JPH07143883A - Lipase gene and mutant lipase - Google Patents

Lipase gene and mutant lipase

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Publication number
JPH07143883A
JPH07143883A JP5293631A JP29363193A JPH07143883A JP H07143883 A JPH07143883 A JP H07143883A JP 5293631 A JP5293631 A JP 5293631A JP 29363193 A JP29363193 A JP 29363193A JP H07143883 A JPH07143883 A JP H07143883A
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Prior art keywords
lipase
amino acid
gene
seq id
set forth
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Japanese (ja)
Inventor
Yoshiaki Miyoda
Katsura Ono
Junji Takaya
Tadashi Yoneda
桂 大野
喜昭 御代田
正 米田
潤治 貴家
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Showa Denko Kk
昭和電工株式会社
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Priority to JP5293631A priority Critical patent/JPH07143883A/en
Publication of JPH07143883A publication Critical patent/JPH07143883A/en
Application status is Pending legal-status Critical

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/16Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18Carboxylic ester hydrolases (3.1.1)
    • C12N9/20Triglyceride splitting, e.g. by means of lipase

Abstract

PURPOSE: To provide a new lipase gene separated from a chromosome DNA of Pseudorxnas mendocina SD702 strain, having improved thermal stability, specific activity, etc., and coding a mutant lipase useful in industrial field as detergent, etc.
CONSTITUTION: Pseudomonas mendocina SD702 strain (FERM P-12544) is inoculated to a medium, cultured for a night at 37°C and subjected to centrifugal separation to collect the cultured cells. The cells are suspended in a buffer solution and lysed by adding lysozyme and RNase A. The lysed product is mixed with phenol and subjected to centrifugal separation. The water layer is separated and mixed with ethanol and the produced precipitate is separated to obtain a chromosome DNA. The DNA is subjected to restriction enzyme treatment and a chromosome DNA library is prepared by conventional method. The library is screened with a probe to select a positive clone and the gene recovered from the clone is subjected to partial specific mutation treatment to obtain the objective new lipase gene coding a mutant lipase having improved thermal stability, specific activity, etc.
COPYRIGHT: (C)1995,JPO

Description

【発明の詳細な説明】 DETAILED DESCRIPTION OF THE INVENTION

【0001】 [0001]

【産業上の利用分野】本発明は洗剤用、食品加工用、製紙工業用等産業上有用な脂質分解酵素である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、さらに該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対応する変異体リパーゼに関する。 The present invention relates to a detergent, a food processing, gene and its nucleotide sequence encoding a novel lipase which is industrially useful lipolytic enzymes such as for the paper industry, further physical properties or chemical of the lipase about mutations and corresponding mutant lipase genes to alter the properties.

【0002】 [0002]

【従来の技術】リパーゼは、産業上、油脂改質、洗剤での配合など種々の分野で利用されており、例えば洗剤組成物の成分として脂質性の汚垢を分解除去する効果が従来から知られている。 BACKGROUND ART Lipases, industrial, oil modification, have been utilized in various fields such as the formulation of a detergent, for example, the effect of decomposing and removing the lipids of soiled as a component of a detergent composition known from the prior art It is. リパーゼを生産する微生物としては、シュードモナス( Pseudomonas )属、アルカリゲネス( Alcaligenes )属、ムコール( Mucor )属、キャンディダ( Candida )属、フミコーラ( Humicola )属などが知られている。 The microorganisms to produce lipase, Pseudomonas (Pseudomonas) genus Alcaligenes (Alcaligenes) genus, Mucor (Mucor) genus Candida (Candida) genus, Humicola (Humicola) genus and the like are known. 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、中でもシュードモナス( 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。 Among these are some things that the lipase gene has been acquired, but is acquired inter alia Pseudomonas (Pseudomonas) lipase gene of a microorganism belonging to numerous genera. これまでに知られているものとしては、シュードモナス・フラジ( Pseudomonas frag As what is known so far, Pseudomonas fragi (Pseudomonas frag
i )(特開昭62-228279 、特開平2-39890 )、シュードモナス・セパシア( Pseudomonas cepacia )(特開平3- i) (JP-A-62-228279, JP-A-2-39890), Pseudomonas cepacia (Pseudomonas cepacia) (JP-A-3
87187 、特開平3-47079 )、シュードモナス・プチダ( Pseudomonas putida )、シュードモナス・シュードアルカリゲネス( Pseudomonas pseudoalcaligenes )(特開平3-500845)、シュードモナス・アエルギノサ( Pseu 87187, JP-A-3-47079), Pseudomonas putida (Pseudomonas putida), Pseudomonas shoe de alkali monocytogenes (Pseudomonas pseudoalcaligenes) (JP-A-3-500845), Pseudomonas aeruginosa (Pseu
domonas aeruginosa )(J.Gen.Microbiol.(1992) 138 ,1 domonas aeruginosa) (J.Gen.Microbiol. (1992 ) 138, 1
325-1335)、シュードモナスsp. 325-1335), Pseudomonas sp. Pseudomonas sp. (Pseudomonas sp.
)109(J.Biol.Chem.(1991) 266 ,18135-18140)、 ) 109 (J.Biol.Chem. (1991) 266, 18135-18140),
シュードモナス・グルマエ( Pseudomonas glumae )(Ap Pseudomonas Gurumae (Pseudomonas glumae) (Ap
pl.Envir.Microbiol.(1992) 58 ,12,3787-3791 )がある。 pl.Envir.Microbiol. (1992) 58, 12,3787-3791 ) there is.

【0003】シュードモナス( 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。 [0003] Pseudomonas (Pseudomonas) lipase bacteria secrete production detergents, food processing, are used in industrial fields, such as for the paper industry. これらの分野で利用するリパーゼは、温度、pH、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。 Lipases utilized in these fields, temperature, pH, it is necessary to be stable to conditions of use of the oxygen, the solvent, such as pressure. 特に洗剤補助剤として配合されるリパーゼには、洗剤に添加してその洗浄効果を高めるために、アルカリ域でのpH、 Particularly lipase formulated as a detergent adjunct, to enhance its cleaning effect is added to the detergent, pH in an alkaline region,
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。 Temperature, oxygen, high stability to additives in detergents, including oxidizing agent is required. この様な化学的に安定な特性を得る方法として、酵素のアミノ酸配列を変えることが知られている。 As a method for obtaining such chemically stable properties, it is known to alter the amino acid sequence of the enzyme. その例としては、特開平4-500608におけるプロテアーゼ及び酸化剤に対する改良などがある。 Examples include and the like improvements to proteases and oxidants in JP-A-4-500608.

【0004】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に記載のアミノ酸配列を有する新規リパーゼ(以下リパーゼLPと略記する。)を見いだし、先に特許出願(特願平4−1603 [0004] The Applicant has excellent properties for use in industrial fields such as detergent, it found a new lipase (hereinafter abbreviated as lipase LP.) Having the amino acid sequence set forth in SEQ ID NO: 1, above patent application (Japanese Patent application No. 4-1603
53)を行った。 53) were carried out.

【0005】 [0005]

【本発明が解決しようとする課題】本発明の目的は、リパーゼLPをコードする遺伝子及びそのヌクレオチド配列、及びリパーゼLPに比べて物理的性質もしくは化学的性質の変化した変異体リパーゼ、及び該変異体リパーゼをコードするように該ヌクレオチド配列が変更された遺伝子を提供することにある。 An object of the present invention The present invention is to provide a gene and its nucleotide sequence encoding the lipase LP, and altered variant lipase of the physical properties or chemical properties compared to the lipase LP, and the mutation and to provide a gene in which the nucleotide sequence is modified to encode the body lipase.

【0006】 [0006]

【課題を解決するための手段】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LPを生産するシュードモナス属細菌の1つであり、本出願人により工業技術院生命工学工業技術研究所に寄託されているシュードモナス・メンドシナ( Pseudomonas mendocina )SD7 The present inventors have SUMMARY OF THE INVENTION In order to achieve the above object, firstly one of the bacteria belonging to the genus Pseudomonas that produces lipase LP, Agency of Industrial Science Technology by the present applicant Pseudomonas mendocina, which have been deposited with the Institute (Pseudomonas mendocina) SD7
02株(生命研菌寄FERM P−12544)に由来するリパーゼ遺伝子を取得した。 To obtain a lipase gene derived from a 02 share (life KenkinYadoriki FERM P-12544). 更にこの遺伝子の特異的な変異により、リパーゼLPのアミノ酸配列の1個以上のアミノ酸を他のアミノ酸に置換した変異体リパーゼをコー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。 Further the specific mutations of this gene was acquired genes nucleotide sequence is modified to encode a variant lipase with substitution of one or more amino acids in an amino acid sequence of a lipase LP with another amino acid. そ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。 The gene was introduced into a host bacterium, after culturing the resulting transformant to obtain the various lipase by conventional separation methods. これらのリパーゼの中からリパーゼL Lipase L from these lipase
Pに比べて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有用な変異体リパーゼが得られることを確認し、本発明を完成するに至った。 Physical properties or chemical properties change compared to the P, to confirm that the useful variant lipase is obtained in industrial fields such as detergent applications, and have completed the present invention.

