JP2000210081A - Heat-resistant alkali cellulase gene - Google Patents
Heat-resistant alkali cellulase geneInfo
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- JP2000210081A JP2000210081A JP11013049A JP1304999A JP2000210081A JP 2000210081 A JP2000210081 A JP 2000210081A JP 11013049 A JP11013049 A JP 11013049A JP 1304999 A JP1304999 A JP 1304999A JP 2000210081 A JP2000210081 A JP 2000210081A
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Abstract
Description
【０００１】[0001]
【発明の属する技術分野】本発明は、衣料用洗剤等に配
合可能な耐熱性アルカリセルラーゼをコードする遺伝子
に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gene encoding a heat-resistant alkaline cellulase which can be added to a detergent for clothing and the like.
【０００２】[0002]
【従来の技術】従来、セルラーゼは、耐熱性が低く、か
つ酸性から中性付近で活性を有するいわゆる酸性或いは
中性セルラーゼと称されるものがほとんどであり、衣料
用洗剤等に配合可能な耐熱性を有するアルカリセルラー
ゼとしては、これまでにBacillus属に属する中温性の好
アルカリ性細菌（Bacillus sp. ＫＳＭ−Ｓ２３７と命
名されＦＥＲＭ Ｐ−１６０６７として寄託された）に
より産生される耐熱性アルカリセルラーゼが唯一のもの
であった（特開平１０−３１３８５９号公報）。2. Description of the Related Art Conventionally, most cellulase is so-called acidic or neutral cellulase which has low heat resistance and has an activity in the range from acidic to neutral. As the alkaline cellulase having a property, only a thermostable alkaline cellulase produced by a mesophilic alkalophilic bacterium belonging to the genus Bacillus (named Bacillus sp. KSM-S237 and deposited as FERM P-16067) has been described. (JP-A-10-313859).
【０００３】[0003]
【発明が解決しようとする課題】しかし、このBacillus
属細菌による耐熱性アルカリセルラーゼの生産性は充分
とはいえず、より効率的な生産技術の開発が切望されて
いた。また、当該耐熱性アルカリセルラーゼ遺伝子のク
ローニングは未だ報告されていない。However, this Bacillus
The productivity of thermostable alkaline cellulase by genus bacteria is not sufficient, and there has been a long-awaited desire to develop more efficient production techniques. In addition, the cloning of the thermostable alkaline cellulase gene has not been reported yet.
【０００４】[0004]
【課題を解決するための手段】本発明者は、上記の耐熱
性アルカリセルラーゼについて、その遺伝子のクローニ
ング、遺伝子組換えによる生産技術の確立に成功し、本
発明を完成した。Means for Solving the Problems The present inventors have succeeded in cloning the gene and establishing a production technique by genetic recombination for the above-mentioned thermostable alkaline cellulase, and completed the present invention.
【０００５】すなわち、本発明は、配列番号１に示すア
ミノ酸配列又は、該アミノ酸配列の１若しくは数個のア
ミノ酸が欠失、置換若しくは付加されたアミノ酸配列を
有する耐熱性アルカリセルラーゼをコードする遺伝子を
提供するものである。また、本発明は、上記の耐熱性ア
ルカリセルラーゼ遺伝子を含有する組換えベクター、高
発現させる為に必要な発現領域（配列番号２の塩基番号
１〜５７２）を含む組換えベクター、及び該組換えベク
ターを含む形質転換体を提供するものである。That is, the present invention relates to a gene encoding a thermostable alkaline cellulase having the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence are deleted, substituted or added. To provide. In addition, the present invention provides a recombinant vector containing the above-mentioned thermostable alkaline cellulase gene, a recombinant vector containing an expression region (base numbers 1 to 572 of SEQ ID NO: 2) required for high expression, and the recombinant vector. It is intended to provide a transformant containing the vector.
