JP2000245466A - Mutant alpha-amylase - Google Patents
Mutant alpha-amylaseInfo
- Publication number
- JP2000245466A JP2000245466A JP11048213A JP4821399A JP2000245466A JP 2000245466 A JP2000245466 A JP 2000245466A JP 11048213 A JP11048213 A JP 11048213A JP 4821399 A JP4821399 A JP 4821399A JP 2000245466 A JP2000245466 A JP 2000245466A
- Authority
- JP
- Japan
- Prior art keywords
- amylase
- gly
- amino acid
- asn
- mutant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、アルカリ側に至適
pHを有し、かつ優れた熱安定性を有し、特に洗剤用酵素
として有用な変異α−アミラーゼ及びその遺伝子並びに
該変異α−アミラーゼを含有する洗浄剤組成物等に関す
る。BACKGROUND OF THE INVENTION The present invention
The present invention relates to a mutant α-amylase having a pH and excellent heat stability and particularly useful as a detergent enzyme, a gene thereof, and a detergent composition containing the mutant α-amylase.
【０００２】[0002]
【従来の技術】α−アミラーゼ［ＥＣ３．２．１．１］
は、衣料や食器等の澱粉汚れや、糊剤の除去に有効であ
ることが知られているが、洗剤においては、洗浄水がア
ルカリとなるため、アルカリ側に至適pHを有するものが
好ましい。その例として、好アルカリ性Bacillus sp. K
SM-AP1378（FERM BP-3048）の生産する、配列番号１で
示されるアミノ酸配列を有するアルカリ液化型α−アミ
ラーゼが知られており（WO94/26881）、洗浄剤用として
極めて有用である。2. Description of the Related Art α-amylase [EC 3.2.1.1]
Is known to be effective in removing starch stains and clothes from tableware, etc., but in detergents, since washing water is alkali, those having an optimum pH on the alkali side are preferable. . An example is the alkaliphilic Bacillus sp. K.
An alkaline liquefied α-amylase having the amino acid sequence represented by SEQ ID NO: 1 produced by SM-AP1378 (FERM BP-3048) is known (WO94 / 26881) and is extremely useful as a detergent.
【０００３】[0003]
【発明が解決しようとする課題】しかし、衣料や食器の
洗浄は、１０〜５０℃で行われるのが一般的であり、洗
剤に配合するα−アミラーゼは、室温〜５０℃で熱安定
性に優れていることが好ましいが、上記アルカリ液化型
α−アミラーゼは、耐熱性がやや低かった。However, clothes and dishes are generally washed at 10 to 50 ° C., and α-amylase to be added to the detergent is not heat-stable at room temperature to 50 ° C. Although it is preferably excellent, the alkali liquefied α-amylase had a slightly lower heat resistance.
【０００４】α−アミラーゼの耐熱性を向上させる方法
として、特定位置のアミノ酸を欠失又は他のアミノ酸に
置換する方法があり、例えば１３３位のヒスチジンをチ
ロシンに置換したBacillus licheniformis由来のα−ア
ミラーゼが知られている（J.Biol. Chem., 15481-1548
8, 1990）。As a method for improving the heat resistance of α-amylase, there is a method of deleting an amino acid at a specific position or replacing it with another amino acid. For example, α-amylase derived from Bacillus licheniformis in which histidine at position 133 is substituted with tyrosine Is known (J. Biol. Chem., 15481-1548).
8, 1990).
【０００５】しかし、配列番号１で示されるアミノ酸配
列を有するα−アミラーゼでは、１３３位に相当する位
置にヒスチジンは存在せず、かかる方法を用いることは
できなかった。このため配列番号１で示されるアミノ酸
配列を有するα−アミラーゼの優れた特性を維持しつ
つ、耐熱性に優れたα−アミラーゼが求められていた。However, in the α-amylase having the amino acid sequence represented by SEQ ID NO: 1, histidine was not present at the position corresponding to position 133, and such a method could not be used. For this reason, there has been a demand for an α-amylase having excellent heat resistance while maintaining the excellent properties of the α-amylase having the amino acid sequence represented by SEQ ID NO: 1.
【０００６】[0006]
【課題を解決するための手段】本発明者は、これまで全
く注目されなかった１９３位のイソロイシンに着目し
た。そして該α−アミラーゼの１９３位のイソロイシン
を欠失又は他のアミノ酸に置換することにより、該α−
アミラーゼの有する優れた特性を維持しながら、耐熱性
が向上することを見出した。Means for Solving the Problems The present inventors have focused on isoleucine at position 193, which has never been noticed before. Then, the isoleucine at position 193 of the α-amylase is deleted or substituted with another amino acid, thereby obtaining the α-amylase.
It has been found that the heat resistance is improved while maintaining the excellent properties of amylase.
【０００７】すなわち、本発明は、配列番号１で示され
るアミノ酸配列又は該アミノ酸配列に対して７０％以上
の相同性を有するアミノ酸配列における１９３位のイソ
ロイシンに相当するアミノ酸が欠失又は他のアミノ酸に
置換されたアミノ酸配列を有する変異α−アミラーゼを
提供するものである。本発明はまた、かかる変異α−ア
ミラーゼをコードする遺伝子を提供するものである。本
発明はまた、かかる遺伝子を含有する組換えベクターを
提供するものである。本発明はまた、かかる組換えベク
ターで形質転換又は染色体相同組換えされた形質転換細
胞を提供するものである。本発明はまた、かかる変異α
−アミラーゼを含有する洗浄剤組成物を提供するもので
ある。[0007] That is, the present invention relates to an amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having 70% or more homology to the amino acid sequence, wherein the amino acid corresponding to isoleucine at position 193 is deleted or other amino acids are deleted. And a mutant α-amylase having an amino acid sequence substituted by The present invention also provides a gene encoding such a mutant α-amylase. The present invention also provides a recombinant vector containing such a gene. The present invention also provides a transformed cell transformed or chromosomally homologously transformed with the recombinant vector. The present invention also relates to such a mutant α
-To provide a detergent composition containing amylase.
【０００８】[0008]
【発明の実施の形態】本発明の変異α−アミラーゼは、
配列番号１に示すアミノ酸配列又は配列番号１に示すア
ミノ酸配列と７０％以上の相同性を有する液化型アルカ
リα−アミラーゼ（以下、「配列番号１等で示されるα
−アミラーゼ」という。）を欠失又は他のアミノ酸に置
換して得られるものである。配列番号１で示される液化
型アルカリα−アミラーゼとしては、例えば本発明者ら
が先に報告したWO94/26881記載の、Bacillus sp. KSM-A
P1378(FERM BP-3048）から得られる野生型液化型α−ア
ミラーゼが挙げられる。相同性は７０％以上であること
が必要であり、８０％以上が好ましく、９０％以上がよ
り好ましく、９５％以上が特に好ましく、９９％以上が
最も好ましい。なお相同性は、Lipman-Pearson法（Scie
nce, 227, 1435(1985)）により計算される。また配列番
号１に示すアミノ酸配列と７０％以上の相同性を示すア
ミノ酸配列を有するα−アミラーゼとしては、例えばWO
95/26397記載のNCIB 12512、NCIB 12513由来のα−アミ
ラーゼ等がある。BEST MODE FOR CARRYING OUT THE INVENTION The mutant α-amylase of the present invention comprises
The amino acid sequence represented by SEQ ID NO: 1 or a liquefied alkaline α-amylase having 70% or more homology with the amino acid sequence represented by SEQ ID NO: 1 (hereinafter referred to as “α represented by SEQ ID NO: 1 or the like”
-Amylase ". ) Is deleted or substituted with another amino acid. The liquefied alkaline α-amylase represented by SEQ ID NO: 1 includes, for example, Bacillus sp. KSM-A described in WO94 / 26881 previously reported by the present inventors.