【0007】すなわち、本発明は、 1)配列番号2に記載された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、 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、 3)遺伝子が、配列番号2に記載されたヌクレオチド配列を含むリパーゼ遺伝子である前記1記載の遺伝子、 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アミノ酸残基が他のアミノ酸残基で置換された変異体リパーゼ、 Accordingly, the present invention provides 1) a gene comprising all or part of the nucleotide sequence encoding the lipase described in SEQ ID NO: 2, 2) the containing portion of the nucleotide sequences set forth in SEQ ID NO: 2 gene 1, 3) gene, the gene of the 1, wherein the lipase gene comprising the nucleotide sequence set forth in SEQ ID NO: 2, 4) amino acid residues of at least one portion of the amino acid sequence set forth in SEQ ID NO: 1 variant lipase which group is substituted with another amino acid residue,

【0008】5)配列番号1に記載されたアミノ酸配列もしくは該アミノ酸配列と少なくとも50%以上の相同性を有するアミノ酸配列における次の位置:18、2 [0008] 5) the following positions in the amino acid sequence having at least 50% or more homology to the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 1: 18,2
1、25、31、43、60、75、79、93、10 1,25,31,43,60,75,79,93,10
4、107、125、142、145、148、15 4,107,125,142,145,148,15
5、156、159、164、183、198、20 5,156,159,164,183,198,20
3、210、216、222、223、224、24 3,210,216,222,223,224,24
2、246、247、257、266、267、27 2,246,247,257,266,267,27
6、または292のうちの少なくとも一箇所のアミノ酸残基が他のアミノ酸残基に置換された変異体リパーゼ、 6 variant lipase which amino acid residues of at least one place of or 292 has been substituted with another amino acid residue,

【0009】6)次の置換:G18A;M21L;D2 [0009] 6) the following substitutions: G18A; M21L; D2
5L;D25R;N31D;D43N;A60V;I7 5L; D25R; N31D; D43N; A60V; I7
5L;G79P;T93V;V104I;V107A; 5L; G79P; T93V; V104I; V107A;
G125Q;I142L;G145S;T148A;G G125Q; I142L; G145S; T148A; G
155S;S156G;T159G;A164S;F1 155S; S156G; T159G; A164S; F1
83Y;S198K;R203S;T210S;V21 83Y; S198K; R203S; T210S; V21
6F;L222A;L223F;M224L;R242 6F; L222A; L223F; M224L; R242
T;R246H;M247L;M257L;T266 T; R246H; M247L; M257L; T266
V;L267F;D276S;またはG292Sのうちの少なくとも1つを含む前記5記載の変異体リパーゼ、 7)前記4ないし前記6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、 V; L267F; D276S; or variant lipase of the 5 further comprising at least one of G292S, 7) gene containing all or part of the nucleotide sequence encoding the variant lipase of the 4 to the 6,

【0010】8)配列番号2に記載されたヌクレオチド配列が、配列番号1に記載されたアミノ酸配列における次の位置:18、21、25、31、43、60、7 [0010] 8) a nucleotide sequence set forth in SEQ ID NO: 2, the following positions in the amino acid sequence set forth in SEQ ID NO: 1: 18,21,25,31,43,60,7
5、79、93、104、107、125、142、1 5,79,93,104,107,125,142,1
45、148、155、156、159、164、18 45,148,155,156,159,164,18
3、198、203、210、216、222、22 3,198,203,210,216,222,22
3、224、242、246、247、257、26 3,224,242,246,247,257,26
6、267、276、または292のうちの少なくとも1箇所のアミノ酸残基が他のアミノ酸残基に置換されたリパーゼをコードするように、対応する部位が変更された遺伝子、及び 6,267,276 or to encode a lipase amino acid residues of at least one location has been substituted with another amino acid residue of 292, the gene corresponding site is changed, and,

【0011】9)配列番号2に記載されたヌクレオチド配列が、配列番号1に記載されたアミノ酸配列において次のアミノ酸残基の置換:G18A;M21L;D25 [0011] 9) a nucleotide sequence set forth in SEQ ID NO: 2, substitution of the following amino acid residues in the amino acid sequence set forth in SEQ ID NO 1: G18A; M21L; D25
L;D25R;N31D;D43N;A60V;I75 L; D25R; N31D; D43N; A60V; I75
L;G79P;T93V;V104I;V107A;G L; G79P; T93V; V104I; V107A; G
125Q;I142L;G145S;T148A;G1 125Q; I142L; G145S; T148A; G1
55S;156G;T159G;A164S;F183 55S; 156G; T159G; A164S; F183
Y;S198K;R203S;T210S;V216 Y; S198K; R203S; T210S; V216
F;L222A;L223F;M224L;R242 F; L222A; L223F; M224L; R242
T;R246H;M247L;M257L;T266 T; R246H; M247L; M257L; T266
V;L267F;D276S;またはG292Sのうちの少なくとも1つが為されたリパーゼをコードするように、対応する部位が変更された遺伝子を提供したものである。 V; L267F; D276S; or to encode at least one has been made lipase of the G292S, is obtained by providing a corresponding site is changed gene.

【0012】特許請求の範囲及び明細書の別の箇所において、アミノ酸残基は以下の1文字表記または3文字表記の略号で示される。 [0012] elsewhere claims and specification, amino acid residues are indicated by abbreviations of the following single letter or 3-letter code. A=Ala=アラニン V=Val=バリン L=Leu=ロイシン I=Ile=イソロイシン P=Pro=プロリン F=Phe=フェニルアラニン W=Trp=トリプトファン M=Met=メチオニン G=Gly=グリシン S=Ser=セリン T=Thr=トレオニン C=Cys=システイン Y=Tyr=チロシン N=Asn=アスパラギン Q=Gln=グルタミン D=Asp=アスパラギン酸 E=Glu=グルタミン酸 K=Lys=リジン R=Arg=アルギニン H=His=ヒスチジン A = Ala = Alanine V = Val = Valine L = Leu = Leucine I = Ile = Isoleucine P = Pro = Proline F = Phe = Phenylalanine W = Trp = Tryptophan M = Met = Methionine G = Gly = Glycine S = Ser = Serine T = Thr = threonine C = Cys = cysteine ​​Y = Tyr = tyrosine N = Asn = asparagine Q = Gln = glutamine D = Asp = aspartic acid E = Glu = glutamic acid K = Lys = lysine R = Arg = arginine H = His = histidine

【0013】アミノ酸の置換及びア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。 [0013] Variants lipase substitutions and amino acids are substituted is represented as follows. すなわち、変異によって置換される元のアミノ酸残基の名称、ア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の順に表記することにより示す。 That indicates by notation in order of the name of the amino acid residues after position, which is substituted is substituted in the name, the amino acid sequence of the original amino acid residues which are substituted by mutation. 例えば、18の位置のグリシンがアラニンで置換された突然変異体は、 For example, 18 glycine mutants replaced with alanine at position of,
G18AまたはGly18Alaと表記される。 It is referred to as the G18A or Gly18Ala.

【0014】本発明において、リパーゼをコードする遺伝子は、コロニーハイブリダイゼーションや平板培地上でのクリアゾーンの形成といった公知の方法で染色体D [0014] In the present invention, the gene encoding the lipase, chromosome D by a known method such as formation of a clear zone on colony hybridization or plate medium
NAから単離することができる。 It can be isolated from NA. すなわち、まず染色体DNAライブラリーを作製する。 That is, first to prepare a chromosomal DNA library. 次に、リパー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、相同なオ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、リパーゼをコードする遺伝子を単離することができる。 Then, if the whole or part of the amino acid sequence of a lipase is known, to prepare homologous oligonucleotide probes, it allows to isolate the gene encoding the lipase to perform colony hybridization using it that can. あるいは、リパーゼを産生しない細菌で染色体DNA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。 Alternatively, lipase to produce a chromosomal DNA library in bacteria that do not produce and plated on agar containing a substrate for lipase. リパーゼ遺伝子を含む染色体DNA断片を持つ細菌は、そのコロニーの回りに、リパー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。 Bacteria with chromosomal DNA fragment containing the lipase gene, about its colony, forming a clear zone produced for lipase to degrade substrates. この方法によってもリパーゼをコードする遺伝子を単離することができるが、いずれの方法でも良い。 Can be to isolate the gene encoding the lipase by this method may be any method.