【０００６】[0006]
【発明の実施の形態】本発明の遺伝子は、配列番号１に
示すアミノ酸配列、又は該アミノ酸配列の１若しくは数
個のアミノ酸が欠失、置換若しくは付加されたアミノ酸
配列をコードする配列を有する。耐熱性アルカリセルラ
ーゼ活性を失わない限り、該アミノ酸配列中のアミノ酸
の欠失置換又は付加（以下、変異ということがある）は
特に制限されない。また、配列番号１のアミノ酸配列に
おけるＮ−末端には、１〜数個のアミノ酸が付加・欠失
していてもよい。BEST MODE FOR CARRYING OUT THE INVENTION The gene of the present invention has a sequence encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted or added. As long as the thermostable alkaline cellulase activity is not lost, deletion substitution or addition (hereinafter sometimes referred to as mutation) of an amino acid in the amino acid sequence is not particularly limited. Further, at the N-terminal in the amino acid sequence of SEQ ID NO: 1, one to several amino acids may be added or deleted.
【０００７】本発明の遺伝子によりコードされる耐熱性
アルカリセルラーゼのアミノ酸配列（配列番号１に示す
アミノ酸番号１〜８２４）と従来公知のセルラーゼとの
相同性を比較すると、Bacillus sp. No.1139由来のセル
ラーゼ（Fukumoriら、J.General Microbiology,131,333
9-3345,1985)とのアミノ酸相同性が８９．１％、またＫ
ＳＭ−６４セルラーゼ（Sumitomoら、Biosci.Biotech.B
iochem.,56,872-877,1992)とのアミノ酸相同性が９１．
７％である。ＫＳＭ−６４セルラーゼの耐熱性は低く、
７０℃、１０分間の熱処理でその残存活性は約１０％で
あるのに対し、本発明の耐熱性アルカリセルラーゼは、
７０℃、１０分間の熱処理での残存活性は、約５０％で
ある。このように、本発明により得られる耐熱性アルカ
リセルラーゼは、ＫＳＭ−６４セルラーゼとのアミノ酸
相同性が９０％以上あるにもかかわらず、耐熱性を有し
ている。即ち本発明の耐熱性アルカリセルラーゼ遺伝子
は、従来のものと異なり、耐熱性を有するアルカリセル
ラーゼをコードする新規な遺伝子である。従って、本遺
伝子と９２％以上相同性を有する耐熱性アルカリセルラ
ーゼ遺伝子は本発明に含まれる。[0007] The homology between the amino acid sequence of the thermostable alkaline cellulase encoded by the gene of the present invention (amino acids 1 to 824 shown in SEQ ID NO: 1) and a conventionally known cellulase was compared with that of Bacillus sp. Cellulase (Fukumori et al., J. General Microbiology, 131, 333).
9-3345, 1985) with an amino acid homology of 89.1%.
SM-64 cellulase (Sumitomo et al., Biosci. Biotech. B
iochem., 56, 872-877, 1992).
7%. The heat resistance of KSM-64 cellulase is low,
While the residual activity is about 10% after heat treatment at 70 ° C. for 10 minutes, the thermostable alkaline cellulase of the present invention comprises:
The residual activity after heat treatment at 70 ° C. for 10 minutes is about 50%. Thus, the thermostable alkaline cellulase obtained according to the present invention has heat resistance despite having 90% or more amino acid homology with KSM-64 cellulase. That is, the thermostable alkaline cellulase gene of the present invention is a novel gene that encodes a thermostable alkaline cellulase, unlike the conventional gene. Therefore, a thermostable alkaline cellulase gene having 92% or more homology with the present gene is included in the present invention.
【０００８】本発明の耐熱性アルカリセルラーゼ遺伝子
は、配列番号１のアミノ酸配列又はその前記変異体をコ
ードするものであればよいが、配列番号２の塩基番号５
７３〜３０４４で示される塩基配列又は該塩基配列の１
若しくは数個の塩基が欠失、置換若しくは付加された塩
基配列を有するものが好ましい。[0008] The thermostable alkaline cellulase gene of the present invention may be any as long as it encodes the amino acid sequence of SEQ ID NO: 1 or the above-mentioned mutant thereof.
