JPH09503916A - アミラーゼ変異体 - Google Patents
アミラーゼ変異体Info
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- JPH09503916A JPH09503916A JP7511196A JP51119695A JPH09503916A JP H09503916 A JPH09503916 A JP H09503916A JP 7511196 A JP7511196 A JP 7511196A JP 51119695 A JP51119695 A JP 51119695A JP H09503916 A JPH09503916 A JP H09503916A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
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- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
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- D06L—DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
- D06L1/00—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods
- D06L1/12—Dry-cleaning or washing fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods using aqueous solvents
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Abstract
Description
Claims (1)
- 【特許請求の範囲】 1.親酵素と比べて改良された洗浄及び/又は皿洗い性能をもつその親α−ア ミラーゼ酵素の変異体であって、その親酵素の1以上のアミノ酸残基が異なるア ミノ酸残基により置換されており、そして/又は、その親α−アミラーゼの1以 上のアミノ酸残基が欠失されており、そして/又は1以上のアミノ酸残基がその 親α−アミラーゼ酵素に付加されているような変異体。但し、その変異体は、親 バチルス・リケニフォルミス(B.Licheniformis)α−アミラーゼの197位にお けるメチオニン残基が、その修飾だけが行われるとき、アラニン又はトレオニン により置換されているものとは異なる。 2.親α−アミラーゼ酵素が微生物の酵素である、請求項1に記載の変異体。 3.親α−アミラーゼ酵素がバクテリア又は真菌の酵素である、請求項1又は 2のいずれかに記載の変異体。 4.親α−アミラーゼがバチルス(Bacillus)の株由来である、請求項3に記 載の変異体。 5.親α−アミラーゼがバチルス・リケニフォルミス(B.licheniformis)、 バチルス・アミロリクエファシエンス(B.amyloliquefaciens)、バチルス・ス テアロサーモフィラス(B.stearothermophilus)又はバチルス・サブチリス(B .subtilis )由来である、請求項4に記載の変異体。 6.親α−アミラーゼが、配列番号:2に示すアミノ酸配列をもつバチルス・ リケニフォルミス(B.licheniformis)α−アミラーゼ、又はそのα−アミラー ゼのアナログであって、 i)配列番号:2に示す配列と少なくとも6%の相同性をもち、 ii)そのα−アミラーゼに対して生じた抗体と反応し、そして/ 又は、 iii)そのα−アミラーゼをコードするDNA配列であってそのDNA配列が配列番 号:1に示されるのと同一プローブとハイブリダイズするDNA配列によりコード される、ようなアナログである、請求項5に記載の変異体。 7.親α−アミラーゼのN−末端内に少なくとも1の置換、欠失又は付加を含 んで成る、請求項6に記載の変異体。 8.親α−アミラーゼの1,2,3,15又は17−35位にある少なくとも1のア ミノ酸残基が置換又は欠失されており、又は少なくとも1のアミノ酸が17−35位 にあるアミノ酸セグメント内でその親α−アミラーゼに付加されている、請求項 7に記載の変異体。 9.親α−アミラーゼの29−35位にある少なくとも1のアミノ酸残基が置換又 は欠失されており、又は少なくとも1のアミノ酸が、29−35位にあるアミノ酸セ グメント内でその親α−アミラーゼに付加されている、請求項8に記載の変異体 。 10.以下のアミノ酸残基の中の少なくとも1:A1,N2,L3又はM15,N 17,R23,S29,A30,Y31,A33,E34,H35,が修飾されている、請求項7 又は8に記載の変異体。 