Wild-type liquefied α-amylase obtained from P1378 (FERM BP-3048). The homology needs to be 70% or more, preferably 80% or more, more preferably 90% or more, particularly preferably 95% or more, and most preferably 99% or more. Homology was determined by the Lipman-Pearson method (Scie
nce, 227, 1435 (1985)). Examples of α-amylase having an amino acid sequence showing 70% or more homology with the amino acid sequence shown in SEQ ID NO: 1 include, for example, WO
95/26397, α-amylase derived from NCIB 12513, and the like.
【０００９】欠失又は他のアミノ酸への置換のうち、他
のアミノ酸に置換することが好ましく、アスパラギン酸
に置換することがより好ましい。これにより耐熱性が向
上する。さらに、１８１位のアルギニン及び１８２位の
グリシンに相当するアミノ酸が欠失したものが特に好ま
しい。これにより耐熱性がさらに向上し、また通常洗剤
に含まれるキレート剤に対する耐性も向上する。[0009] Of the deletions or substitutions with other amino acids, substitution with other amino acids is preferred, and substitution with aspartic acid is more preferred. Thereby, heat resistance is improved. Furthermore, those in which amino acids corresponding to arginine at position 181 and glycine at position 182 have been deleted are particularly preferred. Thereby, heat resistance is further improved, and resistance to a chelating agent usually contained in a detergent is also improved.
【００１０】本発明の変異α−アミラーゼは、例えば配
列番号１等で示されるα−アミラーゼをコードするＤＮ
Ａに部位特異的変異を導入して、変異α−アミラーゼを
コードする遺伝子を得、次いでこれを含有する組換えベ
クター、好ましくはプラスミドを作成し、該ベクターを
用いて宿主を形質転換するか、染色体相同組換えにより
形質転換体を得、これを培養することによって製造され
る。部位特異的変異の方法としては一般的に行なわれて
いる方法であればいずれも採用できるが、例えばTakara
社のSite-Directed Mutagenesis System Mutan Super E
xpress Km Kit等を用いて行なうことができる。本発明
の変異α−アミラーゼをコードする遺伝子としては、例
えば配列番号２で示される塩基配列を有するものが挙げ
られる。The mutant α-amylase of the present invention is, for example, a DNA encoding α-amylase represented by SEQ ID NO: 1 or the like.
A site-specific mutation is introduced into A to obtain a gene encoding the mutant α-amylase, and then a recombinant vector, preferably a plasmid, containing the gene is prepared, and a host is transformed with the vector, It is produced by obtaining a transformant by homologous chromosome recombination and culturing it. Any site-specific mutation can be used as long as it is a commonly used method. For example, Takara
Site-Directed Mutagenesis System Mutan Super E
It can be performed using the xpress Km Kit or the like. The gene encoding the mutant α-amylase of the present invention includes, for example, a gene having a base sequence represented by SEQ ID NO: 2.
【００１１】なお配列番号１で示されるアミノ酸配列と
７０％以上の相同性を有する他のアミノ酸配列を、一文
字表記で図１及び図２に示した。The other amino acid sequence having a homology of 70% or more with the amino acid sequence represented by SEQ ID NO: 1 is shown in single letter notation in FIGS.
【００１２】かくして得られる本発明変異α−アミラー
ゼは、熱に対する安定性が向上し、洗浄剤組成物、澱粉
液化、糖化用組成物、繊維糊抜き剤等として有用であ
る。ここで本発明の洗浄剤組成物には、本発明変異α−
アミラーゼ以外に、さらに、枝切り酵素（例えばプルラ
ナーゼ、イソアミラーゼ、ネオプルラナーゼなど）、α
−グルコシダーゼ、グルコアミラーゼ、プロテアーゼ、
セルラーゼ、リパーゼ、ペクチナーゼ、プロトペクチナ
ーゼ、ペクチン酸リアーゼ、パーオキシダーゼ、ラッカ
ーゼ及びカタラーゼからなる群より選ばれる１種以上の
酵素を配合することができる。また、洗浄剤組成物に通
常配合される界面活性剤、キレート剤、アルカリ剤、無
機塩、再汚染防止剤、塩素捕捉剤、還元剤、漂白剤、蛍
光染料可溶化剤、香料、ケーキング防止剤、酵素の活性
化剤、酸化防止剤、防腐剤、色素、青味付け剤、漂白活
性化剤、酵素安定化剤、調節剤等を配合することができ
る。The thus obtained mutant α-amylase of the present invention has improved stability against heat and is useful as a detergent composition, a starch liquefaction, a saccharification composition, a fiber desizing agent and the like. Here, the detergent composition of the present invention contains the mutant α-
In addition to amylase, further, a debranching enzyme (eg, pullulanase, isoamylase, neopurulanase, etc.), α
-Glucosidase, glucoamylase, protease,
One or more enzymes selected from the group consisting of cellulase, lipase, pectinase, protopectinase, pectate lyase, peroxidase, laccase and catalase can be blended. Also, a surfactant, a chelating agent, an alkali agent, an inorganic salt, a redeposition inhibitor, a chlorine scavenger, a reducing agent, a bleaching agent, a fluorescent dye solubilizing agent, a fragrance, an anti-caking agent which are usually blended in a detergent composition , An enzyme activator, an antioxidant, a preservative, a pigment, a bluing agent, a bleach activator, an enzyme stabilizer, a regulator and the like.
【００１３】本発明の洗浄剤組成物は、本発明変異α−
アミラーゼ及び上記公知の洗浄成分を配合し、常法に従
い製造できる。洗浄剤の形態は、用途に応じて選択する
ことができ、例えば、液体、粉末、顆粒等にすることが
できる。また、本発明洗浄剤組成物は、衣料用洗浄剤、
漂白洗浄剤、自動食器洗浄機用洗浄剤、配水管洗浄剤、
義歯洗浄剤等として使用できるが、特に衣料用洗浄剤、
漂白洗浄剤、自動食器洗浄機用洗浄剤として好適に使用
することができる。[0013] The detergent composition of the present invention comprises the mutant α-form of the present invention.
Amylase and the above-mentioned known cleaning components are blended and can be produced according to a conventional method. The form of the cleaning agent can be selected according to the use, and for example, can be liquid, powder, granules, and the like. Further, the cleaning composition of the present invention is a cleaning agent for clothing,
Bleaching detergent, detergent for automatic dishwasher, drain cleaner,
Can be used as a denture cleaner, etc., especially for clothes,
It can be suitably used as a bleaching detergent and a detergent for automatic dishwashers.
【００１４】また、本発明の変異α−アミラーゼを用い
た澱粉液化・糖化用組成物は、本発明の変異α−アミラ
ーゼ以外に必要に応じてさらにグルコアミラーゼ、マル
ターゼ、プルラナーゼ、イソアミラーゼ、ネオプルラナ
ーゼからなる群より選ばれる１種以上の酵素を配合して
得ることができる。該組成物を用いて常法に従い澱粉を
液化・糖化することができる。The starch liquefaction / saccharification composition using the mutant α-amylase of the present invention may further comprise, if necessary, glucoamylase, maltase, pullulanase, isoamylase, neo pullulanase in addition to the mutant α-amylase of the present invention. And one or more enzymes selected from the group consisting of: Using the composition, starch can be liquefied and saccharified according to a conventional method.
