21Leu; Asp25Leu; Asp25Arg; Asn31Asp; Asp43Asn; A 1 a60Va1; Ile75Leu; Gly79Pro; Thr93Va1; Va1104Ile; Vall07Ala; Glyl25Gln Glyl 45 Ser; Thrl 48 Ala; Glyl 55 Ser; Serl 56 Gly; Thrl 59 Gly; Alal 64 Ser; Phel 8 3 Tyr; Serl98 Lys; Arg203Ser; Thr210Ser; Val216Phe; Leu222A1a; Leu223Phe; M Agr 246His; Met247 Leu; Met257 Leu; Thr266Va1; Leu267P et 2 24 Leu; he; Asp2776Ser; and Giy292Ser, the mutant strain according to the above 5, which comprises at least one of the following: Eighty- zero
7) 前記 4乃至 6のいずれかに記載の変異体リパ一ゼをコードするヌ クレオチド配列の全部もしくは一部を含む遺伝子、  7) a gene comprising all or part of a nucleotide sequence encoding the mutant lipase according to any of 4 to 6 above,
8) 配列番号 1に記載されたァミノ酸配列における次の位置： 1 8、 2 1、 2 5、 3 1、 4 3、 6 0、 7 5、 7 9、 9 3、 1 04、 1 0 7、 8) The following positions in the amino acid sequence described in SEQ ID NO: 1: 18, 21, 25, 31, 43, 60, 75, 79, 93, 104, 107 ,
1 2 5、 1 42、 1 4 5、 1 48、 1 5 5、 1 5 6、 1 5 9、 1 64、 1 8 3、 1 9 8、 2 0 3、 2 1 0、 2 1 6、 2 2 2、 2 2 3、 22 4、 242、 24 6、 24 7、 2 5 7、 2 6 6、 2 6 7、 2 7 6、 および 2 92のうちの少なく とも 1箇所のァミノ酸残基が他のァミ ノ酸残基に置 換されたリパーゼをコ一ドするように、 配列番号 2に記載されたヌクレ ォチド配列の対応する部位が変更された遺伝子、 および 1 2 5, 1 42, 1 4 5, 1 48, 1 5 5, 1 5 6, 1 5 9, 1 64, 1 8 3, 1 9 8, 2 0 3, 2 1 0, 2 1 6, 2 At least one of the amino acid residues of 2, 2, 2, 3, 224, 242, 246, 247, 25 7, 2 66, 2 67, 2 76, and 2 92 A gene in which the corresponding site in the nucleotide sequence set forth in SEQ ID NO: 2 has been changed so as to encode a lipase replaced with another amino acid residue; and
9) 配列番号 1に記載されたァミノ酸配列において次のアミノ酸残基 の置換： G l y l 8 A l a ； M e t 2 1 L e u ； A s p 2 5 L e u ； A s p 2 5 A r g ； A s n 3 1 A s p ； A s p 4 3 A s n ； A 1 a 6 0 V a 1 ； I l e 7 5 L e u ; G l y 79 P r o ； T h r 9 3 V a l ； V a 1 1 04 I l e ; V a l l 0 7 A l a ; G l y l 2 5 G l n ; I l e l 42 L e u ; G l y l 4 5 S e r ； T h r l 4 8 A l a ； G 1 y 1 5 5 S e r ; S e r l 5 6 G l y ; T h r l 5 9 G l y ; A l a l 64 S e r ； P h e l 8 3 T y r ； S e r l 9 8 L y s ； A r g 2 0 3 S e r ； T h r 2 1 0 S e r ； V a l 2 1 6 P h e ； L e u 22 2 A l a ； L e u 22 3 P h e ； M e t 224 L e u ； A r g 2 42 T h r ； A r g 2 46 H i s ； M e t 2 4 7 L e u ； M e t 2 5 7 L e u ； T h r 2 6 6 V a 1 ； L e u 2 6 7 P h e ； A s p 2 7 6 S e r ； および G l y 2 9 2 S e rのうちの少なく とも 1つがなされたリパーゼをコ一ドするよう に、 配列番号 2に記載されたヌクレオチ ド配列の対応する部位が変更さ れた前記に記載の遺伝子を提供するものである。 図面の簡単な説明 9) Substitution of the following amino acid residues in the amino acid sequence described in SEQ ID NO: 1: Glyl8Ala; Met21Leu; Asp25Leu; Asp25Arg; Asn A1a60Va1; Ile75Leu; Gly79Pro; Thr93Val; Va1104Ile; V All 0 7 A la; G lyl 25 G ln; Ilel 42 Leu; G lyl 45 Ser; T hrl 48 A la; G 1 y 1 55 Ser; Serl 56 G ly; T hrl 59 Gly; Alal 64 Ser; Phel 83 Tyr; Serl 98 Lys; Arg 203 Ser; Thr 210 Ser; Val 211 Pa He; M 224 L eu; Arg 242 T hr; Arg 246His; Met 247 L eu; M et 257 L eu; At least one of Thr266Va1, Leu267Phe, Asp276Ser; and Gly2992Ser should be encoded. The above-described gene wherein the corresponding site of the nucleotide sequence set forth in SEQ ID NO: 2 has been changed is provided. It is intended to. BRIEF DESCRIPTION OF THE FIGURES
図 1は、 プラスミ ド p S D L 2 3の制限酵素地図を示す。 図中の矢印 は、 リパーゼの構造遺伝子の位置を示す。 発明の詳細な説明  FIG. 1 shows a restriction map of plasmid pSDL23. Arrows in the figure indicate the positions of lipase structural genes. Detailed description of the invention
請求の範囲および明細書において、 ァミノ酸の置換およびァミノ酸が 置換された変異体リパーゼは次のように表される。 すなわち、 変異によ つて置換される元のァミノ酸残基の名称 （3文字表記） 、 ァミノ酸配列 内の置換される位置、 置換された後のアミノ酸残基の名称 （3文字表記） の順に表記することにより示される。 例えば、 1 8の位置のグリシンが ァラニンで置換された突然変異体は、 G l y l 8 A l aと表記される。 本発明において、 リパーゼをコー ドする遺伝子は、 コロニーハイプリ ダイゼ一ション法ゃ平板培地上でのク リァゾーンの形成法等の公知の方 法で染色体 D N Aから単離することができる。  In the claims and description, the amino acid substitution and the amino acid substituted mutant lipase are represented as follows. That is, the name of the original amino acid residue to be replaced by the mutation (three-letter notation), the position of substitution in the amino acid sequence, and the name of the amino acid residue after the substitution (three-letter notation). It is indicated by notation. For example, a mutant in which the glycine at position 18 has been replaced with alanine is denoted Gly18Ala. In the present invention, a lipase-encoding gene can be isolated from chromosomal DNA by a known method such as colony hybridization method or formation of a clear zone on a plate medium.
すなわち、 コロニーハイプリダイゼ一シヨ ン法では、 まず染色体 D N Aライブラリ一を作製する。 次に、 リバ一ゼの全部または一部のアミノ 酸配列が判っている場合は、 相同なォリゴヌクレオチ ドプローブを作製 し、 それを用いてコロニーハイブリダイゼーショ ンを行うことにより、 リパーゼをコ一ドする遺伝子を単離することができる。  That is, in the colony hybridization method, first, a chromosomal DNA library is prepared. Next, when the amino acid sequence of all or a part of the lipase is known, a homologous oligonucleotide probe is prepared, and colony hybridization is carried out using the probe to obtain a gene encoding lipase. Can be isolated.
あるいは、 ク リアゾーンの形成法では、 リパーゼを産生しない細菌で 染色体 D N Aライブラリーを作製し、 リパーゼの基質を含む寒天上で平 板培養する。 リパーゼ遺伝子を含む染色体 D N A断片を持つ細菌は、 そ のコロニーの回りに、 リパ一ゼが基質を分解するために生じるク リア ゾーンを形成するので、 それを利用してリパーゼをコ一ドする遺伝子を 単離することができる。  Alternatively, in the clear zone formation method, a chromosomal DNA library is prepared using a lipase-producing bacterium and cultured on agar containing a lipase substrate. Bacteria that have a chromosomal DNA fragment containing the lipase gene form a clear zone around the colony where the lipase is degraded by the substrate, and use that to encode the lipase. Can be isolated.