11.以下の突然変異の中の少なくとも1:A1V;M15T,L;N2*;L3 V;A1*+N2;S29A;A30E,N;Y31H,N;A33S;E34D,S;H3 5I,L;or R23K,T、を含んで成り、特に変異体A1*+N2*+L3V+ M15T+R23K+S29A+A30E+Y31H+A33S+E34D+H35Iである、請 求項10に記載の変異体。 12.b)アミノ酸残基51−58にわたる領域にあるアミノ酸残基の中の少なくと も1、特にその51,52及び/又は58にあるアミノ酸残基が、例えば以下の突然変 異:Q51R,A52S,A58P,Vの中の 少なくとも1により修飾されており; c)アミノ酸残基H68が、例えば以下の突然変異:H68N,Qの中の1により 修飾されており; d)85及び/又は88位にあるアミノ酸残基の中の少なくとも1が、特に突然変 異S85Q,K88Qの中の少なくとも1により修飾されており; e)94−104領域内にあるアミノ酸残基の中の少なくとも1、特に94,95,96 ,99,103及び/又は104位にあるアミノ酸残基が、例えば以下の突然変異:N96 Q,G99A,I 103F及び/又はN 104Dの中の少なくとも1により修飾されて おり; f)121−136領域内にあるアミノ酸残基の中の少なくとも1、特に121,127, 128,131,132,133,及び/又は134位にあるアミノ酸残基、例えば、突然変異 D121 N,R 127Q,V 128E,G 131E,E 132T,H 133Y,L 134Q及び /又はK 136Qの中の少なくとも1; g)140,142,148及び/又は152位にあるアミノ酸残基の中の少なくとも1、 例えば以下の突然変異:H 140K,H 142D,D 152S,S 148Nの中の少なく とも1; h)142−148領域内にあるアミノ酸残基の中の少なくとも1、特にその領域内 のアミノ酸残基の全て又は実質的な部分の欠失; i)172−178領域内にあるアミノ酸残基の中の少なくとも1、特に172,175, 177及び/又は178位にあるアミノ酸残基、例えば突然変異N 172S,F177FRG, Q 178I,Eの中の少なくとも1; j)アミノ酸残基S187,A209及び/又はT217の中の少なくとも1、特に突 然変異S 187D,A 209V及び/又はT 217Kの中の少なくとも1; k)アミノ酸残基L230,V233又はR242の中の少なくとも1 、特に突然変異L 230I,V 233A及び/又はR 242P; l)246−251領域内にある少なくとも1のアミノ酸残基、特にアミノ酸残基24 6,247,250及び/又は251、特にH 247A,Y,E 250Q,S及び/又はK 251 A,Qの中の少なくとも1; m)アミノ酸残基E255、特に突然変異E 255P; n)260−269領域内にあるアミノ酸残基の中の少なくとも1、特に260,264, 265,267,268及び/又は269にあるアミノ酸残基、例えば以下の突然変異:A 2 60G,N 265Y,A 269Kの中の少なくとも1; o)290−293領域内にあるアミノ酸残基の中の少なくとも1、特に290,291及 び/又は293位にあるアミノ酸残基、例えば突然変異Y 290F,N,Q 291K, H 293Q,Yの中の少なくとも1; p)314−320領域内にあるアミノ酸残基の中の少なくとも1、例えば315,318 及び/又は320位にあるアミノ酸残基、例えば以下の突然変異:K 315D,L 31 8T及び/又はS 320A; q)アミノ酸残基T341及び/又はQ360の中の少なくとも1、特に突然変異T 341P及び/又はQ 360C; r)369−383領域内にあるアミノ酸残基の中の少なくとも1、例えば370,371 ,372,373,374,375,376,379及び/又は382位にあるアミノ酸残基、例えば 以下の突然変異370*,371*,372*,(370−372)*,S 373P,Q 374P,R 375Y ,A 379S,H 382Sの中の少なくとも1; s)393,398及び/又は409位にあるアミノ酸残基の中の少なくとも1、例え ば以下の突然変異Q 393D,A 398T,P及び/又はV 409I; t)416−421領域内にあるアミノ酸残基の中の少なくとも1、例えば419,420 及び/又は421位にあるアミノ酸残基、例えは突然 変異V 419K,A 420P,N 421Gの中の少なくとも1; u)アミノ酸残基A435及び/又はH450、特に以下の突然変異A 435S及び/ 又はH 450Y;又は v)458−465領域内にあるアミノ酸残基の中の少なくとも1、例えは458,459 及び/又は461位にあるアミノ酸残基、例えば以下の突然変異:P 459T,V 46 1K,Tの中の少なくとも1; である、請求項6に記載の変異体。 