73 to 3044 or one of said base sequences
Alternatively, those having a base sequence in which several bases are deleted, substituted or added are preferable.
【０００９】本発明の耐熱性アルカリセルラーゼ遺伝子
供与体微生物としては、土壌から分離したBacillus属に
属する微生物、例えばBacillus sp. ＫＳＭ−Ｓ２３７
株が挙げられる。該微生物を培養して得られた培養液か
ら遠心分離により菌体を得ることができ、該菌体からの
耐熱性アルカリセルラーゼ遺伝子を含んだ染色体ＤＮＡ
の調製は、例えば、マーマーの方法（Marmur,J.Mol.Bio
l.3,208,1961）や斉藤と三浦の方法（Saito & Miura.Bi
ochim.Biophys.Acta,72,619,1963）等で行うことができ
る。また、耐熱性アルカリセルラーゼ遺伝子を含む染色
体ＤＮＡ断片の調製は、ショットガンクローニング法や
ＰＣＲ法を用いて行うことができる。The heat-resistant alkaline cellulase gene donor microorganism of the present invention is a microorganism belonging to the genus Bacillus isolated from soil, for example, Bacillus sp. KSM-S237.
Strains. Cells can be obtained by centrifugation from a culture solution obtained by culturing the microorganism, and a chromosomal DNA containing a thermostable alkaline cellulase gene from the cells can be obtained.
Can be prepared, for example, by the method of Marmer (Marmur, J. Mol. Bio
l.3,208,1961) and the method of Saito and Miura (Saito & Miura.Bi
ochim. Biophys. Acta, 72, 619, 1963). The preparation of a chromosomal DNA fragment containing a thermostable alkaline cellulase gene can be performed using a shotgun cloning method or a PCR method.
【００１０】一方、ベクターとしては、宿主菌内で複製
維持可能であり、耐熱性アルカリセルラーゼ遺伝子を発
現させることができ、組み込まれた該遺伝子を安定に保
持できれば如何なるものも使用可能である。例えば、Ba
cillus属細菌を宿主とする場合、ｐＵＢ１１０やｐＨＹ
３００ＰＬＫ等が挙げられ、大腸菌を宿主とする場合、
ｐＵＣ１８、ｐＵＣ１９、ｐＢＲ３２２或いはｐＨＹ３
００ＰＬＫ等が挙げられる。[0010] On the other hand, any vector can be used as long as it can maintain replication in a host bacterium, can express a thermostable alkaline cellulase gene, and can stably maintain the integrated gene. For example, Ba
When a bacterium of the genus cillus is used as a host, pUB110 or pHY
300PLK and the like. When Escherichia coli is used as a host,
pUC18, pUC19, pBR322 or pHY3
00PLK and the like.
【００１１】かくしてクローニングされた染色体ＤＮＡ
断片と制限酵素で切断したベクターとの組換えが、ＤＮ
Ａリガーゼを用いて行われる。The chromosomal DNA thus cloned
Recombination of the fragment with the vector cut with restriction enzymes
This is performed using A ligase.
【００１２】得られた組換え体ＤＮＡによる宿主菌の形
質転換は、コンピテント・セル法、プロトプラスト法及
びエレクトロポレーション等により行うことができる。
宿主菌としては、特に制限されないが、Bacillus属（枯
草菌）、Streptomyces属等のグラム陽性菌、E.coli等の
グラム陰性菌、Saccharomyces属、Aspergillus属等の真
菌類が挙げられる。Transformation of a host bacterium with the obtained recombinant DNA can be carried out by a competent cell method, a protoplast method, electroporation, or the like.
The host bacteria are not particularly limited, but include Bacillus (Bacillus subtilis), Gram-positive bacteria such as Streptomyces , Gram-negative bacteria such as E. coli, and fungi such as Saccharomyces and Aspergillus .