【００１５】また本発明の変異α−アミラーゼを用いた
繊維糊抜き剤は、本発明の変異α−アミラーゼ以外に、
さらに必要に応じてプルラナーゼ、イソアミラーゼ、ネ
オプルラナーゼ等を配合して得ることができる。該糊抜
き剤は、常法に従い使用できる。[0015] The fiber desizing agent using the mutant α-amylase of the present invention is, in addition to the mutant α-amylase of the present invention,
Further, if necessary, it can be obtained by blending pullulanase, isoamylase, neo pullulanase and the like. The desizing agent can be used according to a conventional method.
【００１６】[0016]
【実施例】各酵素のアミラーゼ活性及び蛋白量は以下に
示す方法で測定した。アミラーゼ活性測定は、３，５−
ジニトロサリチル酸法（ＤＮＳ法）で測定した。すなわ
ち、５０mM Tris-HCl 緩衝液（pH８．５）中に可溶性澱
粉を溶解した反応液を用い、４０℃で１５分間の反応を
行なった後、生成した還元糖をＤＮＳ法で定量すること
によって測定した。酵素の力価は１分間に１μmol のグ
ルコースに相当する還元糖を生成する酵素量を１単位と
した。蛋白量の測定は、牛血清アルブミンを標準とし
て、Bio-Rad 社のProtein Assay Kit を用いて定量し
た。EXAMPLES The amylase activity and protein content of each enzyme were measured by the following methods. Amylase activity measurement was performed using 3,5-
It was measured by the dinitrosalicylic acid method (DNS method). That is, the reaction was carried out at 40 ° C. for 15 minutes using a reaction solution obtained by dissolving a soluble starch in a 50 mM Tris-HCl buffer (pH 8.5), and the amount of reducing sugar produced was measured by the DNS method. did. The enzyme titer was defined as one unit of the amount of the enzyme producing a reducing sugar corresponding to 1 μmol of glucose per minute. The amount of protein was measured using a protein assay kit from Bio-Rad, using bovine serum albumin as a standard.
【００１７】実施例１ 液化型アルカリα−アミラーゼ
遺伝子の調製 特開平８−３３６３９２号公報の実施例に従い、バチル
ス エスピー KSM-AP1378 株（FERM BP-3048）から、配
列番号２に示す塩基配列を有する遺伝子を調製した。Example 1 Preparation of Liquefied Alkaline α-Amylase Gene According to the examples in JP-A-8-336392, a nucleotide sequence represented by SEQ ID NO: 2 was obtained from Bacillus sp. Strain KSM-AP1378 (FERM BP-3048). The gene was prepared.
【００１８】実施例２ 液化型アルカリα−アミラーゼ
遺伝子への変異導入 Takara社のSite-Directed Mutagenesis System Mutan-S
uper Express Km Kitを用い、変異導入用鋳型プラスミ
ドとしてｐＫＦ１９ＬＡＭＹを用いた。本プラスミド
は、変異用プラスミドベクターｐＫＦ１９ｋのSmaＩ部
位に、我々が独自に開発した発現プロモーター領域ＳＰ
６４及びBacillus sp. KSM-AP1378 株（FERM BP-3048）
由来の配列番号２に示す塩基配列を有するα−アミラー
ゼ遺伝子（約２．１kb）を導入したものである（図３）
（Ikawa et al., Biosci.Biotech. Biochem., 62, 1720
-1725, 1998）。また、変異導入プライマーとして配列
番号３に示すものを用いた。（配列表フリーテキスト：
配列番号２の配列８１２〜８３８において、８２４〜８
２６のata をgat に置換したものである。）配列番号３
の塩基配列は、アニールする対象の鋳型配列の１９３位
のイソロイシンをコードするＡＴＡの位置を、ＡＴＡか
らＧＡＴへ入れ替えたものであり、これを用いて、イソ
ロイシンをアスパラギン酸に置換した。これをＩ１９３
Ｄと略記する。次いで、配列番号４に示す塩基配列を有
する変異導入プライマーを用いて、１８１位のアルギニ
ン及び１８２位のグリシンをコードするＡＧＡ及びＧＧ
Ｔを欠失させ、これにより両アミノ酸が欠失した変異α
−アミラーゼが得られた（RG+I193D）。（配列表フリー
テキスト：配列番号２の配列７７３〜８１４において、
７８８〜７９３のaga ggt を欠失させたものである。） 尚、得られた変異体は塩基配列解析を行なって変異を確
認した。次いで、得られた変異体プラスミド（pKF19LAM
YAA）の変異部位を含む部分を増幅し（図３）、これを
高発現誘導領域SP64を含むプラスミドベクターpHSP64の
SmaＩ部位に導入した（pHSPLAMYAA、図３）。Example 2 Mutagenesis into Liquefied Alkaline α-Amylase Gene Takara's Site-Directed Mutagenesis System Mutan-S
Using the upper Express Km Kit, pKF19LAMY was used as a template plasmid for mutagenesis. This plasmid, to the Sma I site of the mutant plasmid vector for pKF19k, expression promoter region SP that we have independently developed
64 and Bacillus sp. KSM-AP1378 strain (FERM BP-3048)
An α-amylase gene (approximately 2.1 kb) having the nucleotide sequence shown in SEQ ID NO: 2 is introduced (FIG. 3).
(Ikawa et al., Biosci. Biotech. Biochem., 62, 1720
-1725, 1998). The primer shown in SEQ ID NO: 3 was used as a mutation-introducing primer. (Sequence list free text:
In sequences 812 to 838 of SEQ ID NO: 2, 824 to 8
26 in which ata was replaced with gat. ) SEQ ID NO: 3
Is obtained by replacing the position of ATA encoding isoleucine at position 193 of the template sequence to be annealed with ATA to GAT, and using this to replace isoleucine with aspartic acid. This is I193
Abbreviated as D. Next, AGA and GG encoding arginine at position 181 and glycine at position 182 using a mutation-introducing primer having the nucleotide sequence shown in SEQ ID NO: 4.
T, thereby deleting both amino acids
-Amylase was obtained (RG + I193D). (Sequence listing free text: In sequences 773 to 814 of SEQ ID NO: 2,
Aga-ggt of 788 to 793 was deleted. In addition, the obtained mutant was subjected to nucleotide sequence analysis to confirm the mutation. Then, the resulting mutant plasmid (pKF19LAM
(FIG. 3).
It was introduced at the SmaI site (pHSPLAMYAA, FIG. 3).
【００１９】実施例３ 熱安定性変異α−アミラーゼ
（I193D 及びRG+I193D）の大量培養 変異プラスミド（pHSPLAMYAA）をB. subtilis ISW1214
株（leuA metB5 hsdM1）にプロトプラスト法（Chang &
Cohen, Mol. Gen. Genet., 168, 111-115, 1979）によ
り形質転換し、適当な液体培地（コーンスティープリカ
ー、４％；トリプトース、１％；肉エキス、１％；リン
酸１カリウム、０．１％；硫酸マグネシウム０．０１
％；マルトース、２％；塩化カルシウム、０．１％；テ
トラサイクリン、１５μｇ／ml）で３０℃で３日間、坂
口フラスコ中で培養した。得られた培養上清液を硫安分
画（６０％飽和）して２mM CaCl2を含むTris-HCl緩衝液
pH７．５にて透析し、同緩衝液にて平衡化した陰イオン
交換樹脂カラム（DEAE−トヨパール）を通過させた後、
この通過溶液を陽イオン交換樹脂カラム（CM−トヨパー
ル）に吸着させ、塩化ナトリウムの濃度勾配で溶出させ
た。この溶出液を限外濾過膜（アミコン社のPM−10）に
より濃縮し、上記緩衝液にて透析することにより、均一
な精製酵素を得ることができた。得られた、精製品は、
SDS-PAGEにより単一バンドを与え、分子量は約５５kDa
と決定された。Example 3 Large-Scale Culture of Thermostable Mutant α-Amylase (I193D and RG + I193D) The mutant plasmid (pHSPLAMYAA) was isolated from B. subtilis ISW1214.