本発明において、 配列番号 1に記載されたリパーゼ L Pのアミノ酸配 列もしくはそのァミノ酸配列と少なく とも 50%以上の相同性を有する ァミノ酸配列の、 1個以上のァミノ酸を他のァミノ酸に置換した変異体 リパーゼをコ一ドするようにヌクレオチド配列が変更された遺伝子を得 る方法としては、 リパーゼ L Pをコ一 ドするヌクレオチ ド配列を含む遺 伝子に、 リパーゼ L Pのアミノ酸配列中の目的とする部位のアミノ酸の 置換を起こすようなヌクレオチ ド配列の変異を起こすことができるもの であれば如何なる方法を用いてもよい。 In the present invention, the amino acid sequence of lipase LP represented by SEQ ID NO: 1 Amino acid sequence having at least 50% or more homology with the sequence or its amino acid sequence, in which one or more amino acids have been substituted with another amino acid.Nucleotide sequence has been modified to encode lipase. As a method for obtaining a transcribed gene, a gene containing a nucleotide sequence encoding lipase LP may be substituted with a nucleotide sequence that causes amino acid substitution at a target site in the amino acid sequence of lipase LP. Any method can be used as long as it can cause mutation.
かかる変異を高頻度で起こさせる方法の一つに、 部位特異的変異法が ある。 部位特異的変異法とは野生型酵素遺伝子を铸型にして、 変異目的 部位に塩基置換を有する合成ォリゴヌク レオチ ドを変異源とする方法 ( . J. Zoller, M. Smith, "Method in Enzymology"vol.100, R. Wu, L. Grossmann, K. Moldave ed. , P468, Academic Press (1983)) であり、 クンケル （Kunkel) 法 （Kunkel, T. A. (1985) Proc. Natl. Acad. USA. , 82, 488) やギャ ップド ·デュプレックス （Gapped duplex) 法 （Karmer, W. et al (1984) Nucl. AcidsRes. , 12, 9441) などの改良法がある。 またポ リメラ一ゼ ' チェーン ' リアクショ ン （P C R) を用いる方法 （Mullis, K.， Faloona, F.， Scharf, S.， Saiki, R/. Horn, G. and Erlich, H.， (1986) Cold Spring Harber Symp. ： 263)などもある。  One of the methods for causing such mutations at a high frequency is a site-specific mutation method. Site-directed mutagenesis refers to a method in which a wild-type enzyme gene is transformed into type II and a synthetic oligonucleotide having a base substitution at the target site of mutation is used as a mutagen (. J. Zoller, M. Smith, "Method in Enzymology"). vol.100, R. Wu, L. Grossmann, K. Moldave ed., P468, Academic Press (1983)) and the Kunkel method (Kunkel, TA (1985) Proc. Natl. Acad. USA., 82, 488) and improved methods such as the gapped duplex method (Karmer, W. et al (1984) Nucl. Acids Res., 12, 9441). Methods using polymerase 'chain' reaction (PCR) (Mullis, K., Faloona, F., Scharf, S., Saiki, R /. Horn, G. and Erlich, H., (1986) Cold Spring Harber Symp .: 263)
また、 前記変異を起こさせる別の方法として、 変異目的部位を特に定 めずにアミノ酸の置換を起こすランダム変異法がある。 これにはいくつ かの方法が知られている。 例えば、 リパーゼ遺伝子を組み込んだプラス ミ ドを大腸菌の突然変異誘発株に導入する方法がある。 また、 例えば、 マグネシウムイオン、 マンガンイオン等の存在下でポリ メ ラ一ゼ · チェーン ' リアクション （P C R) 反応を行う方法 （Zhou, Y. , Zhang, X. , Ebright, R. H. (1991) Nucleic Acid Research 19, 6052, Tindall, K. R. , Kunkel, T. A. (1988) Biochemistry 27, 6008-6013) や、 修復機能の ない D N Aポリメラ一ゼを用いて D N A増幅反応を行う方法 （Liao, X. , Wise, J. A. (1990) Gene 107 - 111) がある。 As another method for causing the mutation, there is a random mutation method in which amino acid substitution is performed without particularly specifying the target site of the mutation. Several methods are known for this. For example, there is a method of introducing a plasmid into which a lipase gene has been incorporated into a mutagenized strain of Escherichia coli. For example, a method of performing a polymerase chain reaction (PCR) reaction in the presence of magnesium ions, manganese ions, etc. (Zhou, Y., Zhang, X., Ebright, RH (1991) Nucleic Acid Research 19, 6052, Tindall, KR, Kunkel, TA (1988) Biochemistry 27, 6008-6013) There is a method (Liao, X., Wise, JA (1990) Gene 107-111) in which a DNA amplification reaction is performed using a DNA polymerase.
更に、 例えば亜硝酸、 ギ酸、 ヒ ドラジン等の突然変異誘発剤を用いる 方法 (Myers, R. M. , Lerman, L. S. , Maniatis, T. (1985) Science 229, 242- 247) などがある。 変異を起こさせる方法はこれらに限られず他の如何 なる方法を用いてもよい。 例えば、 天然または人工的に紫外線などの突 然変異により、 所望の変異が起こった菌株から本発明に係る遺伝子を得 る方法も考えられる。  Further, for example, there is a method using a mutagen such as nitrous acid, formic acid, hydrazine or the like (Myers, RM, Lerman, L.S., Maniatis, T. (1985) Science 229, 242-247). The method of causing mutation is not limited to these, and any other method may be used. For example, a method for obtaining the gene according to the present invention from a strain in which a desired mutation has occurred due to a natural or artificial mutation such as ultraviolet light or the like can be considered.
変異のための銪型としては、 リパーゼ遺伝子を、 例えば p U C系のよ うな大腸菌のベクタ一に組み込んだものを用いることができる。 上記の ようにして得た変異体リパーゼ遺伝子を宿主菌に導入する。 例えば、 シユードモナス （Pseudomonas) 属細菌を宿主として用いる場合、 R S F 1 0 1 0等の広宿主域プラスミ ド等を用いて染色体外に安定に維持さ せる方法、 宿主菌内で複製できない形の遺伝子を用いて染色体内に組み 込む方法等がある。  As the type III for mutation, a lipase gene obtained by incorporating a lipase gene into an Escherichia coli vector such as a pUC system can be used. The mutant lipase gene obtained as described above is introduced into a host bacterium. For example, when a genus Pseudomonas is used as a host, a method for stably maintaining the gene outside the chromosome by using a broad host range plasmid such as RSF11010, or a method for replicating a gene that cannot be replicated in the host bacterium. And integrate it into the chromosome.
シユー ドモナス (Pseudomonas) 属細菌を宿主と して用いた場合、 変 異体リパーゼは培養液中に分泌される。 培養液からの変異体リパ一ゼの 分離精製は常法に従って行うことができる。 例えば、 培養液に硫安を加 え、 変異型リパーゼを粗分画し、 透析によって硫安を除去後、 C Mセル ロースカラムで分別することにより S D Sポリアク リルアミ ドゲル電気 泳動で単一なバン ドになるまで変異体リパーゼを精製する。 変異体リ パーゼの生産、 および精製法は、 上記方法に限定されるものではなく、 他の方法でも勿論可能である。  When Pseudomonas bacteria are used as a host, mutant lipase is secreted into the culture medium. Separation and purification of the mutant lipase from the culture can be performed according to a conventional method. For example, ammonium sulfate is added to the culture solution, the mutant lipase is roughly fractionated, ammonium sulfate is removed by dialysis, and then fractionated on a CM cellulose column until it becomes a single band by SDS polyacrylamide gel electrophoresis. Purify the mutant lipase. The method for producing and purifying the mutant lipase is not limited to the above-mentioned method, but may be other methods.
本発明の変異体リパーゼはその物理的性質もしくは化学的性質が変化 することにより、 構造が安定化し、 かつ Zまたは機能が強化され、 熱安 定性や比活性などの性質を向上させることができる。 構造の安定化は、 構造内部の疎水的相互作用を強める方向、 表層の正 の荷電を減ずる方向、 二次構造を安定化する方向、 ループ構造を安定化 する方向へアミノ酸残基を変化させることにより達成される。 The mutant lipase of the present invention can have a stable structure, enhanced Z or function, and improved properties such as thermostability and specific activity by changing its physical or chemical properties. Structural stabilization involves changing amino acid residues in a direction that enhances hydrophobic interactions inside the structure, that reduces the positive charge of the surface layer, that stabilizes the secondary structure, and that stabilizes the loop structure. Is achieved by
構造の安定化に好ましいアミノ酸配列の位置は、 1 8、 3 1、 6 0、 7 9、 9 3、 1 04、 1 2 5、 1 45、 1 5 5、 1 64、 1 8 3、 2 0 3、 2 1 6、 2 2 3、 242、 246、 2 6 7、 2 92である。  Preferred positions of the amino acid sequence for stabilizing the structure are: 18, 31, 60, 79, 93, 104, 125, 145, 155, 164, 183, 20 3, 2 16, 2 23, 242, 246, 267, and 292.