【００１３】形質転換体は、資化しうる適当な炭素源、
窒素源やミネラル等を含んだ培地を用いて培養する。酵
素は、各種クロマトグラフィーを用いる一般的な精製法
で得ることができる。[0013] The transformant is a suitable carbon source that can be assimilated,
Culture is performed using a medium containing a nitrogen source, minerals, and the like. The enzyme can be obtained by a general purification method using various types of chromatography.
【００１４】尚、本発明に際し、耐熱性アルカリセルラ
ーゼをコードする遺伝子の上流部には、タンパク質分泌
能が非常に高いプロモーター様領域（配列番号２の塩基
番号１〜５７２）が存在することが判った。この領域に
は、既に論文（Sumitomoら、Biosci.Biotech.Biochem.,
56,872-877,1992 ）にて報告されているＫＳＭ−６４由
来の高いタンパク質分泌能を与える領域と塩基配列で約
９５％の相同性を示したこと、また、実際に本領域を含
む組換えプラスミドを用いた場合の耐熱性セルラーゼの
分泌能が非常に高いことから、本領域は、タンパク質の
高分泌に効果のある領域と判断された。従って、本発明
により、耐熱性アルカリセルラーゼを生産する際には、
本領域を含むように組換えベクターを作製するのが好ま
しい。In the present invention, it has been found that a promoter-like region (base numbers 1 to 572 of SEQ ID NO: 2) having a very high protein secretion ability is present upstream of the gene encoding a thermostable alkaline cellulase. Was. This area already contains papers (Sumitomo et al., Biosci. Biotech. Biochem.,
56,872-877,1992). It showed about 95% homology in base sequence with the KSM-64-derived region giving high protein secretion ability, and the recombinant plasmid actually containing this region. This region was judged to be a region effective for high secretion of protein, since the secretion ability of thermostable cellulase in the case of using was extremely high. Therefore, according to the present invention, when producing thermostable alkaline cellulase,
Preferably, a recombinant vector is prepared to include this region.
【００１５】[0015]
【実施例】実施例１ 菌株の取得 日本国内土壌１ｇを滅菌生理食塩水１０mLに懸濁し、８
０℃で３０分間熱処理した。この熱処理液を適当に希釈
してマスタープレート〔１％肉エキス（オキソイド社
製）、１％バクトペプトン（ディフコ社製）、１％Ｎａ
Ｃｌ、０．１％ＫＨ2ＰＯ4、０．５％Ｎａ2ＣＯ3（別滅
菌）、１．５％寒天〕に塗抹し、３０℃で３日間培養
し、集落を形成させた。レプリカ法により、マスタープ
レートと同じ組成の培地に２％ＣＭＣを加えた滅菌寒天
培地に移植し、３０℃で３〜４日間培養して集落を形成
させた後、コンゴーレッド色素液を流し込み、寒天が赤
色化した中で周囲が染色されない集落を検出した。該当
する集落をマスタープレートから選出し、高力価ＣＭＣ
アーゼ生産菌をスクリーニングした。上記手法により、
本発明のBacillus sp. ＫＳＭ−Ｓ２３７株を取得する
ことができた。Example 1 Acquisition of strain 1 g of soil in Japan was suspended in 10 mL of sterile physiological saline, and
Heat treatment was performed at 0 ° C. for 30 minutes. This heat treatment solution was appropriately diluted and a master plate [1% meat extract (Oxoid), 1% Bactopeptone (Difco), 1% Na
Cl, 0.1% KH 2 PO 4 , 0.5% Na 2 CO 3 (separately sterilized), 1.5% agar], and cultured at 30 ° C. for 3 days to form colonies. By replica method, the cells were transplanted to a sterilized agar medium containing 2% CMC in a medium having the same composition as the master plate, and cultured at 30 ° C. for 3 to 4 days to form colonies. A colony in which the surrounding area was not stained while reddish was detected. Select the appropriate settlement from the master plate, high titer CMC
The ase producing bacteria were screened. By the above method,
The Bacillus sp. KSM-S237 strain of the present invention was able to be obtained.