Strain ( leu A met B5 hsd M1) by protoplast method (Chang &
Cohen, Mol. Gen. Genet., 168, 111-115, 1979) and a suitable liquid medium (corn steep liquor, 4%; tryptose, 1%; meat extract, 1%; 0.1%; magnesium sulfate 0.01
%; Maltose, 2%; calcium chloride, 0.1%; tetracycline, 15 μg / ml) at 30 ° C. for 3 days in a Sakaguchi flask. The obtained culture supernatant is subjected to ammonium sulfate fractionation (60% saturation), and Tris-HCl buffer containing 2 mM CaCl 2
After dialysis at pH 7.5 and passing through an anion exchange resin column (DEAE-Toyopearl) equilibrated with the same buffer,
This passing solution was adsorbed on a cation exchange resin column (CM-Toyopearl) and eluted with a sodium chloride concentration gradient. The eluate was concentrated using an ultrafiltration membrane (PM-10, manufactured by Amicon) and dialyzed against the above buffer to obtain a uniform purified enzyme. The obtained purified product is
A single band was given by SDS-PAGE, and the molecular weight was about 55 kDa.
It was decided.
【００２０】実施例４ 熱安定性の検定 上記で得られたＩ１９３Ｄ及びＲＧ＋Ｉ１９３Ｄについ
て、次に示す手法で熱安定性を検定した。対照として野
生型を用いた。Example 4 Test of Thermal Stability The thermal stability of I193D and RG + I193D obtained above was tested by the following method. Wild type was used as a control.
【００２１】安定性実験１ あらかじめ１０mM Tris-HCl pH８．５緩衝液を５０℃に
てプリインキュベートした中に、約０．２Ｕ／mlとなる
よう酵素を添加後、０分、２０分、４０分、６０分にサ
ンプリングし、上記実施例に示す方法で残存するアミラ
ーゼ活性を測定した。それぞれのスタート時の活性を１
００％として相対活性を求め、アミラーゼ残存活性とし
た。結果を表１に示す。Stability test 1 After pre-incubating 10 mM Tris-HCl pH 8.5 buffer at 50 ° C. and adding enzyme to about 0.2 U / ml, 0 minutes, 20 minutes and 40 minutes Sampling was performed at 60 minutes, and the remaining amylase activity was measured by the method described in the above example. Each activity at the start is 1
The relative activity was determined by setting the activity to 00%, and was defined as the amylase residual activity. Table 1 shows the results.
【００２２】[0022]
【表１】 [Table 1]
【００２３】６０分後の残存活性が、野生型ではほとん
どないのに対し、Ｉ１９３Ｄでは５０％以上であった。
ＲＧ＋Ｉ１９３Ｄではほとんど活性が減少しなかった。The residual activity after 60 minutes was almost non-existent in the wild type, whereas it was 50% or more in I193D.
RG + I193D showed little decrease in activity.
【００２４】安定性実験２ ０．１、０．５、１．０mMの塩化カルシウムを含む１０
mM Tris-HCl pH８．５を予め６０℃で５分間プリインキ
ュベートした。その中に野生型及びＲＧ＋Ｉ１９３Ｄを
約０．２Ｕ／mlとなるように加え、６０℃、３０分間放
置した後、残存するアミラーゼ活性を測定した。残存活
性はインキュベート前の活性を１００％とし、相対活性
で示した。結果を表２に示す。Stability Experiment 2 10 containing 0.1, 0.5 and 1.0 mM calcium chloride
mM Tris-HCl pH 8.5 was pre-incubated at 60 ° C. for 5 minutes in advance. Wild type and RG + I193D were added thereto at a concentration of about 0.2 U / ml, left at 60 ° C. for 30 minutes, and the remaining amylase activity was measured. The residual activity was shown as a relative activity, taking the activity before incubation as 100%. Table 2 shows the results.
【００２５】[0025]
【表２】 [Table 2]
【００２６】ＲＧ＋Ｉ１９３Ｄの熱安定性は、野生型よ
り顕著に向上した。The thermal stability of RG + I193D was significantly improved over the wild type.
【００２７】実施例５ 表３に示す配合で自動食器洗浄機用洗浄剤組成物を製造
した。本洗浄剤に、実施例３で得られた変異α−アミラ
ーゼを配合することにより、優れた洗浄効果を示した。Example 5 A detergent composition for an automatic dishwasher was prepared according to the formulation shown in Table 3. By adding the mutant α-amylase obtained in Example 3 to this detergent, an excellent cleaning effect was exhibited.
【００２８】[0028]
【表３】 [Table 3]
【００２９】[0029]
【発明の効果】本発明の変異α−アミラーゼは、至適pH
がアルカリ側にあり、かつ熱安定性が向上したものであ
る。かかる変異α−アミラーゼは、洗浄剤組成物、液化
・糖化用組成物、繊維糊抜き剤等に配合することにより
効果を発揮する。The mutant α-amylase of the present invention has an optimum pH
Are on the alkaline side and have improved thermal stability. Such a mutant α-amylase exerts its effects by being incorporated into a detergent composition, a liquefaction / saccharification composition, a fiber desizing agent, and the like.