好ましい置換の具体例としては、 G l y l 8 A l a ; A s n 3 1 A s p ； A 1 a 6 0 V a 1 ； G l y 7 9 P r o ; T h r 9 3 V a l ； V a 1 1 04 I l e ; G l y l 2 5 G l n ; G l y l 4 5 S e r ; G l y l 5 5 S- e r ； A l a l 64 S e r ； P h e l 8 3 T y r ； A r g 2 0 3 S e r ； V a l 2 1 6 P h e ； L e u 223 P h e ； A r g 242 Th r ； A r g 2 4 6 H i s ; L e u 2 6 7 P h e, G l y 2 9 2 S e rの置換 のうち少なく とも 1つがなされているものが挙げられる。  Specific examples of preferred substitutions include: Glyl8Ala; Asn31Asp; A1a60Va1; Gly79Pro; Thr93Val; le; Glyl25Gln; Glyl45Ser; Glyl55Ser; Alal64Ser; Phel83Tyr; Arg203Ser; Val216 Phe; Leu223Phe; Arg242Thr; Arg246His; Leu267Phe, Gly292Ser where at least one of the substitutions is made Is mentioned.
また、 機能の強化は、 活性中心付近の正の荷電を増やす方向、 ァミノ 酸残基の崇高さを減ずる方向、 ループ構造の修飾を防ぐ方向、 ループ構 造の構造状の相互作用を減ずる方向ヘアミノ酸残基を変化させることに より達成される。  In addition, the enhancement of the function is to increase the positive charge near the active center, to reduce the height of the amino acid residue, to prevent modification of the loop structure, and to reduce the interaction of the loop structure. Achieved by changing the acid residue.
機能の強化に好ましいアミノ酸配列の位置は、 2 1、 2 5、 4 3、 7 5、 1 0 7、 1 4 2、 1 48、 1 5 6、 1 5 9、 1 9 8、 2 1 0、 22 2、 2 24、 2 4 7、 2 5 7、 2 6 6、 2 7 6である。  Preferred amino acid sequence positions for enhanced function are: 21, 25, 43, 75, 107, 142, 148, 156, 159, 198, 2110, 22 2, 2 24, 2 47, 2 57, 2 66, and 2 76.
好ましい置換の具体例としては、 M e t 2 1 L e u ; A s p 2 5 L e u ; A s p 2 5 A r g ; A s p 4 3 A s n ; I l e 7 5 L e u ; V a l 1 0 7 A l a ; I l e l 42 L e u ; T h r l 4 8 A l a ; S e r l 5 6 G l y ; T h r l 5 9 G l y ; S e r l 9 8 L y s ; T h r 2 1 0 S e r ； L e u 2 2 2 A l a ； M e t 224 L e u ； Me t 247 L e u ； M e t 2 5 7 L e u ; T h r 2 6 6 V a l ； A s p 2 7 6 S e rの置換 のうち少なく とも 1つがなされているものが挙げられる。 Specific examples of preferred substitutions include: Met21Leu; Asp25Leu; Asp25Arg; Asp43Asn; Ile75Leu; Val107Ala ; I lel 42 Leu; T hrl 48 Ala; Serl 56 Gly; Thrl 59 Gly; Serl 98 Lys; Thr 2 10 Ser; Leu 22 2 A la Met224Leu; Met247Leu; Met257Leu; Thr2666Val; Asp2776Ser substitution; At least one of them has been done.
なお、 上記のァミノ酸残基の置換を、 配列番号 1に記載のァミノ酸配 列と少なく とも 50 %以上の相同性を示すァミノ酸配列を有するリパー ゼに適用する場合、 その置換する位置は、 2つのアミノ酸配列の相同性 が最大になるように必要に応じて欠損位置を与えつつ、 配列番号 1に記 載のァミノ酸配列と相同性を有する該リパーゼのァミノ酸配列を並置し た場合に対応する位置を、 配列番号 1に記載のアミノ酸位置にて表した ものである。 発明を実施するための最良の形態 本発明を実施例を挙げて説明するが、 本発明は以下の実施例に限定さ れるものではない。  When the above substitution of an amino acid residue is applied to a lipase having an amino acid sequence having at least 50% homology with the amino acid sequence of SEQ ID NO: 1, the substitution position is as follows. When the amino acid sequence of the lipase having homology with the amino acid sequence described in SEQ ID NO: 1 is juxtaposed, while providing a deletion position as necessary so that the homology of the two amino acid sequences is maximized. Is represented by the amino acid position shown in SEQ ID NO: 1. BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described with reference to examples, but the present invention is not limited to the following examples.
実施例 1 ：染色体 DN Aの調製 Example 1: Preparation of chromosomal DNA
シュ一 ドモナス · メ ン ドシナ (Pseudomonas mendocina) S D 7 0 2 株を L培地 （ポリペプ ト ン 1 %、 酵母エキス 0.5%、 塩化ナ ト リ ウム 0.5%) 3 Om lに植菌し、 37でで1晚培養後、 遠心分離によって上 清を除去し、 菌体を得た。 これを 0.4M塩化ナト リ ウム、 1 0mM E DTAを含む 5 OmMト リス塩酸緩衝液 （p H 8) 5m 1に懸濁した。 これにリゾチーム 2.5m gと R N a s e A 0.25m gを加え、 37 °Cで 30分間穏やかに振とうし、 溶菌させた。 その後、 60°Cで 1 0分間加 温し、 完全に可溶化させた。 この溶液に T E緩衝液 （ 1 mM EDTA を含む 10 mMト リス塩酸緩衝液 （p H 8) ) で飽和させたフヱノール を等量加え、 穏やかに転倒混和し、 遠心分離によって上層の水層を回収 することを 3回繰り返した。 3回目に回収した水層に一 20°Cに冷やし たエタノールを 2倍量加え、 沈澱をプラスチック棒で巻取った。 この沈 澱をエタノールでリ ンスした後、 減圧乾燥し、 TE緩衝液 0.5m 1に再 溶解した。 Pseudomonas mendocina SD702 strain was inoculated into 3 Oml of L medium (1% polypeptone, 0.5% yeast extract, 0.5% sodium chloride) and incubated at 37. After culturing for 1 晚, the supernatant was removed by centrifugation to obtain bacterial cells. This was suspended in 5 ml of a 50 mM Tris-HCl buffer (pH 8) containing 0.4 M sodium chloride and 10 mM EDTA. To this, 2.5 mg of lysozyme and 0.25 mg of RNase A were added, and the cells were lysed by gently shaking at 37 ° C for 30 minutes. Thereafter, the mixture was heated at 60 ° C. for 10 minutes to completely solubilize. To this solution, add an equal volume of phenol saturated with TE buffer (10 mM Tris-HCl buffer (pH 8) containing 1 mM EDTA), mix gently by inversion, and collect the upper aqueous layer by centrifugation. Was repeated three times. To the third aqueous layer collected, twice the amount of ethanol cooled to 120 ° C was added, and the precipitate was wound up with a plastic rod. The precipitate was rinsed with ethanol, dried under reduced pressure, and reconstituted in 0.5 ml of TE buffer. Dissolved.
実施例 2 ：染色体 D N Aライブラリ一の作製 Example 2: Preparation of a chromosome DNA library
染色体 D NAを制限酵素 E c 0 R Iで部分分解した後、 フ ノール · クロ口ホルム抽出、 エタノール沈澱により DNA断片を回収した。 一方、 コスミ ド p WE 1 5を同様に E c o R Iで分解し、 アルカ リホスファ ターゼ処理を行った。 両者を T 4 D N Aリガーゼを用いて連結した。 こ れをファージ粒子にパッケージングした。 このファージを、 マルトース、 硫酸マグネシゥムを含む L培地で培養した大腸菌培養液 6 0 0 μ I に加 え、 3 7°Cで 1 5分間インキュベー トした後、 L培地を l m l になるよ うに加え、 3 7 °Cで 1時間インキュベー ト した。 これをアンピシリ ン 5 0 p p mを含む L平板培地 （L培地をァガロース 1.5%で固めたもの） に塗布し、 3 7°Cで 1晚培養した。 この結果、 約 1000コロニーの形質転 換体を得た。  After partially degrading the chromosomal DNA with the restriction enzyme Ec0RI, a DNA fragment was recovered by extraction with phenol / chloroform and ethanol precipitation. On the other hand, cosmid pWE15 was similarly degraded with EcoRI and treated with alkaline phosphatase. Both were ligated using T4DNA ligase. This was packaged into phage particles. Add the phage to 600 μl of E. coli culture cultured in L medium containing maltose and magnesium sulfate, incubate at 37 ° C for 15 minutes, and add L medium to lml. Incubated at 37 ° C for 1 hour. This was applied to an L plate medium (a L medium solidified with 1.5% agarose) containing 50 ppm of ampicillin, and cultured at 37 ° C for 1 晚. As a result, about 1000 transformants were obtained.