【００１６】実施例２ 菌株の培養 実施例１で得たBacillus sp. ＫＳＭ−Ｓ２３７株を
〔１％肉エキス（オキソイド社製）、２％ポリペプトン
Ｓ（日本製薬社製）、０．１％酵母エキス（ディフコ社
製）、１％ＮａＣｌ、０．１％Ｋ2ＨＰＯ4、０．５％Ｎ
ａ2ＣＯ3（別滅菌）、１％ＣＭＣを加えた液体培地に接
種し、３０℃で３日間振盪培養した。培養後、菌体を遠
心分離により集め、斉藤−三浦の方法に準じてゲノムＤ
ＮＡを調製した。Example 2 Culture of strains Bacillus sp. Strain KSM-S237 obtained in Example 1 was prepared using [1% meat extract (Oxoid), 2% polypeptone S (Nippon Pharmaceutical), 0.1% yeast Extract (Difco), 1% NaCl, 0.1% K 2 HPO 4 , 0.5% N
a 2 CO 3 (separately sterilized) was inoculated into a liquid medium supplemented with 1% CMC, and cultured with shaking at 30 ° C. for 3 days. After the culture, the cells are collected by centrifugation, and the genome D is collected according to the method of Saito-Miura.
NA was prepared.
【００１７】実施例３ Bacillus sp. ＫＳＭ−Ｓ２３
７株の耐熱性アルカリセルラーゼ遺伝子のショットガン
クローニング ゲノムＤＮＡ溶液３μＬ（２．７μｇＤＮＡ）、各種制
限酵素用緩衝液１０μＬ、脱イオン水８５μＬの混合溶
液にそれぞれ、制限酵素（ＢａｍＨＩ、ＥｃｏＲＩ、Ｈ
ｉｎｄIII、ＰｓｔＩ）２μＬを加え、３７℃で２時間
３０分処理した後、６５℃１５分間の熱処理により酵素
を失活させた。エタノール沈澱後、２０μＬをアガロー
スゲル電気泳動により泳動した。ゲルをエチジウムブロ
マイド溶液中に入れ、染色後、約２〜９ｋｂのＤＮＡ断
片を含むゲルを切り出し、G FX PCR DNA and Gel Band
Purification Kit（ファルマシア社製）によりＤＮＡ断
片を精製した。得られたＤＮＡ断片と各種制限酵素（Ｂ
ａｍＨＩ、ＥｃｏＲＩ、ＨｉｎｄIII 、ＰｓｔＩ）で処
理したｐＵＣ１８ベクターとをLigation Kit Ver.2（タ
カラ社製）を用いて連結し（１６℃、２０時間）、Ｅ．
ｃｏｌｉ ＨＢ１０１コンピテントセル（タカラ社製）
に導入した。ＬＢプレート（５０μｇ／mLアンピシリン
含有）上に得られた形質転換体に軟寒天〔０．５％ＣＭ
Ｃ、５０mMグリシン水酸化ナトリウム緩衝液（pH９）、
１％塩化ナトリウム、０．８％寒天〕を重層し、３７℃
で３時間放置後、コンゴーレッド法でハロー形成の有無
を調べた。ハロー形成株のプラスミドを調製し、挿入Ｄ
ＮＡ断片の塩基配列を決定した結果、目的のセルラーゼ
遺伝子は、そのプロモーター様配列上流から、構造遺伝
子内約２．０ｋｂのＨｉｎｄIII 認識部位までの領域が
プラスミドに挿入されており、セルラーゼ遺伝子全域が
挿入されたプラスミドは得られなかった。Example 3 Bacillus sp. KSM-S23
Shotgun of 7 thermostable alkaline cellulase genes
A restriction enzyme ( Bam HI, Eco RI, H) was added to a mixed solution of 3 μL (2.7 μg DNA) of the cloning genomic DNA solution, 10 μL of various restriction enzyme buffers, and 85 μL of deionized water.