【0015】本発明において、配列番号1に記載されたリパーゼLPのアミノ酸配列もしくは該アミノ酸配列と少なくとも50%以上相同性を有するアミノ酸配列の、 [0015] In the present invention, the amino acid sequence having at least 50% or more homology to the amino acid sequence or the amino acid sequence of a lipase LP described in SEQ ID NO: 1,
1個以上のアミノ酸を他のアミノ酸に置換した変異体リパーゼをコー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LPをコードするヌクレオチド配列を含む遺伝子に、リパーゼLPのアミノ酸配列中の目的とする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。 As a method for nucleotide sequence to obtain a modified gene to encode one or more amino acids of the mutant lipase is substituted with other amino acids, the gene comprising a nucleotide sequence encoding a lipase LP, the amino acid sequence of a lipase LP You may use any method as long as it can cause mutation of substitution of causing such nucleotide sequences of amino acids of interest in. 高頻度でかかる変異を起こさせる方法の一つに、部位特異的変異法がある。 One way to cause such mutations frequently, there is a site-directed mutagenesis. 部位特異的変異法とは野生型酵素遺伝子を鋳型にして、変異目的部位に塩基置換を有する合成オリゴヌクレオチドを変異源とする方法(MJZoller,M.Smith,"Meth The site-directed mutagenesis as the template the wild-type enzyme gene, a method of the mutagen synthetic oligonucleotides having the nucleotide substitution mutation target site (MJZoller, M.Smith, "Meth
od in Enzymology",vol.100, R.Wu,L.Grossmann, K.Mol od in Enzymology ", vol.100, R.Wu, L.Grossmann, K.Mol
dave ed., P468, Academic Press(1983) )で、クンケル(Kunkel)法(Kunkel,TA(1985) Proc.Natl.Acad.U dave ed., P468, Academic at the Press (1983)), Kunkel (Kunkel) method (Kunkel, TA (1985) Proc.Natl.Acad.U
SA., 82 ,488. )やギャップド・デュプレックス(Gapped SA., 82, 488.) And gapped duplex (Gapped
duplex )法(Karmer,W. et al(1984)Nucl.AcidsRes., duplex) method (Karmer, W. et al (1984) Nucl.AcidsRes.,
12 , 9441)などの改良法が存在する。 12, 9441) improved methods, such as are present. またポリメラーゼチェーンリアクション(PCR)を用いる方法(Mulli The method using the polymerase chain reaction (PCR) (Mulli
s,K.,Faloona,F.,Scharf,S.,Saiki,R/.Horn,G.,and Erl s, K., Faloona, F., Scharf, S., Saiki, R / .Horn, G., and Erl
ich,H.,(1986)Cold Spring Harber Symp. 51:263)などもある。 . 263), etc. Also: ich, H, (1986) Cold Spring Harber Symp 51..

【0016】また、前記変異を起こさせる別の方法に、 [0016] Also, in another way to cause the mutation,
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。 Without defining a mutation target site especially random mutagenesis causing amino acid substitutions. これにはいくつかの方法が知られている。 This is known in several ways. 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。 For example, there is a method of introducing a plasmid incorporating the lipase gene in mutator strains of E. coli. 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ーゼチェーンリアクション(PCR) Further, for example, magnesium ions, polymerase chain reaction in the presence or manganese ions (PCR)
反応を行う方法(Zhou,Y.,Zhang,X.,Ebright,RH(199 How to carry out the reaction (Zhou, Y., Zhang, X., Ebright, RH (199
1) Nucleic Acid Research 19 ,6052, Tindall,KR,Kun 1) Nucleic Acid Research 19, 6052 , Tindall, KR, Kun
kel,TA(1988) Biochemistry 27 ,6008-6013 )や、修復機能のないDNAポリメラーゼを用いてDNA増幅反応を行う方法(Liao,X.,Wise,JA(1990)Gene 107-11 kel, TA (1988) Biochemistry 27 , 6008-6013) and a method of performing DNA amplification reaction using no repair function DNA polymerase (Liao, X., Wise, JA (1990) Gene 107-11
1)がある。 1) there is.

【0017】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,RM,Lerman,L. Furthermore, for example nitrous acid, formic acid, a method using a mutagenic agent such as hydrazine (Myers, RM, Lerman, L.
S.,Maniatis,T.(1985) Science 229 242-247)などがあるが、これらに限らず如何なる方法を用いてもよい。 S., Maniatis, T. (1985 ) Science 229 242-247) , but there is such, may use any method is not limited thereto. 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。 For example, by mutation, such as natural or artificially UV, from a strain of the desired mutation has occurred, the method of obtaining the gene of the present invention are also contemplated.

【0018】変異のための鋳型としては、リパーゼ遺伝子を、例えばpUC 系のような大腸菌のベクターに組み込んだものを用いることができる。 [0018] As a template for the mutation, it can be used as the lipase gene was incorporated into a vector of E. coli, such as pUC series. 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。 The mutant lipase genes obtained as described above into a host bacterium. 例えば、シュードモナス( Pseudomonas )属細菌を宿主として用いる場合、RSF1010 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。 For example, when using the Pseudomonas (Pseudomonas) bacteria as hosts, incorporated into a chromosome by using a form of a gene can not replicate method, in a host bacterium stably maintained extrachromosomally with broad host range plasmid such as RSF1010 there is a method and the like.

【0019】シュードモナス( 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。 In the case of using a Pseudomonas (Pseudomonas) bacteria as the host, the variant lipase is secreted into the culture medium. 培養液からの変異体リパーゼの分離精製は、 The separation and purification of the variant lipase from the culture medium,
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、CMセルロースカラムで分別することによりSDS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できる。 Ammonium sulfate was added to the culture, the mutant lipase crudely fractionating, after removal of the ammonium sulfate by dialysis, the purified mutant lipase to a single band in SDS polyacrylamide gel electrophoresis by fractionated CM cellulose column it can.
しかし、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。 However, production of the variant lipase, and purification method is not limited to the above method, it is of course possible also in other ways.

【0020】上記のようにして得られた変異体リパーゼのうち、熱安定性もしくは比活性の上昇等、物理的特性もしくは化学的特性の変化したものとしては、G18 [0020] Among the variant lipase obtained as described above, rise or the like of the thermal stability or specific activity, as those that vary in physical properties or chemical properties, G18
A、M21L、D25L、D25R、N31D、D43 A, M21L, D25L, D25R, N31D, D43
N、A60V、I75L、G79P、T93V、V10 N, A60V, I75L, G79P, T93V, V10
4I、V107A、G125Q、I142L、G145 4I, V107A, G125Q, I142L, G145
S、T148A、G155S、S156G、T159 S, T148A, G155S, S156G, T159
G、A164S、F183Y、S198K、R203 G, A164S, F183Y, S198K, R203
S、T210S、V216F、L222A、L223 S, T210S, V216F, L222A, L223
F、M224L、R242T、R246H、M247 F, M224L, R242T, R246H, M247
L、M257L、T266V、L267F、D276 L, M257L, T266V, L267F, D276
S、またはG292Sの置換がなされているものが挙げられる。 S, or substitution of G292S can include those made.

【0021】尚、上記のアミノ酸残基の置換を、配列番号1に記載のアミノ酸配列と少なくとも50%以上の相同性を示すア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、 [0021] Incidentally, the substitution of the amino acid residues, when applied to the lipase having the amino acid sequence of the at least 50% homologous amino acid sequence set forth in SEQ ID NO: 1, the position of the substitution, two while providing defect position as necessary to the amino acid sequence homology is maximized,
配列番号1に記載のアミノ酸配列と相同性を有する該リパーゼのア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。 The position corresponding to when aligned with the amino acid sequence of the lipase having amino acid sequence homology with SEQ ID NO: 1 is a representation in the amino acid position of SEQ ID NO: 1.

【0022】 [0022]

【実施例】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。 EXAMPLES be described by way of embodiments the present invention, but the invention is not limited to the following examples.