実施例 3 ：オリゴヌクレオチドプローブの作製 Example 3: Preparation of oligonucleotide probe
精製したリパーゼの N末端ァミノ酸配列をァミノ酸シーケンサーで分 折し、 配列番号 3に示す結果を得た。 これに基づいて配列番号 4で示す オリゴヌクレオチドプローブを D N A合成機で合成した。 これに、 Ί— 32Ρ— A Τ Ρおよび T 4ポリヌク レオチ ドキナーゼを加え、 3 7でで 1 時間反応させ、 放射能標識した。 The N-terminal amino acid sequence of the purified lipase was analyzed using an amino acid sequencer, and the result shown in SEQ ID NO: 3 was obtained. Based on this, an oligonucleotide probe represented by SEQ ID NO: 4 was synthesized using a DNA synthesizer. Thereto, Ί - 32 Ρ- A Τ Ρ and T 4 Porinuku Reochi Dokinaze was added, reacted at at 37, was radiolabeled.
実施例 4 ： リパーゼをコー ドする遺伝子の単離 Example 4: Isolation of a gene encoding lipase
約 1000コロニーの形質転換体を L平板培地上に置いた二トロセルロー スフィルタ一上で 3 7°Cで 1晚培養した。 フィルターを剥し、 0.5M水 酸化ナト リ ウム Z 1.5M塩化ナト リゥム溶液に浸したろ紙上で 1 0分間 溶菌させ、 1.5M塩化ナト リ ウム， 1 M ト リス塩酸緩衝液 （p H 7) で 浸したろ紙上で 7分間、 2回中和した。 フィルターを 8 0°Cで 2時間焼 成し、 0.5%ドデシル硫酸ナト リウム （S D S) / 6 X S S C ( 1 X S S Cは、 150 mM塩化ナトリウム Zl 5 mMクェン酸ナトリゥム溶液。 n X S S Cは 1 X S S Cの n倍の濃度の塩化ナト リゥム/クェン酸ナト リウム溶液を意味する。 ） 中で菌の残査を洗浄した。 0.1%ドデシル硫 酸ナトリウム （SD S) / 5 Xデンハルト溶液 （Denhardts solution) (0.1 % ί i c o 1 1、 0.1 %ポリ ビニルピロ リ ドン、 0.1 %B S A) / 5 x S S C中でフィルターのプレハイプリダイゼ一シヨ ンを 37°Cで 1時間行った。 同様の溶液中に上記の放射能標識したォリゴヌクレオチ ドプローブを加え、 フィルターのハイプリダイゼ一シヨ ンを 37°Cで 1 晚行った。 その後、 フィルターを 4 X S S Cで 1 0分間、 2 X S S Cで 20分間 2回、 1 X S S Cで 20分間 2回洗浄した。 About 1000 colonies of the transformant were cultured at 37 ° C for 1 晚 on a two-cellulose filter placed on an L plate medium. Remove the filter, lyse for 10 minutes on filter paper soaked in 0.5M sodium hydroxide Z 1.5M sodium chloride solution, and add 1.5M sodium chloride, 1M Tris-HCl buffer (pH 7). Neutralized twice on soaked filter paper for 7 minutes. The filter is calcined at 80 ° C for 2 hours, and 0.5% sodium dodecyl sulfate (SDS) / 6 XSSC (1 XS SC is 150 mM sodium chloride Zl 5 mM sodium citrate solution. n XSSC means sodium chloride / sodium citrate solution with a concentration of n times that of 1 XSSC. ) The bacterial residue was washed in. Prehybridization of the filter in 0.1% sodium dodecyl sulfate (SDS) / 5X Denhardts solution (0.1% icoico11, 0.1% polyvinylpyrrolidone, 0.1% BSA) / 5xSSC One shot was performed at 37 ° C for 1 hour. The above-mentioned radiolabeled oligonucleotide probe was added to the same solution, and the filter was subjected to hybridization for 1 hour at 37 ° C. Thereafter, the filter was washed with 4 XSSC for 10 minutes, 2 XSSC twice for 20 minutes, and 1 XSSC twice for 20 minutes.
このようなコロニーハイブリダィゼーショ ンの結果、 いくつかの陽性 コロニーを得た。 得られたコロニーのうちの 1つからコスミ ドを回収し、 E c 0 R Iで分解した断片をァガロース電気泳動で分離し、 二トロセル ロースフィルターに吸着させた。 コロニーハイプリダイゼーションと同 様の手順でサザンハイプリダイゼーシヨンを行った。 その結果、 18 k b pの断片にハイブリダイズした。  As a result of such colony hybridization, several positive colonies were obtained. Cosmid was recovered from one of the obtained colonies, the fragment digested with Ec0RI was separated by agarose electrophoresis, and adsorbed to a Nitrocellulose filter. Southern hybridization was performed in the same procedure as for colony hybridization. As a result, it hybridized to an 18 kbp fragment.
この断片を回収し、 プラスミ ド P UC 1 1 9の E c o R I部位に連結 し、 大腸菌 J Ml 01を形質転換した。 このプラスミ ドを回収し、 制限 酵素 S a c Iで分解し、 同様の手順でサザンハイプリダイゼ一ションを 行った。 その結果、 2.3k b pの断片を得た。  This fragment was recovered, ligated to the EcoRI site of plasmid PUC119, and transformed into E. coli JMl01. This plasmid was recovered, digested with the restriction enzyme SacI, and subjected to Southern hybridization in the same manner. As a result, a 2.3 kbp fragment was obtained.
実施例 5 ： リパーゼ遺伝子のヌクレオチド配列決定 Example 5: Nucleotide sequencing of lipase gene
実施例 4で得た 2.3k b pの断片を p U C 1 1 9の S a c I部位に連 結し、 大腸菌 J M 1 01を形質転換した。 このプラスミ ドを p S DL 2 3と命名した。 プラスミ ド P SDL 23の制限酵素地図を図 1に示す。 このプラス ミ ドを用いて、 サンガーのジデォキシ法 （Sanger, F., Nicklen, S. , Coulson, A. R. (1977) Pro Natl. Acad. Sci. USA. , 74， 5463) でリパーゼ遺伝子 DN Aのヌクレオチド配列を決定した。 すなわち、 A B I社のダイデォキシターミネ一夕一 · シーゲンシング ·キッ トを用い、 DNAシーケンサーによってヌクレオチ ド配列を解析した。 その結果、 配列番号 2のようなリパ一ゼをコードするヌクレオチ ド配列を得た。 こ の配列から推定されるァミノ酸配列を配列番号 1で示す。 The 2.3 kbp fragment obtained in Example 4 was ligated to the SacI site of pUC119 to transform Escherichia coli JM101. This plasmid was named pSDL23. FIG. 1 shows a restriction map of plasmid PSDL23. Using this plasmid, the Sanger dideoxy method (Sanger, F., Nicklen, S., Coulson, AR (1977) Pro Natl. Acad. Sci. USA., 74, 5463) Determined the nucleotide sequence of the lipase gene DNA. That is, the nucleotide sequence was analyzed by a DNA sequencer using ADI's didoxytermine overnight-sequencing kit. As a result, a nucleotide sequence encoding lipase such as SEQ ID NO: 2 was obtained. The amino acid sequence deduced from this sequence is shown in SEQ ID NO: 1.
実施例 6 ：部位特異的変異による突然変異体リパーゼ遺伝子の構築 Example 6: Construction of mutant lipase gene by site-directed mutation
リパーゼのァミノ酸残基を部位特異的に置換した変異体 （A 1 a 60 Mutants in which the amino acid residue of lipase was site-specifically substituted (A1a60
V a l もしく は L e u 223 P h e) の遺伝子を以下のように構築した。 部分特異的にァミノ酸を変換するため、 配列番号 5および配列番号 6 に示すオリゴヌクレオチドを化学合成し、 T 4ポリヌクレオチドキナー ゼを用いて 5' 末端をリ ン酸化して用いた。 一方、 変異の铸型は、 ブラ ス ミ ド p S D L 23 (図 1) を用いた。 p S D L 23を用いて大腸菌 B W31 3株 （H f r KL 1 6 POZ45 [ l y s A (61 - 62) ] ， d u t 1 , n u 1 , t h y— 1， r e 1 A 1 ) を形質転換し、 この形 質転換体から、 メ ッシングらの方法 （Viera.J. and Messing, J. (1987) Method in Enzymology, 153, 3-11) に従い 1本鎖 D N Aを調製し、 D N A中のデォキシチミ ンの一部がデォキシゥラシルに置き換わった一本 鎖 DNAを得、 これを铸型として用いた。 The V a1 or Le u 223 Phe) gene was constructed as follows. In order to convert amino acids in a partially specific manner, the oligonucleotides shown in SEQ ID NO: 5 and SEQ ID NO: 6 were chemically synthesized and phosphorylated at the 5 'end using T4 polynucleotide kinase before use. On the other hand, for the type I mutation, a plasmid pSDL23 (FIG. 1) was used. E. coli BW31 3 strain (H fr KL16 POZ45 [lysA (61-62)], dut1, nu1, thy-1 and re1A1) was transformed with pSDL23, Single-stranded DNA was prepared from the transformants in accordance with the method of Meshing et al. (Viera. J. and Messing, J. (1987) Method in Enzymology, 153, 3-11), and a part of the deoxythymine in the DNA was prepared. A single-stranded DNA was obtained, which was replaced with deoxyduracil, and this was used as type I.