in dIII, Pst I) the 2μL added, treated for 2 hours 30 minutes at 37 ° C., to inactivate the enzyme by heat treatment of 65 ° C. 15 min. After ethanol precipitation, 20 μL was electrophoresed by agarose gel electrophoresis. The gel was placed in an ethidium bromide solution, and after staining, a gel containing a DNA fragment of about 2 to 9 kb was cut out, and GFX PCR DNA and Gel Band
The DNA fragment was purified using Purification Kit (Pharmacia). The obtained DNA fragment and various restriction enzymes ( B
am HI, Eco RI, Hin dIII , Pst and treated pUC18 vector I) and ligated using Ligation Kit Ver.2 (Takara) (16 ℃, 20 hours), E.
E. coli HB101 competent cell (Takara)
Was introduced. Transformants obtained on LB plates (containing 50 μg / mL ampicillin) were added to soft agar [0.5% CM
C, 50 mM glycine sodium hydroxide buffer (pH 9),
1% sodium chloride, 0.8% agar] and 37 ° C
For 3 hours, and the presence or absence of halo formation was examined by the Congo red method. A plasmid for the halo-forming strain was prepared and inserted into D.
Result of determining the nucleotide sequence of the NA fragment, cellulase gene of interest from the promoter-like sequence upstream, and range up to Hin dIII recognition site of a structural gene which approximately 2.0kb was inserted into a plasmid, the cellulase gene throughout No inserted plasmid was obtained.
【００１８】実施例４ Bacillus sp. ＫＳＭ−Ｓ２３
７セルラーゼ遺伝子のゲノムＰＣＲクローニング ＰｗｏＤＮＡポリメラーゼを用いて、Bacillus sp. Ｋ
ＳＭ−Ｓ２３７セルラーゼ遺伝子のゲノムＰＣＲを行っ
た。実施例３で記載した、ショットガンクローニングよ
り得られたセルラーゼ遺伝子の塩基配列を決定したとこ
ろ、Bacillus sp.No. １１３９セルラーゼとアミノ酸相
同性が高かったので、このBacillus sp.No. １１３９セ
ルラーゼの塩基配列を参考にしてプライマーを構築し
た。脱イオン水６０μＬ、ＰＣＲ用緩衝液１０μＬ、ｄ
ＮＴＰ８μＬ、上流プライマー１０μＬ（０．３μＭ）
（配列番号３）、下流プライマー１０μＬ（０．３μ
Ｍ）（配列番号４）鋳型ＤＮＡ１μＬ（７０μｇ）及び
ＰｗｏＤＮＡポリメラーゼ（ベーリンガーマンハイム社
製）１μＬ（５Ｕ）を混合し、９４℃２分間の熱変成
後、９４℃１分間、５５℃１分間、７２℃３分間（３０
サイクル）及び７２℃５分間の条件でＤＮＡ増幅を行っ
た。得られたＰＣＲ産物（約３ｋｂ）８０μＬと予めＳ
ｍａＩ処理したｐＨＹ３００ＰＬＫ２０μＬ（０．５μ
ｇ）を混和し、ＧＦＸ ＰＣＲ ＤＮＡ ａｎｄ Ｇｅ
ｌ Ｂａｎｄ ＰｕｒｉｆｉｃａｔｉｏｎＫｉｔで精製
後、Ｌｉｇａｔｉｏｎ Ｋｉｔ Ｖｅｒ．２を用いて、
ＰＣＲ産物とｐＨＹ３００ＰＬＫとを連結した（１６
℃、２０時間）。得られたライゲーションミクスチャー
をプロトプラスト法によりBacillus subtilis ＩＳＷ
１２１４株に導入して形質転換体を取得した。形質転換
体の再生培地には、テトラサイクリン塩酸塩（シグマ
製）５μｇ／mLを含むＤＭ３再生寒天培地を用いた（Ch
ang & Cohen,Molec.Gen.Genet.,168,111-115,1979)。Example 4 Bacillus sp. KSM-S23
Genomic PCR cloning of 7 cellulase gene Using Pwo DNA polymerase, Bacillus sp.