【００３０】[0030]
【配列表】 SEQUENCE LISTING <110> KAO CORPORATION <120> Mutant α−Amylase <130> P00741102 <160> 4 <210> 1 <211> 516 <212> PRT <213> Bacillus sp. KSM-AP1378(FERM BP-3048) <400> 1 Met Lys Leu His Asn Arg Ile Ile Ser Val Leu Leu Thr Leu Leu Leu -30 -25 -20 Ala Val Ala Val Leu Phe Pro Tyr Met Thr Glu Pro Ala Gln Ala His -15 -10 -5 His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu 5 10 15 Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala Asn 20 25 30 Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp Lys 35 40 45 Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp 50 55 60 65 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 70 75 80 Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly Ile 85 90 95 Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn Gln 115 120 125 Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp Phe 130 135 140 145 Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr His 150 155 160 Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys Ile 165 170 175 Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Ile 180 185 190 Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp 195 200 205 His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr 210 215 220 225 Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile 230 235 240 Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr 245 250 255 Gly Lys Pro Met Phe Ala Val Ala Alu Phe Trp Lys Asn Asp Leu Ala 260 265 270 Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe 275 280 285 Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Aly Gly 290 295 300 305 Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His 310 315 320 Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly 325 330 335 Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr 340 345 350 Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly 355 360 365 Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser Lys 370 375 380 385 Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr Gln 390 395 400 His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415 Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430 Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly Gln 435 440 445 Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn 450 455 460 465 Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser Val 470 475 480 Trp Val Lys Gln 485 [Sequence List] SEQUENCE LISTING <110> KAO CORPORATION <120> Mutant α-Amylase <130> P00741102 <160> 4 <210> 1 <211> 516 <212> PRT <213> Bacillus sp. KSM-AP1378 (FERM BP -3048) <400> 1 Met Lys Leu His Asn Arg Ile Ile Ser Val Leu Leu Thr Leu Leu Leu -30 -25 -20 Ala Val Ala Val Leu Phe Pro Tyr Met Thr Glu Pro Ala Gln Ala His -15 -10- 5 His Asn Gly Thr Asn Gly Thr Met Met Gln Tyr Phe Glu Trp His Leu 5 10 15 Pro Asn Asp Gly Asn His Trp Asn Arg Leu Arg Asp Asp Ala Ala Asn 20 25 30 Leu Lys Ser Lys Gly Ile Thr Ala Val Trp Ile Pro Pro Ala Trp Lys 35 40 45 Gly Thr Ser Gln Asn Asp Val Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp 50 55 60 65 Leu Gly Glu Phe Asn Gln Lys Gly Thr Val Arg Thr Lys Tyr Gly Thr 70 75 80 Arg Ser Gln Leu Gln Gly Ala Val Thr Ser Leu Lys Asn Asn Gly Ile 85 90 95 Gln Val Tyr Gly Asp Val Val Met Asn His Lys Gly Gly Ala Asp Gly 100 105 110 Thr Glu Met Val Asn Ala Val Glu Val Asn Arg Ser Asn Arg Asn Gln 115 120 125 Glu Ile Ser Gly Glu Tyr Thr Ile Glu Ala Trp Thr Lys Phe Asp Phe 130 135 140 145 Pro Gly Arg Gly Asn Thr His Ser Asn Phe Lys Trp Arg Trp Tyr His 150 155 160 Phe Asp Gly Thr Asp Trp Asp Gln Ser Arg Gln Leu Gln Asn Lys Ile 165 170 175 Tyr Lys Phe Arg Gly Thr Gly Lys Ala Trp Asp Trp Glu Val Asp Ile 180 185 190 Glu Asn Gly Asn Tyr Asp Tyr Leu Met Tyr Ala Asp Ile Asp Met Asp 195 200 205 His Pro Glu Val Ile Asn Glu Leu Arg Asn Trp Gly Val Trp Tyr Thr 210 215 220 225 Asn Thr Leu Asn Leu Asp Gly Phe Arg Ile Asp Ala Val Lys His Ile 230 235 240 Lys Tyr Ser Tyr Thr Arg Asp Trp Leu Thr His Val Arg Asn Thr Thr 245 250 255 Gly Lys Pro Met Phe Ala Val Ala Alu Phe Trp Lys Asn Asp Leu Ala 260 265 270 Ala Ile Glu Asn Tyr Leu Asn Lys Thr Ser Trp Asn His Ser Val Phe 275 280 285 Asp Val Pro Leu His Tyr Asn Leu Tyr Asn Ala Ser Asn Ser Aly Gly 290 295 300 300 305 Tyr Phe Asp Met Arg Asn Ile Leu Asn Gly Ser Val Val Gln Lys His 310 315 320 Pro Ile His Ala Val Thr Phe Val Asp Asn His Asp Ser Gln Pro Gly 325 330 335 Glu Ala Leu Glu Ser Phe Val Gln Ser Trp Phe Lys Pro Leu Ala Tyr 340 345 350 Ala Leu Ile Leu Thr Arg Glu Gln Gly Tyr Pro Ser Val Phe Tyr Gly 355 360 365 Asp Tyr Tyr Gly Ile Pro Thr His Gly Val Pro Ser Met Lys Ser Lys 370 375 380 385 Ile Asp Pro Leu Leu Gln Ala Arg Gln Thr Tyr Ala Tyr Gly Thr Gln 390 395 400 His Asp Tyr Phe Asp His His Asp Ile Ile Gly Trp Thr Arg Glu Gly 405 410 415 Asp Ser Ser His Pro Asn Ser Gly Leu Ala Thr Ile Met Ser Asp Gly 420 425 430 Pro Gly Gly Asn Lys Trp Met Tyr Val Gly Lys His Lys Ala Gly Gln 435 440 445 Val Trp Arg Asp Ile Thr Gly Asn Arg Ser Gly Thr Val Thr Ile Asn 450 455 460 465 Ala Asp Gly Trp Gly Asn Phe Thr Val Asn Gly Gly Ala Val Ser Val 470 475 480 Trp Val Lys Gln 485