上記のように調製した変異源ォリゴヌクレオチドおよび铸型 D N Aを ァニーリ ングバッフ ァー （2 OmMトリス塩酸緩衝液 （p H 8) 、 1 0 mM塩化マグネシウム、 5 OmM塩化ナト リウム、 I mM D TT) 中 で混合し、 65 °Cで 1 5分静置後、 37 °Cで 1 5分静置し、 変異源オリ ゴヌクレオチドを変異の目的部位にァニーリ ングさせた。  Annealing buffer (2 OmM Tris-HCl buffer (pH 8), 10 mM magnesium chloride, 5 OmM sodium chloride, ImM DTT) was prepared by annealing the mutagenic oligonucleotide and type I DNA prepared as described above. The mixture was allowed to stand at 65 ° C for 15 minutes and then at 37 ° C for 15 minutes to allow the mutagenic oligonucleotide to anneal to the target site of the mutation.
次に、 この DN A混合液に 2.5倍量のエクステンショ ンバッファー ( 50 mM T r i s HC l , pH8.0 、 6 OmM CHgCOONH,. 5 mM M g C l 2 、 5 m M D TT、 I mM N A D、 0.5 m M dATP、 0.5 mM d CTP、 0.5 mM dGT P、 0.5 m M d T T P) を加え、 さらに E. c o l i DNAリガーゼと T 4 DNAポリメ ラーゼを加えて 25 °Cで 2時間静置して相補鎖の合成を行った。 その後、 この DN Aを用いて大腸菌 BMH 71 - 1 8 m u t Sを形質転換し、 アンピシリ ン耐性のコロニーを選択した。 上記形質転換体の数個からプ ラスミ ド DN Aを調製し、 実施例 5と同様にジデォキシ法によりヌクレ ォチ ド配列を決定し、 目的のアミノ酸残基に対応するコ ドンに変換して いることを確認した。 Next, the DN A mixture of 2.5 volumes of Extension buffer (50 mM T ris HC l, pH8.0, 6 OmM CHgCOONH ,. 5 mM M g C l 2, 5 m MD TT, I mM NAD, 0.5 m M dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP), and E. coli DNA ligase and T4 DNA polymerase. Was done. Thereafter, Escherichia coli BMH71-18 mut S was transformed with the DNA, and ampicillin-resistant colonies were selected. Plasmid DNA was prepared from several of the above transformants, the nucleotide sequence was determined by the dideoxy method as in Example 5, and converted to codons corresponding to the target amino acid residues. It was confirmed.
上記のように作製したプラスミ ドを S a c Iで完全に分解後、 変異体 リパーゼ遺伝子部分を含む DN A断片をァガロースゲル電気泳動で分画 し、 ァガロースゲル中より抽出精製した。 広宿主域べクタ一である pM F Y 42を S a c Iで完全に分解後、 上記のように精製した変異体リ パーゼ遺伝子部分を含む DN A断片と混合し、 T 4 DN Aリガーゼによ つて連結反応を行い、 大腸菌 J Ml 01株を形質転換し、 カナマイシン 耐性のコロニーを選択した。 これらの形質転換体よりプラスミ ド DNA を抽出、 精製、 分析し、 PMFY 42の S a c I切断部位に変異体リ パーゼ遺伝子を含む S a c I DNA断片が挿入されたプラスミ ドを得た。 実施例 7 ： リパーゼの調製  After the plasmid prepared as described above was completely digested with SacI, the DNA fragment containing the mutant lipase gene portion was fractionated by agarose gel electrophoresis, and extracted and purified from agarose gel. After pM FY42, which is a broad host range vector, is completely digested with SacI, it is mixed with the DNA fragment containing the mutant lipase gene portion purified as described above, and mixed with T4 DNA ligase. A ligation reaction was performed to transform Escherichia coli JMl01 strain, and a kanamycin-resistant colony was selected. Plasmid DNA was extracted, purified, and analyzed from these transformants to obtain a plasmid in which a SacI DNA fragment containing a mutant lipase gene was inserted into the SacI cleavage site of PMFY42. Example 7: Preparation of lipase
実施例 6で得た変異体リパーゼ遺伝子を含むプラスミ ドもしくは野生 型リパーゼ遺伝子を含むプラスミ ドを用い、 リパーゼ欠損株である LD 9株 (シュ一ドモナス · メ ン ドシナ (Pseudomonas mendocina) S D 702株に N—メチルー N， 一ニトロ一 N—ニトロソグァニジンを作用 させ、 リパーゼ基質を含む平板培地にまいて培養し、 ク リアゾーンを形 成しない株を選択することにより取得した変異株） をエレク トロポレー シヨ ン法により形質転換し、 カナマイシン耐性のコロニーを選択した。 すなわち、 まず L D 9株を L培地 5 m 1で O D ==0.7 まで生育させた 後、 遠心分離により菌体を回収した。 この菌体を滅菌水に懸濁し、 再び 回収した後、 5 0 0 1の滅菌水に再懸濁した。 この菌懸濁液にプラス ミ ド D N Aを加え、 電極付き 4 m mセルに移した後、 オラ ッ ド (BI0RAD) 社製ジーン .パルサ一 ·ェレク トロポレーシヨ ン . システム (Gene Pulser electropolation system) により高電圧電気 0ノレス (¾ 圧 2.5k V， 電気容量 2 5 F， 抵抗 1000Ω) を与えた。 その後、 菌懸 濁液に L培地を l m 1加え、 3 7 °Cで 1時間振とう培養した後、 カナマ イシン 2 0 p p mおよびリパーゼの基質であるォリ一ブオイルのェマル ジョンを含む L平板培地に塗布した。 3 7 °Cで 1晚培養し、 生育したコ ロニ一のうち、 コロニーの回りにクリアゾーンを形成したものを選抜し、 形質転換株を得た。 Using the plasmid containing the mutant lipase gene or the plasmid containing the wild-type lipase gene obtained in Example 6, the lipase-deficient strain LD9 (Pseudomonas mendocina) SD702 was used. A mutant strain obtained by reacting with N-methyl-N, mono-nitro-N-nitrosoguanidine, culturing on a plate medium containing a lipase substrate, and selecting a strain that does not form a clear zone) was obtained. Transformation was carried out by the kanamycin method, and kanamycin-resistant colonies were selected. That is, first, 9 strains of LD were grown in 5 ml of L medium until OD = 0.7. Thereafter, the cells were collected by centrifugation. The cells were suspended in sterile water, collected again, and then resuspended in 5001 sterile water. After adding plasmid DNA to the bacterial suspension and transferring it to a 4 mm cell with electrodes, high voltage was applied using Gene Pulser electropolation system (BI0RAD). electrical 0 Noresu (¾ pressure 2.5k V, capacitance 2 5 F, resistor 1000 [Omega]) were given. Then, add 1 lm of L medium to the cell suspension, and culture with shaking at 37 ° C for 1 hour. Was applied. Cultures grown at 37 ° C for 1 晚 were selected from those that formed a clear zone around the colonies among the grown colonies to obtain transformed strains.
この形質転換株を、 ツイーン （Tw e e n) 8 0を 1 %含有し、 炭酸 ナ ト リ ウムで P H 9に調整したリパーゼ生産培地 3 0 O m 1中で 3 5°C. 1 4時間振とう培養し、 培地中に変異体リパーゼを分泌生産させた。 こ の培養液を遠心分離し、 上清をとり、 粗酵素液を調製した。 これを硫安 分画し、 透析によって硫安を除去後、 CMセルロースカラムにて処理し、 S D Sポリアク リルアミ ドゲル電気泳動で単一のバン ドになるまで精製 した。  This transformant was shaken at 35 ° C for 14 hours in 30 Om1 of a lipase production medium containing 1% Tween 80 and adjusted to PH9 with sodium carbonate. After culturing, the mutant lipase was secreted and produced in the medium. The culture was centrifuged, and the supernatant was taken to prepare a crude enzyme solution. This was subjected to ammonium sulfate fractionation, and after removing ammonium sulfate by dialysis, treated with a CM cellulose column and purified by SDS polyacrylamide gel electrophoresis until a single band was obtained.