【0023】実施例1:染色体DNAの調製 シュードモナス・メンドシナ( Pseudomonas mendocina [0023] Example 1: Preparation of chromosomal DNA Pseudomonas mendocina (Pseudomonas mendocina
)SD702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%)30mlに植菌し、37℃で1晩培養後、遠心分離によって上清を除去し、菌体を得た。 ) SD702 strain L medium (polypeptone 1%, 0.5% yeast extract, 0.5% sodium chloride) was inoculated into 30 ml, after overnight incubation at 37 ° C., the supernatant was removed by centrifugation, cells It was obtained. これを0.4M塩化ナトリウム、1 This 0.4M sodium chloride, 1
0mM EDTAを含む50mMトリス塩酸緩衝液(p 50mM Tris-HCl buffer containing 0 mM EDTA (p
H8)5mlに懸濁した。 H8) was suspended in 5ml. これにリゾチーム2.5mg This lysozyme 2.5mg
とRNaseA0.25mgを加え、37℃で30分間穏やかに振とうし、溶菌させた。 And RNaseA0.25mg was added, gently shaking at 37 ° C. 30 minutes to lyse. その後、60℃で10 Then, 10 at 60 ℃
分間加温し、完全に可溶化させた。 Min warmed, completely solubilized. この溶液にTE緩衝液(1mM EDTAを含む10mMトリス塩酸緩衝液(pH8))で飽和させたフェノールを等量加え、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。 The solution in TE buffer (10 mM Tris-HCl buffer containing 1mM EDTA (pH8)) phenol saturated with added equal amounts, gently mix by inverting three times to recover the upper aqueous layer by centrifugation repeated. 3回目に回収した水層に−2 To the aqueous layer was recovered in the third -2
0℃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。 Adding ethanol was cooled to 0 ° C. 2 times, the precipitate was taken up in plastic rods. 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0.5mlに再溶解した。 After rinsing the precipitate with ethanol, vacuum dried and redissolved in TE buffer 0.5 ml.

【0024】実施例2:染色体DNAライブラリーの作製 染色体DNAを制限酵素EcoRIで部分分解した後、 [0024] Example 2: After partially digesting the prepared chromosomal DNA of chromosomal DNA library with the restriction enzymes EcoRI,
フェノール・クロロホルム抽出、エタノール沈澱によりDNA断片を回収した。 Phenol-chloroform extraction, the DNA fragment was recovered by ethanol precipitation. 一方、コスミドpWE15を同様にEcoRIで分解し、アルカリホスファターゼ処理を行った。 On the other hand, it cosmid pWE15 similarly digested with EcoRI, and subjected to alkaline phosphatase treatment. 両者をT4DNA リガーゼを用いて連結した。 Both were ligated using T4DNA ligase. これをファージ粒子にパッケージングした。 This was packaged into phage particles. このファージを、マルトース、硫酸マグネシウムを含むL培地で培養した大腸菌培養液600μlに加え、37℃で15分間インキュベートした後、L培地を1mlになるように加え、37℃で1時間インキュベートした。 The phage, maltose, in addition to the E. coli culture 600μl cultured in L medium containing magnesium sulfate, after incubation for 15 minutes at 37 ° C., was added so that the L medium 1 ml, were incubated for 1 hour at 37 ° C.. これをアンピシリン50ppmを含むL平板培地(L培地をアガロース1.5%で固めたもの)に塗布し、37℃で1晩培養した。 This L plate medium containing ampicillin 50ppm was applied to (the L medium which solidified with 1.5% agarose) and cultured overnight at 37 ° C.. この結果、約1000コロニーの形質転換体を得た。 As a result, transformants were obtained from about 1000 colonies.

【0025】実施例3:オリゴヌクレオチドプローブの作製 精製したリパーゼのN末端アミノ酸配列をアミノ酸シーケンサーで分析し、配列番号3に示す結果を得た。 [0025] Example 3: N-terminal amino acid sequence of the lipase produced purified oligonucleotide probes were analyzed by an amino acid sequencer to obtain the results shown in SEQ ID NO: 3. 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DNA合成機で合成した。 The oligonucleotide probe shown in SEQ ID NO: 4 based on this synthesized in a DNA synthesizer. これに、γ- 32 P-ATP及び To this, γ- 32 P-ATP and
T4ポリヌクレオチドキナーゼを加え、37℃で1時間反応させ、放射能標識した。 T4 polynucleotide kinase was added, reacted at 37 ° C., radiolabeled.

【0026】実施例4:リパーゼをコードする遺伝子の単離 約1000コロニーの形質転換体をL平板培地上に置いたニトロセルロースフィルター上で37℃で1晩培養した。 [0026] Example 4: the transformant of isolated approximately 1000 colonies of the gene encoding the lipase was incubated overnight at 37 ° C. on nitrocellulose filters placed on L plate medium. フィルターを剥し、0.5M水酸化ナトリウム/ Peeled off the filter, 0.5M sodium hydroxide /
1.5M塩化ナトリウム溶液に浸した濾紙上で10分間溶菌させ、1.5M 塩化ナトリウム 1Mトリス塩酸緩衝液(pH7)で浸した濾紙上で7分間、2回中和した。 On filter paper soaked in 1.5M sodium chloride solution lysed 10 min, 1.5M sodium chloride 1M Tris-HCl buffer (pH 7) with soaked on filter paper 7 minutes, neutralized twice. フィルターを80℃で2時間焼成し、0.5%ドデシル硫酸ナトリウム(SDS)/6×SSC(1×SS The filters were baked for 2 hours at 80 ° C., 0.5% sodium dodecyl sulfate (SDS) / 6 × SSC (1 × SS
Cは、150mM塩化ナトリウム/15mMクエン酸ナトリウム溶液。 C is, 150 mM sodium chloride / 15 mM sodium citrate solution. n×SSCは1×SSCのn倍の濃度の塩化ナトリウム/クエン酸ナトリウム溶液。 n × SSC is n times the concentration of 1 × SSC sodium chloride / sodium citrate solution. )中で菌の残査を洗浄した。 ) To wash the residue of bacteria in. 0.1%ドデシル硫酸ナトリウム(S 0.1% sodium dodecyl sulfate (S
DS)/5×デンハルト溶液(Denhardts solution) DS) / 5 × Denhardt's solution (Denhardts solution)
(0.1%ficoll、0.1%ポリビニルピロリドン、0.1%BSA ) (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% BSA)
/5×SSC中でフィルターのプレハイブリダイゼーションを37℃で1時間行った。 / In 5 × SSC in was carried out for 1 hour Prehybridization of filters 37 ° C.. 同様の溶液中に上記の放射能標識したオリゴヌクレオチドプローブを加え、フィルターのハイブリダイゼーションを37℃で1晩行った。 Similar solutions oligonucleotide probes radiolabeled above was added in was carried out overnight at 37 ° C. Hybridization of the filters. その後、フィルターを4×SSCで10分間、2× Then, 10 minutes the filter with 4 × SSC, 2 ×
SSCで20分間2回、1×SSCで20分間2回洗浄した。 2 times 20 minutes SSC, and washed twice for 20 minutes at 1 × SSC.

【0027】このようなコロニーハイブリダイゼーションの結果、いくつかの陽性コロニーを得た。 [0027] As a result of the colony hybridization, we got some positive colonies. 得られたコロニーのうちの1つからコスミドを回収し、EcoRI The resulting cosmid is recovered from one of the colonies, EcoRI
で分解した断片をアガロース電気泳動で分離し、ニトロセルロースフィルターに吸着させた。 In the degraded fragments were separated by agarose electrophoresis and adsorbed on a nitrocellulose filter. コロニーハイブリダイゼーションと同様の手順でサザンハイブリダイゼーションを行った。 Southern hybridization was carried out in the same procedure as colony hybridization. その結果、18kbpの断片にハイブリダイズした。 As a result, it was hybridized to a fragment of 18kbp.

【0028】この断片を回収し、プラスミドpUC11 [0028] and recovering the fragment, plasmid pUC11
9のEcoRI部位に連結し、大腸菌JM101を形質転換した。 Ligated into the EcoRI site of 9, and the E. coli JM101 was transformed. このプラスミドを回収し、制限酵素SacI The plasmid was recovered, restriction enzyme SacI
で分解し、同様の手順でサザンハイブリダイゼーションを行った。 In decomposing, Southern hybridization was performed in the same procedure. その結果、2.3kbpの断片を得た。 As a result, we obtain a fragment of 2.3kbp.