【００３１】 <210> 2 <211> 1786 <212> DNA <213> Bacillus sp. KSM-AP1378(FERM BP-3048) <400> 2 cagcgtgata atataaattt gaaatgaaca cctatgaaaa tatggtagcg attgcgcgac 60 gagaaaaaac ttgggagtta ggaagtgata ttaaaggatt ttttttgact tgttgtgaaa 120 acgcttgcat aaattgaagg agagggtgct tttt atg aaa ctt cat aac cgt ata 175 Met Lys Leu His Asn Arg Ile -30 -25 att agc gta cta tta aca cta ttg tta gct gta gct gtt ttg ttt cca 223 Ile Ser Val Leu Leu Thr Leu Leu Leu Ala Val Ala Val Leu Phe Pro -20 -15 -10 tat atg aCg gaa cca gca caa gcc cat cat aat ggg acg aat ggg acc 271 Tyr Met Thr Glu Pro Ala Gln Ala His His Asn Gly Thr Asn Gly Thr -5 5 atg atg cag tat ttt gaa tgg cat ttg cca aat gac ggg aac cac tgg 319 Met Met Gln Tyr Phe Glu Trp His Leu Pro Asn Asp Gly Asn His Trp 10 15 20 aac agg tta cga gat gac gca gct aac tta aag agt aaa ggg att acc 367 Asn Arg Leu Arg Asp Asp Ala Ala Asn Leu Lys Ser Lys Gly Ile Thr 25 30 35 40 gct gtt tgg att cct cct gca tgg aag ggg act tcg caa aat gat gtt 415 Ala Val Trp Ile Pro Pro Ala Trp Lys Gly Thr Ser Gln Asn Asp Val 45 50 55 ggg tat ggt gcc tat gat ttg tac gat ctt ggt gag ttt aac caa aag 463 Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys 60 65 70 gga acc gtc cgt aca aaa tat ggc aca agg agt cag ttg caa ggt gcc 511 Gly Thr Val Arg Thr Lys Tyr Gly Thr Arg Ser Gln Leu Gln Gly Ala 75 80 85 gtg aca tct ttg aaa aat aac ggg att caa gtt tat ggg gat gtc gtg 559 Val Thr Ser Leu Lys Asn Asn Gly Ile Gln Val Tyr Gly Asp Val Val 90 95 100 atg aat cat aaa ggt gga gca gac ggg aca gag atg gta aat gcg gtg 607 Met Asn His Lys Gly Gly Ala Asp Gly Thr Glu Met Val Asn Ala Val 105 110 115 120 gaa gtg aac cga agc aac cga aac caa gaa ata tca ggt gaa tac acc 655 Glu Val Asn Arg Ser Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Thr 125 130 135 att gaa gca tgg acg aaa ttt gat ttc cct gga aga gga aat acc cat 703 Ile Glu Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg Gly Asn Thr His 140 145 150 tcc aac ttt aaa tgg cgc tgg tat cat ttt gat ggg aca gat tgg gat 751 Ser Asn Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Thr Asp Trp Asp 155 160 165 cag tca cgt cag ctt cag aac aaa ata tat aaa ttc aga ggt acc gga 799 Gln Ser Arg Gln Leu Gln Asn Lys Ile Tyr Lys Phe Arg Gly Thr Gly 170 175 180 aag gca tgg gac tgg gaa gta gat ata gag aac ggc aac tat gat tac 847 Lys Ala Trp Asp Trp Glu Val Asp Ile Glu Asn Gly Asn Tyr Asp Tyr 185 190 195 200 ctt atg tat gca gac att gat atg gat cat cca gaa gta atc aat gaa 895 Leu Met Tyr Ala Asp Ile Asp Met Asp His Pro Glu Val Ile Asn Glu 205 210 215 ctt aga aat tgg gga gtt tgg tat aca aat aca ctt aat cta gat gga 943 Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn Thr Leu Asn Leu Asp Gly 220 225 230 ttt aga atc gat gct gtg aaa cat att aaa tac agc tat acg aga gat 991 Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr Ser Tyr Thr Arg Asp 235 240 245 tgg cta aca cat gtg cgt aac acc aca ggt aaa cca atg ttt gca gtt 1039 Trp Leu Thr His Val Arg Asn Thr Thr Gly Lys Pro Met Phe Ala Val 250 255 260 gca gaa ttt tgg aaa aat gac ctt gct gca atc gaa aac tat tta aat 1087 Ala Alu Phe Trp Lys Asn Asp Leu Ala Ala Ile Glu Asn Tyr Leu Asn 265 270 275 280 aaa aca agt tgg aat cac tcc gtg ttc gat gtt cct ctt cat tat aat 1135 Lys Thr Ser Trp Asn His Ser Val Phe Asp Val Pro Leu His Tyr Asn 285 290 295 ttg tac aat gca tct aat agt ggt ggc tat ttt gat atg aga aat att 1183 Leu Tyr Asn Ala Ser Asn Ser Aly Gly Tyr Phe Asp Met Arg Asn Ile 300 305 310 tta aat ggt tct gtc gta caa aaa cac cct ata cat gca gtc aca ttt 1231 Leu Asn Gly Ser Val Val Gln Lys His Pro Ile His Ala Val Thr Phe 315 320 325 gtt gat aac cat gac tct cag cca gga gaa gca ttg gaa tcc ttt gtt 1279 Val Asp Asn His Asp Ser Gln Pro Gly Glu Ala Leu Glu Ser Phe Val 330 335 340 caa tcg tgg ttc aaa cca ctg gca tat gca ttg att ctg aca agg gag 1327 Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Leu Ile Leu Thr Arg Glu 345 350 355 360 caa ggt tac cct tcc gta ttt tac ggt gat tac tac ggt ata cca act 1375 Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Thr 365 370 375 cat ggt gtt cct tcg atg aaa tct aaa att gat cca ctt ctg cag gca 1423 His Gly Val Pro Ser Met Lys Ser Lys Ile Asp Pro Leu Leu Gln Ala 380 385 390 cgt caa acg tat gcc tac gga acc caa cat gat tat ttt gat cat cat 1471 Arg Gln Thr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp His His 395 400 405 gat att atc ggc tgg acg aga gaa ggg gac agc tcc cac cca aat tca 1519 Asp Ile Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser His Pro Asn Ser 410 415 420 gga ctt gca act att atg tcc gat ggg cca ggg ggt aat aaa tgg atg 1567 Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly Gly Asn Lys Trp Met 425 430 435 440 tat gtc ggg aaa cat aaa gct ggc caa gta tgg aga gat atc acc gga 1615 Tyr Val Gly Lys His Lys Ala Gly Gln Val Trp Arg Asp Ile Thr Gly 445 450 455 aat agg tct ggt acc gtc acc att aat gca gat ggt tgg ggg aat ttc 1663 Asn Arg Ser Gly Thr Val Thr Ile Asn Ala Asp Gly Trp Gly Asn Phe 460 465 470 act gta aac gga ggg gca gtt tcg gtt tgg gtg aag caa taa ataaggaac 1714 Thr Val Asn Gly Gly Ala Val Ser Val Trp Val Lys Gln 475 480 485 aagaggcgaa aattactttc ctacatgcag agctttccga tcactcatac acccaatata 1774 aattggaagc tt 1786 <210> 2 <211> 1786 <212> DNA <213> Bacillus sp. agagggtgct tttt atg aaa ctt cat aac cgt ata 175 Met Lys Leu His Asn Arg Ile -30 -25 att agc gta cta tta aca cta ttg tta gct gta gct gtt ttg ttt cca 223 Ile Ser Val Leu Leu Thr Leu Leu Ala Val Ala Val Leu Phe Pro -20 -15 -10 tat atg aCg gaa cca gca caa gcc cat cat aat ggg acg aat ggg acc 271 Tyr Met Thr Glu Pro Ala Gln Ala His His Asn Gly Thr Asn Gly Thr -5 5 atg atg cag tat ttt gaa tgg cat ttg cca aat gac ggg aac cac tgg 319 Met Met Gln Tyr Phe Glu Trp His Leu Pro Asn Asp Gly Asn His Trp 10 15 20 aac agg tta cga gat gac gca gct aac tta aag agt aaa ggg att acc 367 Asn Arg Leu Arg Asp Asp Ala Ala Asn Leu Lys Ser Lys Gly Ile Thr 25 30 35 40 gct gtt tgg att cct cct gca tgg aag ggg act tcg caa aat gat gtt 415 Ala Val Trp Ile Pro P ro Ala Trp Lys Gly Thr Ser Gln Asn Asp Val 45 50 55 ggg tat ggt gcc tat gat ttg tac gat ctt ggt gag ttt aac caa aag 463 Gly Tyr Gly Ala Tyr Asp Leu Tyr Asp Leu Gly Glu Phe Asn Gln Lys 60 65 70 gga acc gtc cgt aca aaa tat ggc aca agg agt cag ttg caa ggt gcc 511 Gly Thr Val Arg Thr Lys Tyr Gly Thr Arg Ser Gln Leu Gln Gly Ala 75 80 85 gtg aca tct ttg aaa aat aac ggg att caa gtt tat ggg gat gtc gtg 559 Val Thr Ser Leu Lys Asn Asn Gly Ile Gln Val Tyr Gly Asp Val Val 90 95 100 atg aat cat aaa ggt gga gca gac ggg aca gag atg gta aat gcg gtg 607 Met Asn His Lys Gly Gly Ala Asp Gly Thr Glu Met Val Asn Ala Val 105 110 115 