実施例 8 ：変異体リパーゼの比活性 Example 8: Specific activity of mutant lipase
実施例 7で精製した部位特異的変異による変異体リパーゼの分子数を The number of molecules of the mutant lipase due to the site-specific mutation purified in Example 7 was determined.
2 8 0 n mの吸収で一定にして活性を測定し、 野生型リパーゼと比較し た。 活性測定は以下の方法により行った。 Activity was measured at a constant absorbance of 280 nm and compared to wild-type lipase. The activity was measured by the following method.
p—ニトロフヱニルパルミテー ト （以下 p N P Pと略す） を 2 m gZ m 1 になるようにイソプロピルアルコールに溶かす。 この p N P P溶液 と 1 0 0 mMビシン緩衝液 （Bicine buffer) (p H 8.0) を 1 : 1 0 の割合で混ぜ、 基質溶液とする。 基質溶液 5 0 0 μ I に酵素液 2 0 1 を加え、 室温で 1〜 1 0分間反応させた後、 1 N塩酸を 200 1加え て反応を止め、 分光光度計で 405 nmの吸光度を測定する。 Dissolve p-nitrophenyl palmitate (hereinafter abbreviated as p NPP) in isopropyl alcohol to a concentration of 2 mgZm1. This pNPP solution and 100 mM Bicine buffer (pH 8.0) are mixed at a ratio of 1:10 to prepare a substrate solution. Enzyme solution 201 in 500 μl substrate solution After reacting at room temperature for 1 to 10 minutes, add 1N hydrochloric acid (200 1) to stop the reaction, and measure the absorbance at 405 nm using a spectrophotometer.
野生型の酵素の活性を 1 00としたときの比活性の値を表 1に示す。 実施例 9 ：変異体リパーゼの熱安定性  Table 1 shows specific activity values when the activity of the wild-type enzyme is 100. Example 9: Thermostability of mutant lipase
野生型リパーゼと、 上記で調製した部位特異的変異による変異体 （A l a 60V a lおよび L e u 223 P h e) リパーゼの熱安定性を以下 のように測定した。 同活性値量の、 変異体リパーゼ液および野生型リ パーゼ液を 5 OmMほう酸バッファー （p H I O) 中、 60 °Cで 1 0分 間保温した後、 リパーゼ活性を測定した。 野生型の酵素の熱処理前の活 性を 1 00としたときの結果を表 1に示す。  The thermostability of the wild-type lipase and the mutants (Ala60Val and Leu223Phe) produced by the site-specific mutation prepared above were measured as follows. The mutant lipase solution and the wild-type lipase solution having the same activity value were incubated in a 5 OmM borate buffer (pHIO) at 60 ° C for 10 minutes, and then the lipase activity was measured. Table 1 shows the results when the activity of the wild-type enzyme before heat treatment was set to 100.
酵 素 比活性 熱安定性 Enzyme specific activity Thermal stability
野生型 1 00 51  Wild type 1 00 51
Ala 60 Val 98 59  Ala 60 Val 98 59
Leu 223 Phe 64 84 発明の効果  Leu 223 Phe 64 84 Effect of the Invention
本発明の遺伝子により、 本発明に関わるリパーゼ、 すなわち野生型リ パーゼ L Pもしく はその変異体リパーゼの生産および改変が容易となる。  The gene of the present invention facilitates the production and modification of the lipase according to the present invention, that is, the wild-type lipase LP or its mutant lipase.
また、 本発明によるリパーゼ L Pの改変により、 新規のァミノ酸配列 を持ち、 リパーゼ L Pに比べて、 例えば熱安定性もしく は比活性の上昇 等、 物理的性質もしくは化学的性質がその使用分野により適合するよう 変化した変異体リパーゼを得ることができ、 洗剤用、 食品加工用、 製紙 工業用等の工業分野で有用なリパーゼを提供することができる。 配列表 配列番号： 1 In addition, the modified lipase LP according to the present invention has a novel amino acid sequence, and its physical properties or chemical properties, such as an increase in heat stability or specific activity, are different from those of lipase LP depending on the field of use. It is possible to obtain a mutant lipase which has been changed so as to be compatible, and it is possible to provide a lipase useful in industrial fields such as detergents, food processing, and papermaking. Sequence Listing SEQ ID NO: 1
配列の長さ ： 2 9 3 Array length: 2 9 3
配列の型：アミノ酸 Sequence type: amino acid
トポロジー：直鎖状  Topology: linear
配列の種類：タンパク質 Sequence type: protein
起源 Origin
生物名： シュ一トモナス · メンドシナ (Pseudomonas mendocina; 株名： S D 7 0 2  Organism name: Pseudomonas mendocina; strain name: SD 7 0 2
配列 Array
Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15 Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15
Leu Gly His Gly Met Leu Gly Phe Asp Ser He Leu Gly Val Asn Tyr Leu Gly His Gly Met Leu Gly Phe Asp Ser He Leu Gly Val Asn Tyr
20 25 30  20 25 30
Trp Tyr Gly lie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr  Trp Tyr Gly lie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr
35 40 45  35 40 45
Val Thr Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin 50 55 60  Val Thr Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin 50 55 60
Leu Leu Gin Gin Val Glu Asp lie Val Ala lie Ser Gly Lys Gly LysLeu Leu Gin Gin Val Glu Asp lie Val Ala lie Ser Gly Lys Gly Lys
65 70 75 8065 70 75 80
Val Asn Leu He Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val Val Asn Leu He Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val
85 90 95 85 90 95
Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala
100 105 110  100 105 110
Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly lie Ser Asp  Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly lie Ser Asp
115 120 125  115 120 125
Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly lie Val Asn 130 135 140 Gly Leu Gly Thr Leu lie Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly lie Val Asn 130 135 140 Gly Leu Gly Thr Leu lie Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160
Pro Gin Asn Ala Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala Pro Gin Asn Ala Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala
165 170 175 Ala Arg Phe Asn Ala Lys Phe Pro Gin Gly lie Pro Thr Ser Ala Cys  165 170 175 Ala Arg Phe Asn Ala Lys Phe Pro Gin Glylie Pro Thr Ser Ala Cys
180 185 190  180 185 190
Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser  Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser
195 200 205  195 200 205
Gly Thr Ser Pro Leu Thr Asn Val Leu Asp Pro Ser Asp Leu Leu Met 210 215 220  Gly Thr Ser Pro Leu Thr Asn Val Leu Asp Pro Ser Asp Leu Leu Met 210 215 220
Gly Ala Ser Ser Leu Thr Phe Gly Ser Glu Ala Asn Asp Gly Leu Val 225 230 235 240 Gly Ala Ser Ser Leu Thr Phe Gly Ser Glu Ala Asn Asp Gly Leu Val 225 230 235 240
Gly Arg Cys Ser Ser Arg Met Gly Gin Val lie Arg Asp Asn Tyr Arg Gly Arg Cys Ser Ser Arg Met Gly Gin Val lie Arg Asp Asn Tyr Arg
245 250 255 Met Asn His Leu Asp Glu Val Asn Gin Thr Leu Gly Leu Thr Ser Leu  245 250 255 Met Asn His Leu Asp Glu Val Asn Gin Thr Leu Gly Leu Thr Ser Leu
260 265 270  260 265 270
Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin His Ala Asn Arg Leu  Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin His Ala Asn Arg Leu
275 280 285  275 280 285
Lys Asn Ala Gly Leu  Lys Asn Ala Gly Leu
290 配列番号： 2  290 SEQ ID NO: 2
配列の長さ ： 8 7 9 Array length: 8 7 9
配列の型：核酸 Sequence type: nucleic acid
鎖の数：二本鎖 Number of chains: double strand
トポロジー：直鎖状  Topology: linear
配列の種類： Genomic D N A Sequence type: Genomic DNA
起源 Origin