13.プロリンとは異なるアミノ酸残基がプロリン残基により置換されている、 請求項1−12のいずれか1項に記載の変異体。 14.以下の突然変異:R 242P,E 255P,T 341P,S 373P,Q 374P, A 420P,Q 482Pの中の少なくとも1を含んで成る、請求項13に記載の変異体 。 15.M197位又はE255位における突然変異をさらに含んで成る、請求項6−14 のいずれか1項に記載の変異体。 16.以下の突然変異M 197T,G,I,A,L,A,S,N,C又はE 255P の中の1を含んで成る、請求項15に記載の変異体。 17.以下の突然変異: の中の1を含んで成るバチルス・リケニフォルミス(B.Licheniformis)由来の 親α−アミラーゼの変異体。 18.少なくとも2のα−アミラーゼ、例えば微生物と哺乳類α−アミラーゼ由 来の部分的アミノ酸配列の組合せを含んで成るハイブリゥドα−アミラーゼであ る、請求項1に記載の変異体。 19.少なくとも2のバクテリアのα−アミラーゼ、少なくとも1のバクテリア と1の真菌のα−アミラーゼ、又は少なくとも2の真菌のα−アミラーゼの組合 せを含んで成る、請求項18に記載の変異体。 20.バチルス・リケニフォルミス(B.Licheniformis)の株由来のα−アミラ ーゼのC−末端部分及びバチルス・アミロリクエファシエンス(B.amyloliquef aciens )の株又はバチルス・ステアロサーモフィラス(B.stearothermophilus )の株由来のα−アミラーゼのN−末端部分を含んで成る、請求項19に記載の変 異体。 21.バチルス・リケニフォルミス(B.liCheniformis)α−アミラーゼのC− 末端部分の少なくとも430アミノ酸残基を含んで成る、請求項20に記載の変異体 。 22.−配列番号:4に示すアミノ酸配列をもつバチルス・アミロリクエファシ エンス(B.amyloliquefaciens)α−アミラーゼの37 N−末端アミノ酸残基に対応するアミノ酸セグメント及び配列番号:2に示すア ミノ酸配列をもつバチルス・リケニフォルミス(B.Licheniformis)α−アミラ ーゼの445C−末端アミノ酸残基に対応するアミノ酸セグメント、又は −配列番号:6に示すアミノ酸配列をもつバチルス・ステアロサーモフィラス (B.stearothermophilus)α−アミラーゼの68N−末端アミノ酸残基に対応す るアミノ酸セグメント及び配列番号:2に示すアミノ酸配列をもつバチルス・リ ケニフォルミス(B.Licheniformis)α−アミラーゼ415C−末端アミノ酸残基 に対応するアミノ酸セグメント、 を含んで成る、請求項21に記載の変異体。 23.親α−アミラーゼのものよりも高い加水分解速度を示す、先の請求項のい ずれか1項に記載のα−アミラーゼの変異体。 24.請求項1〜22の中のいずれか1項に記載のα−アミラーゼ変異体をコード するDNA配列を含んで成るDNA構築物。 25.請求項24に記載のDNA構築物を担持する組換え発現ベクター。 26.請求項23に記載のDNA構築物又は請求項25に記載のベクターにより形質転 換された細胞。 27.微生物である、請求項26に記載の細胞。 28.バクテリア又は真菌である、請求項27に記載の細胞。 29.グラム陽性バクテリア、例えばバチルス・サブチリス(Bacillus subtil is )、バチルス・リケニフォルミス(Bacillus licheniformis)、バチルス・ レンタス(Bacillus lentus)、バチルス・ブレビス(Bacillus brevis)、バ チルス・ステアロサーモフィラス(Bacillus stearothermophilus)、バチルス ・アルカロフィラス(Bacillus alkalophilus)、バチルス・アミロリクエファ シエンス(Bacillus amyloliquefaciens)、バチルス・コアギュランス(Bacil lus coagulans)、バチルス・サーキュランス(Bacillus circulans)、バチ ルス・ラウタス(Bacillus lautus)、バチルス・チューリンゲンシス(Bacill us thuringiensis)、又はストレプトミセス・リビダンス(Streptomyces liv idans )又はストレプトミセス・ムリナス(Streptomyces murinus)、又はグラ ム陽性バクテリア、例えば大腸菌(E.coli)である請求項28に記載の細胞。 30.請求項1〜23のいずれか1項に記載のα−アミラーゼ変異体の製造方法で あって、請求項26〜29のいずれか1項に記載の細胞を、そのα−アミラーゼ変異 体の生産を誘導する条件下で培養し、そしてそのα−アミラーゼ変異体をその後 にそのカルチャーから回収するような方法。 