120 gaa gtg aac cga agc aac cga aac caa gaa ata tca ggt gaa tac acc 655 Glu Val Asn Arg Ser Asn Arg Asn Gln Glu Ile Ser Gly Glu Tyr Thr 125 130 135 att gaa gca tgg acg aaa ttt gat ttc cct gga aga gga aat acc cat 703 Ile Glu Ala Trp Thr Lys Phe Asp Phe Pro Gly Arg Gly Asn Thr His 140 145 150 tcc aac ttt aaa tgg cgc tgg tat cat ttt gat ggg aca gat tgg gat 751 Ser Asn Phe Lys Trp Arg Trp Tyr His Phe Asp Gly Thr Asp Trp Asp 155 160 165 cag tca cgt cag ctt cag aac aaa ata tat aaa ttc aga ggt acc gga 799 Gln Ser Arg Gln Leu Gln Asn Lys Ile Tyr Lys Phe Arg Gly Thr Gly 170 175 180 aag gca tgg gac tgg gaa gta gat ata gag aac ggc aac tat gat tac 847 Lys Ala Trp Asp Trp Glu Val Asp Ile Glu Asn Gly Asn Tyr Asp Tyr 185 190 195 200 ctt atg tat gca gac att gat atg gat cat cca ga g atc aat gaa 895 Leu Met Tyr Ala Asp Ile Asp Met Asp His Pro Glu Val Ile Asn Glu 205 210 215 ctt aga aat tgg gga gtt tgg tat aca aat aca ctt aat cta gat gga 943 Leu Arg Asn Trp Gly Val Trp Tyr Thr Asn Thr Leu Asn Leu Asp Gly 220 225 230 ttt aga atc gat gct gtg aaa cat att aaa tac agc tat acg aga gat 991 Phe Arg Ile Asp Ala Val Lys His Ile Lys Tyr Ser Tyr Thr Arg Asp 235 240 245 tgg cta aca cat gtg cgt aac acc aca ggt aaa cca atg ttt gca gtt 1039 Trp Leu Thr His Val Arg Asn Thr Thr Gly Lys Pro Met Phe Ala Val 250 255 260 gca gaa ttt tgg aaa aat gac ctt gct gca atc gaa aac tat tta aat 1087 Ala Al u Phe Trp Lys Asn Asp Leu Ala Ala Ile Glu Asn Tyr Leu Asn 265 270 275 280 aaa aca agt tgg aat cac tcc gtg ttc gat gtt cct ctt cat tat aat 1135 Lys Thr Ser Trp Asn His Ser Val Phe Asp Val Pro Leu His Tyr Asn 285 290 295 ttg tac aat gca tct aat agt ggt ggc tat ttt gat atg aga aat att 1183 Leu Tyr Asn Ala Ser Asn Ser Aly Gly Tyr Phe Asp Met Arg Asn Ile 300 305 310 tta aat ggt tct gtc gta caaaaa cct ata cat gca gtc aca ttt 1231 Leu Asn Gly Ser Val Val Gln Lys His Pro Ile His Ala Val Thr Phe 315 320 325 gtt gat aac cat gac tct cag cca gga gaa gca ttg gaa tcc ttt gtt 1279 Val Asp Asn His Asp Ser Gln Pro Gly Glu Ala Leu Glu Ser Phe Val 330 335 340 caa tcg tgg ttc aaa cca ctg gca tat gca ttg att ctg aca agg gag 1327 Gln Ser Trp Phe Lys Pro Leu Ala Tyr Ala Leu Ile Leu Thr Arg Glu 345 350 355 360 caa ggt tac cct tcc gta ttt tac ggt gat tac tac ggt ata cca act 1375 Gln Gly Tyr Pro Ser Val Phe Tyr Gly Asp Tyr Tyr Gly Ile Pro Thr 365 370 375 cat ggt gtt cct tcg atg aaa tct aaa att gat cca ctt ctg cag gca 1423 His Gly Val Pro Ser Met Lys Ser Lys Ile Asp Pro Leu Leu Gln Ala 380 385 390 cgt caa acg tat gcc tac gga acc caa cat gat tat ttt gat cat cat 1471 Arg Gln Thr Tyr Ala Tyr Gly Thr Gln His Asp Tyr Phe Asp His His 395 400 405 gat att atc ggc tgg acg aga gaa ggg gac agc tcc cac cca aat tca 1519 Asp Ile Ile Gly Trp Thr Arg Glu Gly Asp Ser Ser His Pro Asn Ser 410 415 420 gga ctt gca act att atg tcc gat ggg cca ggg ggt aat aaa tgg atg 1567 Gly Leu Ala Thr Ile Met Ser Asp Gly Pro Gly Gly Asn Lys Trp Met 425 430 435 440 tat gtc ggg aaa cat aaa gct ggc caa gta tgg aga gat atc acc gga 1615 Tyr Val Gly Lys His Lys Ala Gly Gln Val Trp Arg Asp Ile Thr Gly 445 450 455 aat agg tct ggt acc gtc acc att aat gca gat ggt tgg ggg aat ttc 1663 Asn Arg Ser Gly Thr Val Thr Ile Asn Ala Asp Gly Trp Gly Asn Phe 460 465 470 act gta aac gga ggg gca gtt tcg gtt tgg gtg aag caa taa ataaggaac 1714 Thr Val Asn Gly Gly Ala Val Ser Val Trp Val Lys Gln 475 480 485 aagaggcgaa aattactttc ctacatgcag agctttccga tcactcatac acccaatata 1774 aattggaagc tt 1786
【００３２】 <210> 3 <211> 27 <212> DNA <213> artificial sequence <220> <223> In No. 812〜838 of Sequence ID No. 2, substituting gat for ata of No. 824〜826. <400> 3 tgggaagtag atgatgagaa cggcaac 27<210> 3 <211> 27 <212> DNA <213> artificial sequence <220> <223> In No. 812-838 of Sequence ID No. 2, substituting gat for ata of No. 824-826. <400> 3 tgggaagtag atgatgagaa cggcaac 27
【００３３】 <210> 4 <211> 36 <212> DNA <213> artificial sequence <220> <223> In No. 773〜814 of Sequence ID No. 2, deleting aga ggt of No. 788〜793. <400> 4 aaaatatata aattcaccgg aaaggcatgg gactgg 36<210> 4 <211> 36 <212> DNA <213> artificial sequence <220> <223> In No. 773-814 of Sequence ID No. 2, deleting aga ggt of No. 788-793. < 400> 4 aaaatatata aattcaccgg aaaggcatgg gactgg 36
【図１】Bacillus sp. KSM-AP1378 の生産するα−アミ
ラーゼ（KSMAP1378）の成熟酵素アミノ酸配列（配列番
号１と同じ）とその他のBacillus属細菌α−アミラーゼ
の成熟酵素アミノ酸配列との相同性を示す図である。NC
IB 12512とNCIB 12513はWO9526397 記載のα−アミラー
ゼ、B. amyloはB. amyloliquefaciens、B. stearo はB.
stearothermophilus、B. lichen はB. licheniformis
の生産するα−アミラーゼ。下記した＊は全てのα−ア
ミラーゼで相同性があることを示している。FIG. 1 shows the homology between the amino acid sequence of the mature enzyme of α-amylase (KSMAP1378) produced by Bacillus sp. KSM-AP1378 (same as SEQ ID NO: 1) and the mature enzyme amino acid sequences of α-amylase of other Bacillus bacteria. FIG. NC
IB 12512 and NCIB 12513 is described WO9526397 alpha-amylase, B. Amylo is B. amyloliquefaciens, B. Stearo is B.
stearothermophilus , B. lichen is B. licheniformis
Α-amylase produced by The * below indicates that all α-amylases have homology.
【図２】Bacillus sp. KSM-AP1378 の生産するα−アミ
ラーゼ（KSMAP1378）の成熟酵素アミノ酸配列（配列番
号１と同じ）とその他のBacillus属細菌α−アミラーゼ
の成熟酵素アミノ酸配列との相同性を示す図である。NC
IB 12512とNCIB 12513はWO9526397 記載のα−アミラー
ゼ、B. amyloはB. amyloliquefaciens、B. stearo はB.
stearothermophilus、B. lichen はB. licheniformis
の生産するα−アミラーゼ。下記した＊は全てのα−ア
ミラーゼで相同性があることを示している。FIG. 2 shows the homology between the amino acid sequence of the mature enzyme of α-amylase (KSMAP1378) produced by Bacillus sp. KSM-AP1378 (same as SEQ ID NO: 1) and the amino acid sequence of the mature enzyme of α-amylase of other Bacillus bacteria. FIG. NC
IB 12512 and NCIB 12513 is described WO9526397 alpha-amylase, B. Amylo is B. amyloliquefaciens, B. Stearo is B.
stearothermophilus , B. lichen is B. licheniformis
Α-amylase produced by The * below indicates that all α-amylases have homology.