生物名： シユードモナス · メンドシナ (Pseudomonas mendocina) 株名： S D 7 0 2 Organism name: Pseudomonas mendocina Stock Name: SD 7 0 2
配列の特徴 Array features
特徴を表す記号： mat peptide  Characteristic symbol: mat peptide
存在位置： 1. . 879  Location: 1. .879
特徴を決定した方法： S  How the features were determined: S
配列 Array
GCC TGG TTC GGC TCC TCC GGC TAC ACC CAG ACC AAG TAC CCC ATC GTC 48 Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15  GCC TGG TTC GGC TCC TCC GGC TAC ACC CAG ACC AAG TAC CCC ATC GTC 48 Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15
CTC GGC CAC GGC ATG CTC GGC TTC GAC AGC ATC CTC GGC GTC AAT TAC 96CTC GGC CAC GGC ATG CTC GGC TTC GAC AGC ATC CTC GGC GTC AAT TAC 96
Leu Gly His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val Asn Tyr Leu Gly His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val Asn Tyr
20 25 30  20 25 30
TGG TAT GGC ATC CCG GCC GCC CTG CGC CGC GAC GGT GCC AGC GTC TAC 144 Trp Tyr Gly lie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr  TGG TAT GGC ATC CCG GCC GCC CTG CGC CGC GAC GGT GCC AGC GTC TAC 144 Trp Tyr Glylie Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr
35 40 45  35 40 45
GTC ACC GAG GTC AGT CAG CTC GAT ACC TCC GAA GCG CGC GGC GAG CAG 192 Val Thr Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin  GTC ACC GAG GTC AGT CAG CTC GAT ACC TCC GAA GCG CGC GGC GAG CAG 192 Val Thr Glu Val Ser Gin Leu Asp Thr Ser Glu Ala Arg Gly Glu Gin
50 55 60  50 55 60
TTG CTG CAG CAA GTC GAG GAT ATC GTC GCC ATC AGC GGC AAA GGC AAG 240 Leu Leu Gin Gin Val Glu Asp lie Val Ala lie Ser Gly Lys Gly Lys TTG CTG CAG CAA GTC GAG GAT ATC GTC GCC ATC AGC GGC AAA GGC AAG 240 Leu Leu Gin Gin Val Glu Asp lie Val Ala lie Ser Gly Lys Gly Lys
65 70 75 80 65 70 75 80
GTC AAT CTG ATC GGC CAC AGC CAC GGC GGC CCC ACC ACG CGC TAC GTT 288 Val Asn Leu lie Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val  GTC AAT CTG ATC GGC CAC AGC CAC GGC GGC CCC ACC ACG CGC TAC GTT 288 Val Asn Leulie Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val
85 90 95  85 90 95
GCC GCC GTG CGG CCG GAC CTG GTC GCC TCG GTC ACC AGC GTC GGT GCA 336GCC GCC GTG CGG CCG GAC CTG GTC GCC TCG GTC ACC AGC GTC GGT GCA 336
Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala
100 105 110  100 105 110
CCG CAC AAG GGC TCG GCC GCT GCC GAC TTC CTC AAG GGC ATC AGC GAT 384 Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly He Ser Asp - CCG CAC AAG GGC TCG GCC GCT GCC GAC TTC CTC AAG GGC ATC AGC GAT 384 Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly He Ser Asp -
m 313 393 QVV 339 V0 9V3 100 3VX 3X3 33V 0X9 933 XV9 33V 9V0 OIL m 313 393 QVV 339 V0 9V3 100 3VX 3X3 33V 0X9 933 XV9 33V 9V0 OIL
m 092  m 092
J "31 Π31 iqi ui3 usy ΐ¾ nig dsy ng^ si{{ usy H  J "31 Π31 iqi ui3 usy ΐ¾ nig dsy ng ^ si {{usy H
918 3X3 30V 33V 9X3 330 9X3 33V 9V3 3VV 3X3 3V3 3V0 3X3 0Ώ 3VV 9XV 丄 usy dsy Sjy UI9 ;3M S-iV s-iy 918 3X3 30V 33V 9X3 330 9X3 33V 9V3 3VV 3X3 3V3 3V0 3X3 0Ώ 3VV 9XV 丄 usy dsy Sjy UI9; 3M S-iV s-iy
89L 093 3VI 3VV 3VD 0D3 9V3 309 9XV 333 03X 10V 30X 393 330 89L 093 3VI 3VV 3VD 0D3 9V3 309 9XV 333 03X 10V 30X 393 330
Λ dsy usy ュ io sqd -iqi nai •I3S s BTVΛ dsy usy u io sqd -iqi naiI3S s BTV
310 3X3 300 3V0 OVV 339 3V0 031 3D9 3X1 30V 013 931 33X 339 300 02 n9q dsy JGS OJJ dsy usv I nsq 0Jd S 310 3X3 300 3V0 OVV 339 3V0 031 3D9 3X1 30V 013 931 33X 339 300 02 n9q dsy JGS OJJ dsy usv I nsq 0Jd S
2Z9 DIV 013 9X3 OVO 33V 333 OVO 313 919 3VV 03V 013 933 39V 33V 300  2Z9 DIV 013 9X3 OVO 33V 333 OVO 313 919 3VV 03V 013 933 39V 33V 300
SOS 002 96T  SOS 002 96T
J ュ ΑΐΟ usy m ュ ^TV niO 5ΐ ^9 3DV 901 0。丄 3VI IVl 393 310 390 3VV 013 39V 3VI V3G 990 WO 399 J Interview ΑΐΟ usy m Interview ^ TV niO 5ΐ ^ 9 3DV 901 0.丄 3VI IVl 393 310 390 3VV 013 39V 3VI V3G 990 WO 399
06T 081  06T 081
JRI OJJ 3TI 9¾i sAq BIV usy sqd  JRI OJJ 3TI 9¾i sAq BIV usy sqd
X3X 330 33V 33V 333 OXV 300 9V3 333 3XX 3VV ODD XVV 3XX 303 333  X3X 330 33V 33V 333 OXV 300 9V3 333 3XX 3VV ODD XVV 3XX 303 333
S9I Οΐ eiV ηΐθ usv J3S Aio Π9 ^IV usy u[9 OJJ  S9I eieiV ηΐθ usv J3S Aio Π9 ^ IV usy u [9 OJJ
330 333 VVG 33V 3VV 313 D3I 9V0 913 03X 100 0X0 339 3VV 9V3 933  330 333 VVG 33V 3VV 313 D3I 9V0 913 03X 100 0X0 339 3VV 9V3 933
09T SSI OS!  09T SSI OS!
■I9S J3S usy an nsq •iqi naq Λ ο ■ I9S J3S usy an nsq • iqi naq Λο
08^ 33V 33V 30V 331 XOV 309 331 j OkLtL 3VV OXV 0X3 03V 390 013 390 08 ^ 33V 33V 30V 331 XOV 309 331 j OkLtL 3VV OXV 0X3 03V 390 013 390
O  O
usy A 3Π Βΐν BIV m BTV ojj Ai  usy A 3Π Βΐν BIV m BTV ojj Ai
Z 3VV 310 DIV 309 D 010 0X3 333 VOV 339 310 933 900 330 333 390  Z 3VV 310 DIV 309 D 010 0X3 333 VOV 339 310 933 900 330 333 390
021  021
S96lO/t?6df/XDd £8 I/S6 OAV Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin His Ala Asn Arg LeuS96lO / t? 6df / XDd £ 8 I / S6 OAV Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gin His Ala Asn Arg Leu
275 280 285 275 280 285
AAG AAC GCC GGG CTA 879 Lys Asn Ala Gly Leu  AAG AAC GCC GGG CTA 879 Lys Asn Ala Gly Leu
290 配列番号： 3  290 SEQ ID NO: 3
配列の長さ ： 3 0 Array length: 30
生物名： シュートモナス · メンドシナ (Pseudomonas mendocinaリ  Organism: Shootmonas mendocina (Pseudomonas mendocina
株名： S D 7 0 2  Stock Name: SD 7 0 2
配列 Array
Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15  Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gin Thr Lys Tyr Pro lie Val 1 5 10 15
Leu Gly His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val  Leu Gly His Gly Met Leu Gly Phe Asp Ser lie Leu Gly Val
20 25 30 配列番号： 4  20 25 30 SEQ ID NO: 4
配列の長さ ： 2 0 Array length: 20
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配列の種類：他の核酸 合成 D N A Sequence type: Other nucleic acids Synthetic DNA
配列 Array
CACGGCATGC TCGGCTTCGA 20 配列番号： 5  CACGGCATGC TCGGCTTCGA 20 SEQ ID NO: 5
配列の長さ ： 2 2 Array length: 2 2
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 トポロジー：直鎖状 配列の種類：他の核酸 合成 DNA 配列 Number of chains: single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence
ACCTCCGAAG TCCGCGGCGA GC 22 配列番号： 6  ACCTCCGAAG TCCGCGGCGA GC 22 SEQ ID NO: 6
配列の長さ ： 29 Array length: 29
配列の型：核酸 Sequence type: nucleic acid
鎖の数：一本鎖 Number of chains: single strand
トポロジー：直鎖状  Topology: linear
配列の種類：他の核酸 合成 DNA 配列 Sequence type: Other nucleic acid Synthetic DNA sequence
TCGACCCCAG CGATCTGTTC ATGGGCGCC 29  TCGACCCCAG CGATCTGTTC ATGGGCGCC 29

Claims

請求の範囲 The scope of the claims
1 ) 配列番号 1に記載されたリパーゼをコ一ドする配列番号 2に記載 されたヌクレオチド配列の全部もしく は一部を含む遺伝子。 1) A gene that encodes the lipase described in SEQ ID NO: 1 and includes all or a part of the nucleotide sequence described in SEQ ID NO: 2.