31.その親酵素の中のいずれと比較しても改良された洗浄及び/又は皿洗い性 能をもつハイブリッドα−アミラーゼを製造する方法であって、 a)その親α−アミラーゼの中の1のα−アミラーゼ遺伝子又は対応cDNAのN −末端コーディング領域を、他の親α−アミラーゼのα−アミラーゼ遺伝子又は 対応のcDNAのC−末端コーディング領域と組換えて、組換え体を作り、 b)その親α−アミラーゼのいずれと比較しても改良された洗浄及び/又は皿 洗い性能をもつハイブリッドα−アミラーゼを生産する組換え体を選択し、 c)適当な培養基中で好適な条件下段階b)において選択した組換え体を培養 し、そして d)段階c)において得られたカルチャーからそのハイブリッドα−アミラー ゼを回収する、 ことを含んで成る方法。 32.親α−アミラーゼの中の少なくとも1がハイブリッドα−アミラーゼであ る、請求項31に記載の方法。 33.N−末端コーディング領域がバチルス・アミロリクエファシエンス(Baci llus amyloliquefaciens)の株又はバチルス・ステアロサーモフィラス(Bacil lus stearothermophilus)の株由来であり、そしてそのC−末端コーディング 領域がバチルス・リケニフォルミス(B.Licheniformis)の株由来である、請求 項32に記載の方法。 34.N−末端コーディング領域がバチルス・アミロリクエファシエンス(Baci llus amyloliquefaciens)又はバチルス・ステアロサーモフイラス(Bacillus stearothermophilus)α−アミラーゼのN−末端部分をコードし、そのアミノ 酸配列が、それぞれ、配列番号:4と6に示すものである、請求項33に記載の方 法。 35.C−末端コーディング漁期がバチルス・リケニフォルミス(B.Lichenifo rmis )α−アミラーゼのC−末端部分であり、そのアミノ酸配列が図2に示すも のである、請求項34に記載の方法。 36.親α−アミラーゼに比べたとき改良された洗浄及び/又は皿洗い性能をも つ親α−アミラーゼの変異体の製造方法であって、 a)その親α−アミラーゼの中の変異体をコードする遺伝子を含む細胞の集団 を構築し、 b)少なくとも1の洗浄及び/又は皿洗い条件を刺激する条件下でα−アミラ ーゼ活性についてその細胞集団をスクリーニングし、 c)段階b)において選択された条件下でその親α−アミラーゼと比べて改良 された活性をもつその親α−アミラーゼの変異体をコードする遺伝子を含む細胞 を上記集団から単離し、 d)適当な培養基中で好適な条件下段階c)において単離された 細胞を培養し、そして e)段階d)において得られたカルチャーからそのα−アミラーゼ変異体を回 収する、 ことを含んで成る方法。 37.段階a)において構築された細胞集団を、ランダム又は部位指定突然変異 誘発により構築する、請求項36に記載の方法。 38.段階b)の条件が、洗浄及び/又は皿洗いが行われるところの温度又はpH 及び/又は慣用の洗浄及び/又は皿洗い洗剤組成物の少なくとも1の構成成分の 存在を含む、請求項36に記載の方法。 39.請求項30〜38の中のいずれかに記載の方法により製造されるα−アミラー ゼ変異体。 40.洗浄及び/又は皿洗いのための、請求項1〜23又は39のいずれか1項に記 載のα−アミラーゼ変異体の使用。 41.場合により非発塵粒状物、安定化された液体又は保護された酵素の形態に ある、請求項1〜23又は39のいずれか1項に記載のα−アミラーゼ変異体を含ん で成る洗剤添加物。 42.0.02〜200mgの酵素/添加物1gを含む、請求項41に記載の洗剤添加物。 43.他の酵素、例えばプロテアーゼ、リパーゼ、ペルオキシダーゼ、他のデン プン分解酵素及び/又はセルラーゼをさらに含んで成る、請求項40又は41に記載 の洗剤添加物。 44.請求項1〜23又は39のいずれか1項に記載のα−アミラーゼ変異体を含ん で成る洗剤組成物。 45.他の酵素、例えばプロテアーゼ、リパーゼ、ペルオキシダーゼ、他のデン プン分解酵素及び/又はセルラーゼをさらに含んで成る、請求項44に記載の洗剤 組成物。 46.請求項1〜23又は39のいずれか1項に記載のα−アミラーゼ 変異体を含んで成る手動又は自動皿洗い洗剤組成物。 47.他の酵素、例えばプロテアーゼ、リパーゼ、ペルオキシダーゼ、他のデン プン分解酵素及び/又はセルラーゼをさらに含んで成る、請求項46に記載の皿洗 い洗剤組成物。 48.請求項1〜23又は39のいずれか1項に記載のα−アミラーゼ変異体を含ん で成る手動又は自動洗濯洗浄組成物。 49.他の酵素、例えばプロテアーゼ、リパーゼ、ペルオキシダーゼ、デンプン 分解酵素及び/又はセルラーゼをさらに含んで成る、請求項48に記載の洗濯洗浄 組成物。 50.繊維糊抜きのための、請求項1〜23又は39のいずれか1項に記載のα−ア ミラーゼ変異体の使用。