【図３】α−アミラーゼ遺伝子への変異導入と変異α−
アミラーゼ遺伝子の枯草菌による生産の模式図である。FIG. 3. Mutation introduction into α-amylase gene and mutation α-amylase gene
It is a schematic diagram of the production of the amylase gene by Bacillus subtilis.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 ＦＩ テーマコート゛(参考） Ｃ１２Ｎ 5/10 Ｃ１２Ｎ 9/26 Ａ 9/26 Ｃ１２Ｎ 5/00 Ａ //(Ｃ１２Ｎ 15/09 ＺＮＡ Ｃ１２Ｒ 1:125） (Ｃ１２Ｎ 1/21 Ｃ１２Ｒ 1:125） (Ｃ１２Ｎ 9/26 Ｃ１２Ｒ 1:125） (72)発明者 林 康弘 栃木県芳賀郡市貝町赤羽2606 花王株式会 社研究所内 (72)発明者 萩原 浩 栃木県芳賀郡市貝町赤羽2606 花王株式会 社研究所内 (72)発明者 尾崎 克也 栃木県芳賀郡市貝町赤羽2606 花王株式会 社研究所内 Ｆターム(参考） 4B024 AA03 BA13 CA02 DA07 EA04 GA19 GA21 HA01 4B050 CC03 DD02 FF04E FF05E FF10E LL04 4B065 AA16Y AA19X AC14 AC15 BA02 BC01 BD17 BD22 CA32 CA57 4H003 AC23 BA09 DA17 EA15 EA16 EA20 EA25 EB08 EB34 EB37 EC01 FA47 ──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 9/26 A 9/26 C12N 5/00 A // (C12N 15/09 ZNA C12R 1 : 125) (C12N 1/21 C12R 1: 125) (C12N 9/26 C12R 1: 125) (72) Inventor Yasuhiro Hayashi 2606 Kabanecho, Akamachi, Haga-gun, Tochigi Pref. Kao Corporation Research Laboratory (72) Inventor Hagiwara Hiro 2606 Kabane-cho, Akaga-cho, Haga-gun, Tochigi Pref.Katsuya Ozaki Inventor Katsuya Ozaki 2606 Kabane-cho, Akaga-cho, Haga-gun, Tochigi F-term (reference) 4B024 AA03 BA13 CA02 DA07 EA04 GA19 GA21 HA01 4B050 CC03 DD02 FF04E FF05E FF10E LL04 4B065 AA16Y AA19X AC14 AC15 BA02 BC01 BD17 BD22 CA32 CA57 4H003 AC23 BA09 DA17 EA15 EA16 EA20 EA25 EB08 EB34 EB37 EC01 FA47
Claims (7)
該アミノ酸配列に対して７０％以上の相同性を有するア
ミノ酸配列における１９３位のイソロイシンに相当する
アミノ酸が欠失又は他のアミノ酸に置換されたアミノ酸
配列を有する変異α−アミラーゼ。1. An amino acid corresponding to isoleucine at position 193 in the amino acid sequence represented by SEQ ID NO: 1 or an amino acid sequence having at least 70% homology to the amino acid sequence, has been deleted or replaced with another amino acid. A mutant α-amylase having an amino acid sequence.
位のグリシンに相当するアミノ酸が欠失したものである
請求項１記載の変異α−アミラーゼ。2. Arginine and 182 at position 181
The mutant α-amylase according to claim 1, wherein the amino acid corresponding to glycine at the position is deleted.
請求項１又は２記載の変異α−アミラーゼ。3. The mutant α-amylase according to claim 1, wherein the other amino acid is aspartic acid.
α−アミラーゼをコードする遺伝子。A gene encoding the mutant α-amylase according to any one of claims 1 to 3.
ベクター。5. A recombinant vector containing the gene according to claim 4.
換又は染色体相同組換えされた形質転換細胞。6. A transformed cell transformed or chromosomally homologously transformed with the recombinant vector according to claim 5.
α−アミラーゼを含有する洗浄剤組成物。7. A detergent composition containing the mutant α-amylase according to any one of claims 1 to 3.
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|---|---|---|---|
| JP04821399A JP4220611B2 (en) | 1999-02-25 | 1999-02-25 | Mutant α-amylase |
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|---|---|
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| JP4220611B2 JP4220611B2 (en) | 2009-02-04 |
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| JP04821399A Expired - Fee Related JP4220611B2 (en) | 1999-02-25 | 1999-02-25 | Mutant α-amylase |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008505632A (en) * | 2004-07-07 | 2008-02-28 | ダニスコ エイ／エス | Non-maltogenic exoamylase variant |
| US7803604B2 (en) | 2000-07-28 | 2010-09-28 | Henkel Ag & Co. Kgaa | Amylolytic enzyme extracted from Bacillus sp. A 7-7 (DSM 12368) and washing and cleaning agents containing this novel amylolytic enzyme |
| US8030050B2 (en) | 2005-07-07 | 2011-10-04 | Danisco A/S | Modified amylases from Pseudomonas species |
| US8129511B2 (en) | 2003-07-07 | 2012-03-06 | Danisco A/S | Modified amylases, nucleic acids encoding those amylases and uses thereof |
| US8143048B2 (en) | 2003-07-07 | 2012-03-27 | Danisco A/S | Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof |
| US8178336B2 (en) | 2006-06-19 | 2012-05-15 | Danisco A/S | Polypeptide |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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1999
- 1999-02-25 JP JP04821399A patent/JP4220611B2/en not_active Expired - Fee Related
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7803604B2 (en) | 2000-07-28 | 2010-09-28 | Henkel Ag & Co. Kgaa | Amylolytic enzyme extracted from Bacillus sp. A 7-7 (DSM 12368) and washing and cleaning agents containing this novel amylolytic enzyme |
| US8129511B2 (en) | 2003-07-07 | 2012-03-06 | Danisco A/S | Modified amylases, nucleic acids encoding those amylases and uses thereof |
| US8143048B2 (en) | 2003-07-07 | 2012-03-27 | Danisco A/S | Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof |
| US8663966B2 (en) | 2003-07-07 | 2014-03-04 | Dupont Nutrition Biosciences Aps | Polypeptide |
| US8809032B2 (en) | 2003-07-07 | 2014-08-19 | Dupont Nutrition Biosciences Aps | Exo-specific amylase polypeptides, nucleic acids encoding those polypeptides and uses thereof |
| JP2008505632A (en) * | 2004-07-07 | 2008-02-28 | ダニスコ エイ／エス | Non-maltogenic exoamylase variant |
| US8137944B2 (en) | 2004-07-07 | 2012-03-20 | Danisco A/S | Modified amylases from pseudomonas species, methods of making and uses thereof |
| JP2012070751A (en) * | 2004-07-07 | 2012-04-12 | Danisco As | Polypeptide |
| JP2014221074A (en) * | 2004-07-07 | 2014-11-27 | デュポン ニュートリション バイオサイエンシーズ エーピーエス | Polypeptide |
| US8030050B2 (en) | 2005-07-07 | 2011-10-04 | Danisco A/S | Modified amylases from Pseudomonas species |
| US8178336B2 (en) | 2006-06-19 | 2012-05-15 | Danisco A/S | Polypeptide |
| WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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|---|---|
| JP4220611B2 (en) | 2009-02-04 |
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