【0029】実施例5:リパーゼ遺伝子のヌクレオチド配列決定 この2.3kbpの断片をpUC119のSacI部位に連結し、大腸菌JM101を形質転換した。 [0029] Example 5: a fragment of nucleotide sequencing of lipase gene The 2.3kbp ligated into the SacI site of pUC119, and the E. coli JM101 was transformed. このプラスミドをpSDL23(図1)と命名した。 This plasmid was named PSDL23 (Figure 1). このプラスミドを用いて、サンガー(Sanger)のジデオキシ法(Sa Using this plasmid, the dideoxy method of Sanger (Sanger) (Sa
nger,F.,Nicklen,S.,Coulson,AR(1977) Proc.Natl.Ac nger, F., Nicklen, S., Coulson, AR (1977) Proc.Natl.Ac
ad.Sci.USA., 74 ,5463 )でリパーゼ遺伝子DNAのヌクレオチド配列を決定した。 Ad.Sci.USA., and determining the nucleotide sequence of the lipase gene DNA at 74, 5463). すなわち、ABI社のダイデオキシターミネーターシーケンシングキットを用い、D That is, using the ABI's dideoxy terminator sequencing kit, D
NAシーケンサーによってヌクレオチド配列を解析した。 It was analyzed nucleotide sequences by NA sequencer. その結果、配列番号2のようなリパーゼをコードするヌクレオチド配列を得た。 As a result, to obtain a nucleotide sequence encoding a lipase, such as SEQ ID NO: 2. そこから推定されるアミノ酸配列を配列番号1で示す。 The amino acid sequence deduced therefrom in SEQ ID NO: 1.

【0030】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 リパーゼのアミノ酸残基を部位特異的に置換した変異体(A60VもしくはL223F)の遺伝子を以下のように構築した。 [0030] Example 6: Construction of genes as follows site specific mutation sites of amino acid residues of the building lipase mutant lipase gene by specifically substituted variants (A60V or L223F). 配列番号5もしくは配列番号6記載のアミノ酸を変換するためのオリゴヌクレオチドを化学合成し、T4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。 Chemically synthesized oligonucleotides for converting the amino acid of SEQ ID NO: 5 or SEQ ID NO: 6, was used to phosphorylate the 5 'ends using T4 polynucleotide kinase. 一方、変異の鋳型は、プラスミドp On the other hand, mutations in template plasmid p
SDL23(図1)を用いた。 SDL23 (Figure 1) was used. pSDL23を用いて大腸菌BW313株(HfrKL16PO/45[lysA(61-62)], dut pSDL23 using E. coli BW313 shares (HfrKL16PO / 45 [lysA (61-62)], dut
1,nug1,thy-1 ,relA1)を形質転換し、この形質転換体から、メッシング(Messing )らの方法(Viera,J. a 1, nug1, thy-1, relA1) were transformed, from this transformant, meshing (Messing) et al. Method (Viera, J. A
nd Messing,J. (1987) Method in Enzymology, 153 ,3-1 nd Messing, J. (1987) Method in Enzymology, 153, 3-1
1 )に従い1本鎖DNAを調製しDNA中のデオキシチミンの一部がデオキシウラシルに置き換わった一本鎖D Single strand D of single-stranded DNA was prepared part of deoxy thymine in DNA is replaced with deoxyuracil according 1)
NAを得、これを鋳型として用いた。 Give the NA, was used as a template.

【0031】上記のように調製した変異源オリゴヌクレオチドおよび鋳型DNAをアニーリングバッファー(2 The mutagen oligonucleotide and template DNA annealing buffer were prepared as described above (2
0mMトリス塩酸緩衝液(pH8)、10mM塩化マグネシウム、50mM塩化ナトリウム、1mM DTT) 0mM Tris-HCl buffer (pH 8), 10 mM magnesium chloride, 50 mM sodium chloride, 1 mM DTT)
中で混合し、65℃で15分静置後、37℃で15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アニーリングさせた。 It was mixed at medium, 15 minutes after standing at 65 ° C., standing for 15 minutes at 37 ° C., were annealed to mutagens oligonucleotide target site mutations.

【0032】次に、このDNA混合液に2.5 倍量のエクステンションバッファー(50mMTrisHCl pH8.0、60mM CH Next, this DNA mixture 2.5 times the extension buffer (50mMTrisHCl pH8.0,60mM CH
3 COONH 4 、5mM MgCl 2 、5mM DTT 、1mM NAD 、0.5mM dAT 3 COONH 4, 5mM MgCl 2, 5mM DTT, 1mM NAD, 0.5mM dAT
P、0.5mM dCTP、0.5mMdGTP 、0.5mMdTTP )を加え、さらにE.coli DNAリガーゼとT4DNA ポリメラーゼを加えて25℃で2時間静置して相補鎖の合成を行った。 P, 0.5mM dCTP, 0.5mMdGTP, 0.5mMdTTP) was added, was synthesized complementary strand to stand 2 hours at 25 ° C. further added to E. coli DNA ligase and T4DNA polymerase. その後、このDNAを用いて大腸菌BMH71ー18 mutS を形質転換し、アンピシリン耐性のコロニーを選択した。 Then, this DNA was transformed into E. coli BMH71 over 18 mutS was used to select ampicillin-resistant colonies. 上記形質転換体の数個からプラスミドDNAを調製し、実施例5と同様にジデオキシ法によりヌクレオチド配列を決定し、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。 The transformant several Plasmid DNA was prepared from, to determine the nucleotide sequence similarly by the dideoxy method as in Example 5, it was confirmed that by converting the codon corresponding to amino acid residues of interest.

【0033】上記のように作製したプラスミドをSac [0033] Sac a plasmid prepared as described above
Iで完全に分解後、変異体リパーゼ遺伝子部分を含むD After complete degradation in I, D comprising a mutant lipase gene portion
NA断片をアガロースゲル電気泳動で分画し、アガロースゲル中より抽出精製した。 The NA fragments fractionated by agarose gel electrophoresis, and extracted and purified from the agarose gel. 広宿主域ベクターであるp p is a broad-host-range vector
MFY42をSacI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DNA断片と混合し、T4DNA リガーゼによって連結反応を行い、大腸菌JM101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。 After completely decompose MFY42 with SacI, mixed with the DNA fragment containing the purified mutant lipase gene portion as described above, it performs ligation reaction by T4DNA ligase, and E. coli strain JM101 was transformed, selecting kanamycin-resistant colonies did. これらの形質転換体よりプラスミドDN Plasmid DN from these transformants
Aを抽出、精製、分析し、pMFY42のSacI切断部位に変異体リパーゼ遺伝子を含むSacIDNA断片が挿入されたプラスミドを得た。 Extract A, purified, analyzed, to obtain a plasmid SacIDNA fragment was inserted containing the mutant lipase gene into the SacI cleavage site of PMFY42.

【0034】実施例7:リパーゼの調製 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LD9株(シュードモナス・ [0034] Example 7: using a plasmid containing a plasmid or wild type lipase gene including the variant lipase gene obtained in Preparation Example 6 of lipase, a lipase-deficient strain LD9 strain (Pseudomonas
メンドシナ( Pseudomonas mendocina )SD702株にN−メチル−N'−ニトロ−N−ニトロソグアニジンを作用させ、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ョ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。 Mendocina (Pseudomonas mendocina) to SD702 strains reacted with N- methyl -N'- nitro -N- nitrosoguanidine, culturing plated on plate medium containing lipase substrate was obtained by selecting a strain which does not form a clear zone the mutant) was transformed by electroporation method to select kanamycin-resistant colonies.

【0035】すなわち、まずLD9株をL培地5mlでOD=0.7まで生育させた後、遠心分離により菌体を回収した。 [0035] That is, after the first LD9 strain was grown in L medium 5ml until OD = 0.7, the cells were collected by centrifugation. この菌体を滅菌水に懸濁し、再び回収した後、500μlの滅菌水に再懸濁した。 The cells were suspended in sterilized water, was recovered again, and resuspended in 500μl of sterile water. この菌懸濁液にプラスミドDNAを加え、電極付き4mmセルに移した後、BIORAD社製Gene Pulser ele Plasmid DNA was added to the cell suspension, after transfer to the electrode with 4mm cell, BIORAD Co. Gene Pulser ele
ctropolation systemにより高電圧電気パルス(電圧2.5kV、電気容量25μF、抵抗1000Ω)を与えた。 High voltage electrical pulses by ctropolation system (voltage 2.5 kV, capacitance 25 [mu] F, resistor 1000 [Omega]) were given. その後、菌懸濁液にL培地を1 Thereafter, the L medium cell suspension 1
ml加え、37℃で1時間振とう培養した後、カナマイシン20ppm及びリパーゼの基質であるオリーブオイルのエマルジョンを含むL平板培地に塗布した。 ml was added and after 1 hour shaking culture at 37 ° C., was applied to L plate medium containing an emulsion of olive oil which is a substrate for kanamycin 20ppm and lipase. 37℃ 37 ℃
で1晩培養し、生育したコロニーのうち、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。 In overnight culturing, among the grown colonies were selected those forming a clear zone around the colonies to obtain transformant strains.