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DK1133/93 | 1993-10-08 | ||
DK113393A DK113393D0 (ja) | 1993-10-08 | 1993-10-08 | |
DK0140/94 | 1994-02-02 | ||
DK14094 | 1994-02-02 | ||
PCT/DK1994/000370 WO1995010603A1 (en) | 1993-10-08 | 1994-10-05 | Amylase variants |
US08/720,899 US5753460A (en) | 1993-10-08 | 1996-10-10 | Amylase variants |
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JP2007187147A Division JP4510856B2 (ja) | 1993-10-08 | 2007-07-18 | アミラーゼ変異体 |
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- 1994-10-05 BR BR9407767A patent/BR9407767A/pt not_active IP Right Cessation
- 1994-10-05 WO PCT/DK1994/000370 patent/WO1995010603A1/en active Application Filing
- 1994-10-05 AU AU78074/94A patent/AU7807494A/en not_active Abandoned
-
1996
- 1996-04-04 FI FI961524A patent/FI119996B/fi not_active IP Right Cessation
- 1996-10-10 US US08/720,899 patent/US5753460A/en not_active Expired - Lifetime
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2001520006A (ja) * | 1997-10-13 | 2001-10-30 | ノボ ノルディスク アクティーゼルスカブ | α−アミラーゼ変異体 |
JP2002530072A (ja) * | 1998-11-16 | 2002-09-17 | ノボザイムス アクティーゼルスカブ | α−アミラーゼ変異体 |
JP2005245453A (ja) * | 1999-02-04 | 2005-09-15 | Diversa Corp | 遺伝的ワクチンおよび酵素の非確率論的生成 |
JP2002540785A (ja) * | 1999-03-30 | 2002-12-03 | ノボザイムス アクティーゼルスカブ | α−アミラーゼ変異体 |
JP4668426B2 (ja) * | 1999-03-30 | 2011-04-13 | ノボザイムス アクティーゼルスカブ | α−アミラーゼ変異体 |
JP2011525350A (ja) * | 2008-06-06 | 2011-09-22 | ダニスコ・ユーエス・インク | バシラス・スブチリス由来のアルファ−アミラーゼ変異体とその使用方法 |
Also Published As
Publication number | Publication date |
---|---|
FI119996B (fi) | 2009-05-29 |
US5753460A (en) | 1998-05-19 |
FI961524A (fi) | 1996-05-30 |
WO1995010603A1 (en) | 1995-04-20 |
CN1189558C (zh) | 2005-02-16 |
FI961524A0 (fi) | 1996-04-04 |
EP0722490B2 (en) | 2013-10-23 |
BR9407767A (pt) | 1997-03-18 |
EP0722490A1 (en) | 1996-07-24 |
CN1134725A (zh) | 1996-10-30 |
EP0722490B1 (en) | 2006-02-22 |
AU7807494A (en) | 1995-05-04 |
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