2) 配列番号 2に記載されたヌクレオチド配列の一部を含む請求の範 囲第 1項記載の遺伝子。  2) The gene according to claim 1, comprising a part of the nucleotide sequence set forth in SEQ ID NO: 2.
3) 遺伝子が、 配列番号 2に記載されたヌクレオチド配列の全部を含 むリパ-ゼ遺伝子である請求の範囲第 1項記載の遺伝子。  3) The gene according to claim 1, wherein the gene is a lipase gene containing the entire nucleotide sequence of SEQ ID NO: 2.
4) 配列番号 1に記載されたァミノ酸配列のうち少なく とも一箇所の ァミノ酸残基が他のァミノ酸残基で置換された変異体リパーゼ。  4) A mutant lipase in which at least one amino acid residue in the amino acid sequence described in SEQ ID NO: 1 has been substituted with another amino acid residue.
5) 配列番号 1に記載されたァミノ酸配列もしく はそのァミノ酸配列 と少なく とも 5 0 %以上の相同性を有するアミノ酸配列における次の位 置 ： 1 8、 2 1、 2 5、 3 1、 43、 60、 75、 79、 93、 1 04、 1 0 7、 1 2 5、 1 42、 1 4 5、 1 4 8、 1 5 5、 1 5 6、 1 5 9、 5) The amino acid sequence described in SEQ ID NO: 1 or the following position in the amino acid sequence having at least 50% homology with the amino acid sequence: 18, 21, 25, 31 , 43, 60, 75, 79, 93, 104, 107, 1 25, 142, 1 45, 1 48, 1 55, 1 56, 1 59,
1 64、 1 8 3、 1 9 8、 2 0 3 2 1 0、 2 1 6、 22 2、 22 3、 2 2 4、 242、 2 46、 2 4 7 2 5 7、 2 6 6、 2 6 7、 2 7 6、 および 2 9 2のうちの少なく とも 箇所のァミノ酸残基が他のァミ ノ酸 残基に置換された変異体リパーゼ。 1 64, 1 8 3, 1 9 8, 2 0 3 2 1 0, 2 1 6, 22 2, 22 3, 2 2 4, 242, 2 46, 2 4 7 2 5 7, 2 6 6, 2 6 A mutant lipase in which at least one amino acid residue among 7, 276, and 292 is substituted with another amino acid residue.
6) 次の置換： G 1 y 1 8 A 1 a M e t 2 1 L e u ; A s p 2 5 L e u ； A s p 2 5 A r g ； A s n 3 1 A s p ； A s p 4 3 A s n ； A 1 a 6 0 V a 1 ； I l e 7 5 L e u ; G l y 7 9 P r o ; T h r 9 3 V a 1 ； V a 1 1 0 4 I l e ; V a l l 0 7 A l a ; G l y l 2 5 G l n ; I l e l 42 L e u ; G l y l 4 5 S e r ; T h r l 4 8 A l a ; G l y 1 5 5 S e r ； S e r 1 5 6 G 1 y ； T h r 1 5 9 G 1 y ； A 1 a 16) The following substitutions: G1y18A1aMet21Leu; Asp25Leu; Asp25Arg; Asn31Asp; Asp43Asn; A 1 a 60 V a 1; I le 75 Leu; Gly 79 Pro; T hr 93 V a 1; V a 1104 I le; V all 07 A la; G lyl 25 G ln; I lel 42 Leu; G lyl 45 Ser; T hrl 48 A la; Gly 15 55 Ser; Ser 15 56 G 1 y; T hr 15 G 1 y; A 1 a 1
64 S e r ； P h e l 8 3 T y r ； S e r 1 9 8 L y s ； A r g 2 0 364 S er; P hel 83 Tyr; S er 198 Lys; Arg 203
S e r ； T h r 2 1 0 S e r ； V a l 2 1 6 P h e ； L e u 222 A 1 a ； L e u 223 P h e ； Me t 224 L e u ； A r g 242 Th r ； A r g 246 H i s ； Me t 247 L e u ； Me t 257 L e u ； T h r 266 V a 1 ； L e u 267 P h e ； A s p 276 S e r ； および G 1 y 292 S e rのうちの少なく とも 1つを含む請求の範囲第 5項記載 の変異体リパーゼ。 Ser; Thr 210 Ser; Val 2 16 P he; Leu 222 A 1 A; Leu223Phe; Met224Leu; Arg242Thr; Arg246His; Met247Leu; Met257Leu; Thr266Va1; Leueu267Phe A mutant lipase according to claim 5, comprising at least one of: Asp276Ser; and Gy292Ser.
7) 請求の範囲第 4項乃至第 6項のいずれかの項に記載の変異体リ パーゼをコー ドするヌクレオチド配列の全部もしくは一部を含む遺伝子。 7) A gene comprising all or a part of the nucleotide sequence encoding the mutant lipase according to any one of claims 4 to 6.
8) 配列番号 1に記載されたァミノ酸配列における次の位置： 18、 21、 25、 31、 43、 60、 75、 79、 93、 104、 1 07、 125、 142、 145、 148、 155、 156、 159、 164、 183、 198、 203、 210、 216、 222、 223、 224、 242、 246、 247、 257、 266、 267、 276、 または 2 92のうちの少なく とも 1箇所のァミノ酸残基が他のァミノ酸残基に置 換されたリパーゼをコ一 ドするように、 配列番号 2に記載されたヌクレ ォチド配列の対応する部位が変更された遺伝子。 8) The following positions in the amino acid sequence described in SEQ ID NO: 1, 18, 21, 25, 31, 43, 60, 75, 79, 93, 104, 107, 125, 142, 145, 148, 155, 156, 159, 164, 183, 198, 203, 210, 216, 222, 223, 224, 242, 246, 247, 257, 266, 267, 276, or 292 at least one amino acid residue A gene in which the corresponding site in the nucleotide sequence set forth in SEQ ID NO: 2 has been changed so that it encodes a lipase whose group has been replaced with another amino acid residue.
9) 配列番号 1に記載されたァミノ酸配列において次のァミノ酸残基 の置換： G l y l 8 A l a ； Me t 21 L e u ； A s p 25 L e u ； A s p 25A r g ; A s n 31 A s p ; A s p 43 A s n ; A l a 60 V a 1 ； I l e 75 L e u ; G l y 79 P r o ; Th r 93 V a l ； V a 1 104 1 l e ； V a l l 07A l a ； G l y l 25 G l n ; I l e i 42 L e u ; G l y l 45 S e r ; Th r l 48 A l a ; G l y l 55 S e r ; S e r l 56 G l y ; Th r l 59 G l y ; A l a l 64 S e r ； P h e l 83 Ty r ； S e r l 98 L y s ； A r g 203 S e r ； Th r 21 0 S e r ； V a l 216 P h e ; L e u 222 A l a ； L e u 223 P h e ； Me t 224 L e u ; A r g 242 Th r ； A r g 2 46 H i s ； Me t 247 L e u ； Me t 257 L e u ； T h r 266 V a 1 ； L e u 2 6 7 P h e ； A s p 2 7 6 S e r ； および G l y 2 9 2 S e rのうちの少なく とも 1つがなされたリパーゼをコ一ドするよう に、 配列番号 2に記載されたヌクレオチ ド配列の対応する部位が変更さ れた請求の範囲第 8項に記載の遺伝子。 9) Substitution of the following amino acid residue in the amino acid sequence described in SEQ ID NO: 1: Glyl8Ala; Met21Leu; Asp25Leu; Asp25Arg; Asn31Asp A sp60 A sn; A la 60 V a 1; I le 75 Leu; G ly 79 Pro; Th r 93 V al; V a 1104 1 le; V all 07A la; G lyl 25 G ln; I lei 42 Leu; G lyl 45 Ser; Thrl 48 A la; Glyl 55 Ser; Serl 56 Gly; Thrl 59 Gly; Alal 64 Ser; Phel 83 Tyr; Serl 98 Lys; Arg 203 Ser; Thr 210 Ser; Val 216 Phe; Leu 222 A la; Leu 223 Phe; Met 224 Leu; Arg242 Thr; Arg2 46 H is; Me t 247 L eu; Me t 257 L eu; T hr 266 SEQ ID NO: 2 to encode a lipase in which at least one of V a1; Leu267Phe; Asp276Ser; and Gly2992Ser is encoded. 9. The gene according to claim 8, wherein a site corresponding to the described nucleotide sequence is changed.
PCT/JP1994/001965 1993-11-24 1994-11-21 Lipase gene and variant lipase WO1995014783A1 (en)

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