【0036】この形質転換株を、ツイーン80を1%含有し、炭酸ナトリウムでpH9に調整したリパーゼ生産培地300ml中で35℃、14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。 [0036] The transformed strain, containing Tween 80 1%, 35 ° C. lipase production medium in 300ml adjusted to pH9 with sodium carbonate, and cultured with shaking for 14 hours, allowed to secretory production of mutant lipase in a medium It was. 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。 The culture was centrifuged, The supernatant was the crude enzyme solution was prepared. 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、SDS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 fractionated ammonium sulfate precipitation, after removal of the ammonium sulfate by dialysis, were treated with CM cellulose column and purified to a single band in SDS-polyacrylamide gel electrophoresis.

【0037】実施例8:変異体リパーゼの比活性 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280nm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。 [0037] Example 8: a constant number of molecules of the variant lipase by site-specific mutation was purified by specific activity Example 7 variant lipase at 280nm absorption was measured activity, compared to wild-type lipase. 活性測定は以下の方法により行った。 Activity measurements were carried out by the following methods.

【0038】p−ニトロフェニルパルミテート(以下p [0038] p- nitrophenyl palmitate (hereinafter p
NPPと略す)を2mg/mlになるようにイソプロピルアルコールに溶かす。 Dissolve abbreviated as NPP) in isopropyl alcohol so that the 2 mg / ml. このpNPP溶液と100mM The pNPP solution and 100mM
ビシン緩衝液(Bicine buffer )(pH8.0)を1: Bicine buffer (Bicine buffer) (pH8.0) 1:
10の割合で混ぜ、基質溶液とする。 Mix ten, and substrate solution. 基質溶液500μ Substrate solution 500μ
lに酵素液20μlを加え、室温で1〜10分間反応させた後、1N塩酸を200μl加えて反応を止め、分光光度計で405nmの吸光度を測定する。 The enzyme solution 20μl added to l, after reacting for 1 to 10 minutes at room temperature, 1N hydrochloric acid to stop the reaction by adding 200 [mu] l, measuring the absorbance at 405nm in a spectrophotometer.

【0039】野生型の酵素の活性を100としたときの比活性の値を表1に示す。 [0039] Table 1 shows the values ​​of specific activity when the 100 the activity of the wild-type enzyme.

【0040】 [0040]

【表1】 [Table 1]

【0041】実施例9:変異体リパーゼの熱安定性 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リパーゼの熱安定性を以下のように測定した。 [0041] Example 9 was measured as a thermal stability Wild-type lipase variant lipase, the thermal stability of the variant lipase by site-directed mutagenesis the above prepared below.
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50mMほう酸バッファー(pH10)中、60℃ During the activity value amount, 50 mM boric acid buffer variant lipase solution and wild-type lipase solution (pH10), 60 ℃
で10分間保温した後、リパーゼ活性を測定した。 After incubation in 10 minutes, the lipase activity. 熱処理前の活性を100としたときの結果を表1に示す。 The results when the 100 active before the heat treatment shown in Table 1.

【0042】 [0042]

【発明の効果】本発明の遺伝子により、本発明に関わるリパーゼ、すなわちリパーゼLPもしくは変異体リパーゼの生産及び改変が容易となる。 The genes of the present invention, the lipase according to the present invention, i.e., the production and modification of lipase LP or variant lipase is facilitated. また、本発明によるリパーゼLPの改変により、新規のアミノ酸配列を持ち、 Moreover, by modification of lipase LP according to the invention has a novel amino acid sequence,
リパーゼLPに比べて例えば熱安定性もしくは比活性の上昇等の物理的性質もしくは化学的性質が変化した変異体リパーゼが得られ、洗剤用途等の工業分野での利用に適したリパーゼを提供することができる。 Physical properties or chemical properties such as increase of such as thermal stability or specific activity as compared to the lipase LP is the variant lipase which changes obtained, to provide a lipase suitable for use in industrial fields such as detergent applications can.

【0043】 [0043]

【配列表】 [Sequence Listing]

【0044】配列番号:1 配列の長さ:293 配列の型:アミノ酸 トポロジー:直鎖状 配列の種類:タンパク質 起源 生物名:シュードモナス・メンドシナ( Pseudomonas men [0044] SEQ ID NO: 1 the length of the sequence: 293 array type: amino acid Topology: linear sequence of types: protein source organism name: Pseudomonas mendocina (Pseudomonas men
docina ) 株名:SD702 配列 Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gln Thr Lys Tyr Pro Ile Val 1 5 10 15 Leu Gly His Gly Met Leu Gly Phe Asp Ser Ile Leu Gly Val Asn Tyr 20 25 30 Trp Tyr Gly Ile Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr 35 40 45 Val Thr Glu Val Ser Gln Leu Asp Thr Ser Glu Ala Arg Gly Glu Gln 50 55 60 Leu Leu Gln Gln Val Glu Asp Ile Val Ala Ile Ser Gly Lys Gly Lys 65 70 75 80 Val Asn Leu Ile Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val 85 90 95 Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala 100 105 110 Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly Ile Ser Asp 115 120 125 Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly Ile Val Asn 130 135 140 Gly Leu Gly Thr Leu Ile Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160 Pro Gln 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 Gln Gly Ile Pro Thr Ser Ala Cys 180 185 190 Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser 195 docina) Ltd. name: SD702 array Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gln Thr Lys Tyr Pro Ile Val 1 5 10 15 Leu Gly His Gly Met Leu Gly Phe Asp Ser Ile Leu Gly Val Asn Tyr 20 25 30 Trp Tyr Gly Ile Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr 35 40 45 Val Thr Glu Val Ser Gln Leu Asp Thr Ser Glu Ala Arg Gly Glu Gln 50 55 60 Leu Leu Gln Gln Val Glu Asp Ile Val Ala Ile Ser Gly Lys Gly Lys 65 70 75 80 Val Asn Leu Ile Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val 85 90 95 Ala Ala Val Arg Pro Asp Leu Val Ala Ser Val Thr Ser Val Gly Ala 100 105 110 Pro His Lys Gly Ser Ala Ala Ala Asp Phe Leu Lys Gly Ile Ser Asp 115 120 125 Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly Ile Val Asn 130 135 140 Gly Leu Gly Thr Leu Ile Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160 Pro Gln 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 Gln Gly Ile Pro Thr Ser Ala Cys 180 185 190 Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser 195 200 205 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 Arg Cys Ser Ser Arg Met Gly Gln Val Ile Arg Asp Asn Tyr Arg 245 250 255 Met Asn His Leu Asp Glu Val Asn Gln Thr Leu Gly Leu Thr Ser Leu 260 265 270 Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gln His Ala Asn Arg Leu 275 280 285 Lys Asn Ala Gly Leu 290 200 205 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 Arg Cys Ser Ser Arg Met Gly Gln Val Ile Arg Asp Asn Tyr Arg 245 250 255 Met Asn His Leu Asp Glu Val Asn Gln Thr Leu Gly Leu Thr Ser Leu 260 265 270 Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gln His Ala Asn Arg Leu 275 280 285 Lys Asn Ala Gly Leu 290

【0045】配列番号:2 配列の長さ:879 配列の型:核酸 鎖の数:二本鎖 トポロジー:直鎖状 配列の種類:Genomic DNA 起源 生物名:シュードモナス・メンドシナ( Pseudomonas men [0045] SEQ ID NO: 2 sequence Length: 879 SEQ types: the number of nucleic acid strands: double-stranded Topology: linear sequence type: Genomic DNA Origin Organism: Pseudomonas mendocina (Pseudomonas men
docina ) 株名:SD702 配列の特徴 特徴を表す記号:mat peptide 存在位置:1..879 特徴を決定した方法:S 配列 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 Gln Thr Lys Tyr Pro Ile Val 1 5 10 15 CTC 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 Ile Leu Gly Val Asn Tyr 20 25 30 TGG TAT GGC ATC CCG GCC GCC CTG CGC CGC GAC GGT GCC AGC GTC TAC 144 Trp Tyr Gly Ile Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr 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 Gln Leu Asp Thr Ser Glu Ala Arg Gly Glu Gln 50 55 60 TTG CTG CAG CAA GTC GAG GAT ATC GTC GCC ATC AGC GGC AAA GGC AAG 240 Leu Leu Gln Gln Val Glu Asp Ile Val Ala Ile Ser Gly Lys Gly Lys 65 70 75 80 GTC AAT CTG ATC GGC CAC AGC CAC GGC GGC CCC ACC ACG CGC TAC GTT 288 Val Asn Leu Ile Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val 85 90 95 GCC GCC GTG CGG Docina) strain name: SD702 symbols have the features characteristic of the sequence:: mat peptide presence position: 1..879 method to determine the characteristics: S sequence 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 Gln Thr Lys Tyr Pro Ile Val 1 5 10 15 CTC 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 Ile Leu Gly Val Asn Tyr 20 25 30 TGG TAT GGC ATC CCG GCC GCC CTG CGC CGC GAC GGT GCC AGC GTC TAC 144 Trp Tyr Gly Ile Pro Ala Ala Leu Arg Arg Asp Gly Ala Ser Val Tyr 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 Gln Leu Asp Thr Ser Glu Ala Arg Gly Glu Gln 50 55 60 TTG CTG CAG CAA GTC GAG GAT ATC GTC GCC ATC AGC GGC AAA GGC AAG 240 Leu Leu Gln Gln Val Glu Asp Ile Val Ala Ile Ser Gly Lys Gly Lys 65 70 75 80 GTC AAT CTG ATC GGC CAC AGC CAC GGC GGC CCC ACC ACG CGC TAC GTT 288 Val Asn Leu Ile Gly His Ser His Gly Gly Pro Thr Thr Arg Tyr Val 85 90 95 GCC 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 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 Ile Ser Asp 115 120 125 GGC CCC GCC GGG CCG GTC GCC ACA CCG CTG CTG GCG GGC ATC GTC AAC 432 Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly Ile Val Asn 130 135 140 GGC CTG GGC ACG CTG ATC AAC TTC CTC TCC GGC AGT TCC AGC ACC ACC 480 Gly Leu Gly Thr Leu Ile Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160 CCG CAG AAC GCC CTG GGT TCG CTG GAG TCG CTC AAC AGC GAA GGC GCC 528 Pro Gln Asn Ala Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala 165 170 175 GCG CGC TTC AAT GCC AAG TTC CCG CAG GGC ATC CCC ACC AGC GCC TGT 576 Ala Arg Phe Asn Ala Lys Phe Pro Gln Gly Ile Pro Thr Ser Ala Cys 180 185 190 GGC GAA GGG GCA TAC AGC GTG AAC GGC GTG CGC TAT TAC TCC TGG AGC 624 Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser 195 200 205 GGC 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 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 Ile Ser Asp 115 120 125 GGC CCC GCC GGG CCG GTC GCC ACA CCG CTG CTG GCG GGC ATC GTC AAC 432 Gly Pro Ala Gly Pro Val Ala Thr Pro Leu Leu Ala Gly Ile Val Asn 130 135 140 GGC CTG GGC ACG CTG ATC AAC TTC CTC TCC GGC AGT TCC AGC ACC ACC 480 Gly Leu Gly Thr Leu Ile Asn Phe Leu Ser Gly Ser Ser Ser Thr Thr 145 150 155 160 CCG CAG AAC GCC CTG GGT TCG CTG GAG TCG CTC AAC AGC GAA GGC GCC 528 Pro Gln Asn Ala Leu Gly Ser Leu Glu Ser Leu Asn Ser Glu Gly Ala 165 170 175 GCG CGC TTC AAT GCC AAG TTC CCG CAG GGC ATC CCC ACC AGC GCC TGT 576 Ala Arg Phe Asn Ala Lys Phe Pro Gln Gly Ile Pro Thr Ser Ala Cys 180 185 190 GGC GAA GGG GCA TAC AGC GTG AAC GGC GTG CGC TAT TAC TCC TGG AGC 624 Gly Glu Gly Ala Tyr Ser Val Asn Gly Val Arg Tyr Tyr Ser Trp Ser 195 200 205 GGC ACC AGC CCG CTG ACC AAC GTG CTC GAC CCC AGC GAC CTG CTG ATG 672 Gly Thr Ser Pro Leu Thr Asn Val Leu Asp Pro Ser Asp Leu Leu Met 210 215 220 GGC GCC TCC TCG CTG ACC TTC GGC TCC GAG GCC AAC GAC GGC CTG GTC 720 Gly Ala Ser Ser Leu Thr Phe Gly Ser Glu Ala Asn Asp Gly Leu Val 225 230 235 240 GGC CGC TGC AGT TCG CGC ATG GGC CAG GTG ATC CGC GAC AAC TAC CGG 768 Gly Arg Cys Ser Ser Arg Met Gly Gln Val Ile Arg Asp Asn Tyr Arg 245 250 255 ATG AAC CAC CTC GAC GAG GTC AAC CAG ACC CTG GGG CTG ACC AGC CTG 816 Met Asn His Leu Asp Glu Val Asn Gln Thr Leu Gly Leu Thr Ser Leu 260 265 270 TTC GAG ACC GAT CCG GTG ACC GTC TAC CGT CAG CAC GCC AAC CGC CTG 864 Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gln His Ala Asn Arg Leu 275 280 285 AAG AAC GCC GGG CTA 879 Lys Asn Ala Gly Leu 290 ACC AGC CCG CTG ACC AAC GTG CTC GAC CCC AGC GAC CTG CTG ATG 672 Gly Thr Ser Pro Leu Thr Asn Val Leu Asp Pro Ser Asp Leu Leu Met 210 215 220 GGC GCC TCC TCG CTG ACC TTC GGC TCC GAG GCC AAC GAC GGC CTG GTC 720 Gly Ala Ser Ser Leu Thr Phe Gly Ser Glu Ala Asn Asp Gly Leu Val 225 230 235 240 GGC CGC TGC AGT TCG CGC ATG GGC CAG GTG ATC CGC GAC AAC TAC CGG 768 Gly Arg Cys Ser Ser Arg Met Gly Gln Val Ile Arg Asp Asn Tyr Arg 245 250 255 ATG AAC CAC CTC GAC GAG GTC AAC CAG ACC CTG GGG CTG ACC AGC CTG 816 Met Asn His Leu Asp Glu Val Asn Gln Thr Leu Gly Leu Thr Ser Leu 260 265 270 TTC GAG ACC GAT CCG GTG ACC GTC TAC CGT CAG CAC GCC AAC CGC CTG 864 Phe Glu Thr Asp Pro Val Thr Val Tyr Arg Gln His Ala Asn Arg Leu 275 280 285 AAG AAC GCC GGG CTA 879 Lys Asn Ala Gly Leu 290

【0046】配列番号:3 配列の長さ:30 配列の型:アミノ酸 トポロジー:直鎖状 配列の種類:ペプチド フラグメント型:N 末端フラグメント 起源 生物名:シュードモナス・メンドシナ( Pseudomonas men [0046] SEQ ID NO: 3 sequence Length: 30 sequence types: amino acid Topology: linear sequence type: peptide Fragment type: N-terminal fragment Origin Organism: Pseudomonas mendocina (Pseudomonas men
docina ) 株名:SD702 配列 Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gln Thr Lys Tyr Pro Ile Val 1 5 10 15 Leu Gly His Gly Met Leu Gly Phe Asp Ser Ile Leu Gly Val 20 25 30 docina) Ltd. name: SD702 array Ala Trp Phe Gly Ser Ser Gly Tyr Thr Gln Thr Lys Tyr Pro Ile Val 1 5 10 15 Leu Gly His Gly Met Leu Gly Phe Asp Ser Ile Leu Gly Val 20 25 30

【0047】配列番号:4 配列の長さ:20 配列の型:核酸 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 CACGGCATGC TCGGCTTCGA 20 [0047] SEQ ID NO: 4 sequence Length: 20 Type of sequence: number of a nucleic acid strand: single strand Topology: linear sequence type: other nucleic acid synthetic DNA sequence CACGGCATGC TCGGCTTCGA 20

【0048】配列番号:5 配列の長さ:22 配列の型:核酸 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 ACCTCCGAAG TCCGCGGCGA GC [0048] SEQ ID NO: 5 Length of sequence: 22 SEQ types: the number of nucleic acid strands: single strand Topology: linear sequence type: other nucleic acid synthetic DNA sequence ACCTCCGAAG TCCGCGGCGA GC
22 22

【0049】配列番号:6 配列の長さ:30 配列の型:核酸 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 TCGACCCCAG AGATCTGTTC ATGGGCGCC 30 [0049] SEQ ID NO: 6 SEQ Length: 30 Type of sequence: number of a nucleic acid strand: single strand Topology: linear sequence type: other nucleic acid synthetic DNA sequence TCGACCCCAG AGATCTGTTC ATGGGCGCC 30

【図面の簡単な説明】 BRIEF DESCRIPTION OF THE DRAWINGS

【図1】プラスミドpSDL23の制限酵素地図を示す。 FIG. 1 shows a restriction enzyme map of plasmid pSDL23. (矢印は、リパーゼの構造遺伝子の位置を示す。) (Arrows indicate the location of the structural gene of the lipase.)

───────────────────────────────────────────────────── ────────────────────────────────────────────────── ───

【手続補正書】 [Procedure amendment]

【提出日】平成6年1月13日 [Filing date] 1994 January 13

【手続補正1】 [Amendment 1]

【補正対象書類名】明細書 [Correction target document name] specification

【補正対象項目名】0006 [Correction target item name] 0006

【補正方法】変更 [Correction method] change

【補正内容】 [Correction contents]

【0006】 [0006]

【課題を解決するための手段】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LPを生産するシュードモナス属細菌の1つであり、本出願人により工業技術院生命工学工業技術研究所に寄託されているシュードモナス・メンドシナ( Pseudomonas mendocina )SD7 The present inventors have SUMMARY OF THE INVENTION In order to achieve the above object, firstly one of the bacteria belonging to the genus Pseudomonas that produces lipase LP, Agency of Industrial Science Technology by the present applicant Pseudomonas mendocina, which have been deposited with the Institute (Pseudomonas mendocina) SD7
02株(受託番号FERM P−12944)に由来するリパーゼ遺伝子を取得した。 To obtain a lipase gene derived from the 02 strain (accession number FERM P-12944). 更にこの遺伝子の特異的な変異により、リパーゼLPのアミノ酸配列の1個以上のアミノ酸を他のアミノ酸に置換した変異体リパーゼをコー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。 Further the specific mutations of this gene was acquired genes nucleotide sequence is modified to encode a variant lipase with substitution of one or more amino acids in an amino acid sequence of a lipase LP with another amino acid. そ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。 The gene was introduced into a host bacterium, after culturing the resulting transformant to obtain the various lipase by conventional separation methods. これらのリパーゼの中からリパーゼLP Lipase LP from these lipase
に比べて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有用な変異体リパーゼが得られることを確認し、本発明を完成するに至った。 Physical properties or chemical properties change compared to, verify that the useful variant lipase is obtained in industrial fields such as detergent applications, and have completed the present invention.

【書類名】 受託番号変更届 [Document name] accession number notification of change

【提出日】 平成6年9月19日 [Filing date] 1994 September 19,

【旧寄託機関の名称】 通商産業省工業技術院生命工学工業技術研究所 [The former name of the depositary institution] National Institute of Bioscience and Human-Technology

【旧受託番号】 微工研菌寄第P−12944 [Old] accession number FERM No. P-12944
issue

【新寄託機関の名称】 通商産業省工業技術院生命工学工業技術研究所 [New name of the depositary institution] National Institute of Bioscience and Human-Technology

【新受託番号】 FERM BP−4291 [New] accession number FERM BP-4291

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl. 6識別記号 庁内整理番号 FI 技術表示箇所 C11D 7/42 (C12N 15/09 ZNA C12R 1:38) (C12N 9/20 C12R 1:38) C12R 1:38) (72)発明者 貴家 潤治 東京都大田区多摩川2丁目24番25号 昭和 電工株式会社生化学研究所内 ────────────────────────────────────────────────── ─── front page continued (51) Int.Cl. 6 in identification symbol Agency Docket No. FI art display portion C11D 7/42 (C12N 15/09 ZNA C12R 1:38 ) (C12N 9/20 C12R 1:38) C12R 1:38) (72) inventor Kiya Junji Ota-ku, Tokyo Tama 2-chome 24th No. 25 Showa Denko Co., Ltd. Institute of biochemistry in

Claims (9)

    【特許請求の範囲】 [The claims]
  1. 【請求項1】 配列番号2に記載された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。 1. A gene comprising all or part of the nucleotide sequence encoding the lipase described in SEQ ID NO: 2.
  2. 【請求項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項1記載の遺伝子。 2. A gene according to claim 1 comprising a portion of the nucleotide sequences set forth in SEQ ID NO: 2.
  3. 【請求項3】 遺伝子が、配列番号2に記載されたヌクレオチド配列を含むリパ−ゼ遺伝子である請求項1記載の遺伝子。 3. A gene, lipase comprising a nucleotide sequence set forth in SEQ ID NO: 2 - gene of claim 1 wherein the peptidase gene.
  4. 【請求項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アミノ酸残基が他のアミノ酸残基で置換された変異体リパーゼ。 4. SEQ ID NO: variant lipase which amino acid residues of at least one portion of the amino acid sequences described are substituted with other amino acid residues 1.
  5. 【請求項5】 配列番号1に記載されたアミノ酸配列もしくは該アミノ酸配列と少なくとも50%以上の相同性を有するアミノ酸配列における次の位置:18、21、 5. The following positions in the amino acid sequence having at least 50% or more homology to the amino acid sequence or the amino acid sequence set forth in SEQ ID NO: 1: 18, 21,
    25、31、43、60、75、79、93、104、 25,31,43,60,75,79,93,104,
    107、125、142、145、148、155、1 107,125,142,145,148,155,1
    56、159、164、183、198、203、21 56,159,164,183,198,203,21
    0、216、222、223、224、242、24 0,216,222,223,224,242,24
    6、247、257、266、267、276、または292のうちの少なくとも一箇所のアミノ酸残基が他のアミノ酸残基に置換された変異体リパーゼ。 6,247,257,266,267,276 or variant lipase amino acid residues of at least one position has been substituted with another amino acid residue of 292,.
  6. 【請求項6】 次の置換:G18A;M21L;D25 6. The following substituted: G18A; M21L; D25
    L;D25R;N31D;D43N;A60V;I75 L; D25R; N31D; D43N; A60V; I75
    L;G79P;T93V;V104I;V107A;G L; G79P; T93V; V104I; V107A; G
    125Q;I142L;G145S;T148A;G1 125Q; I142L; G145S; T148A; G1
    55S;S156G;T159G;A164S;F18 55S; S156G; T159G; A164S; F18
    3Y;S198K;R203S;T210S;V216 3Y; S198K; R203S; T210S; V216
    F;L222A;L223F;M224L;R242 F; L222A; L223F; M224L; R242
    T;R246H;M247L;M257L;T266 T; R246H; M247L; M257L; T266
    V;L267F;D276S;またはG292Sのうちの少なくとも1つを含む請求項5記載の変異体リパーゼ。 V; L267F; D276S; or variant lipase of claim 5 further comprising at least one of the G292S.
  7. 【請求項7】 請求項4ないし請求項6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 containing all or part of the nucleotide sequence encoding the variant lipase of the claims 4 to 6, wherein.
  8. 【請求項8】 配列番号2に記載されたヌクレオチド配列が、配列番号1に記載されたアミノ酸配列における次の位置:18、21、25、31、43、60、75、 8. A nucleotide sequence of SEQ ID NO: 2, the following positions in the amino acid sequence set forth in SEQ ID NO: 1: 18,21,25,31,43,60,75,
    79、93、104、107、125、142、14 79,93,104,107,125,142,14
    5、148、155、156、159、164、18 5,148,155,156,159,164,18
    3、198、203、210、216、222、22 3,198,203,210,216,222,22
    3、224、242、246、247、257、26 3,224,242,246,247,257,26
    6、267、276、または292のうちの少なくとも1箇所のアミノ酸残基が他のアミノ酸残基に置換されたリパーゼをコードするように、対応する部位が変更された遺伝子。 6,267,276 or as amino acid residues of at least one portion in 292 encodes a lipase has been substituted with another amino acid residue, the corresponding sites were modified gene.
  9. 【請求項9】 配列番号2に記載されたヌクレオチド配列が、配列番号1に記載されたアミノ酸配列において次のアミノ酸残基の置換:G18A;M21L;D25 9. Nucleotide sequence of SEQ ID NO: 2, substitution of the following amino acid residues in the amino acid sequence set forth in SEQ ID NO 1: G18A; M21L; D25
    L;D25R;N31D;D43N;A60V;I75 L; D25R; N31D; D43N; A60V; I75
    L;G79P;T93V;V104I;V107A;G L; G79P; T93V; V104I; V107A; G
    125Q;I142L;G145S;T148A;G1 125Q; I142L; G145S; T148A; G1
    55S;156G;T159G;A164S;F183 55S; 156G; T159G; A164S; F183
    Y;S198K;R203S;T210S;V216 Y; S198K; R203S; T210S; V216
    F;L222A;L223F;M224L;R242 F; L222A; L223F; M224L; R242
    T;R246H;M247L;M257L;T266 T; R246H; M247L; M257L; T266
    V;L267F;D276S;またはG292Sのうちの少なくとも1つが為されたリパーゼをコードするように、対応する部位が変更された遺伝子。 V; L267F; D276S; or to encode at least one has been made lipase of the G292S, corresponding sites has been changed gene.
JP5293631A 1993-11-24 1993-11-24 Lipase gene and mutant lipase Pending JPH07143883A (en)

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