Genomic PCR of the SM-S237 cellulase gene was performed. When the nucleotide sequence of the cellulase gene obtained by shotgun cloning described in Example 3 was determined, and the amino acid homology was high with Bacillus sp. No. 1139 cellulase, the nucleotide sequence of this Bacillus sp. Primers were constructed with reference to the sequence. 60 μL of deionized water, 10 μL of buffer for PCR, d
NTP 8μL, upstream primer 10μL (0.3μM)
(SEQ ID NO: 3), 10 μL of downstream primer (0.3 μL
M) (SEQ ID NO: 4) 1 μL (70 μg) of template DNA and 1 μL (5 U) of Pwo DNA polymerase (manufactured by Boehringer Mannheim) were mixed, and after thermal denaturation at 94 ° C. for 2 minutes, 94 ° C. for 1 minute, 55 ° C. for 1 minute, 72 ° C. 3 minutes (30
Cycle) and DNA amplification was performed at 72 ° C. for 5 minutes. 80 μL of the obtained PCR product (about 3 kb) and S
ma I treated pHY300PLK20μL (0.5μ
g), and mix GFX PCR DNA and Ge
l After purifying with Band Purification Kit, Ligation Kit Ver. Using 2,
The PCR product and pHY300PLK were ligated (16
° C, 20 hours). The obtained ligation mixture was subjected to Bacillus subtilis ISW by protoplast method.
The transformant was introduced into strain 1214 to obtain a transformant. As a regeneration medium for the transformant, a DM3 regeneration agar medium containing 5 μg / mL of tetracycline hydrochloride (manufactured by Sigma) was used (Ch).
ang & Cohen, Molec. Gen. Genet., 168, 111-115, 1979).
【００１９】実施例５ ＤＮＡシークエンス セルラーゼ遺伝子の塩基配列解析は、３７７ＤＮＡシー
クエンサー（アプライド バイオシステム社製）を用い
て行った。決定した塩基配列の中から次のプライマー配
列を決め順次シークエンスを行い、耐熱性アルカリセル
ラーゼの構成アミノ酸（８２４アミノ酸）を決定した。
ターミネーター領域を含むBacillus sp.ＫＳＭ−Ｓ２３
７セルラーゼ遺伝子の塩基配列及び推定されるプロモー
ター領域を配列番号２に記載した。組換えプラスミドを
保持する枯草菌（Bacillus subtilis ＩＳＷ１２１
４）をマルトース、ポリペプトンを主成分とする培地
（pH６．８、１５μｇ／mLのテトラサイクリンを含有す
る）に接種し、３０℃で４８時間振盪培養（１２５rp
m）した結果、培養液中に、５５，０００単位／Ｌの耐
熱性アルカリセルラーゼを生産した。Example 5 DNA sequence Analysis of the base sequence of the cellulase gene was performed using a 377 DNA sequencer (manufactured by Applied Biosystems). The next primer sequence was determined from the determined base sequence, and the sequence was sequentially performed to determine the constituent amino acids (824 amino acids) of the thermostable alkaline cellulase.
Bacillus sp. KSM-S23 containing a terminator region
The nucleotide sequence of the 7-cellulase gene and the putative promoter region are described in SEQ ID NO: 2. Bacillus subtilis ISW121 carrying the recombinant plasmid
4) was inoculated into a medium containing maltose and polypeptone as main components (pH 6.8, containing 15 μg / mL tetracycline), and cultured at 30 ° C. for 48 hours with shaking (125 rp).
m) As a result, 55,000 units / L of heat-resistant alkaline cellulase was produced in the culture solution.
【００２０】〔セルラーゼ活性測定法（３，５−ジニト
ロサリチル酸（ＤＮＳ）法）〕終濃度１％ＣＭＣ（日本
製紙製）と０．１Ｍグリシン−水酸化ナトリウム（pH
９）緩衝液を含んだ基質溶液０．９mLに、適当な濃度に
希釈した酵素溶液０．１mLを加え、４０℃で２０分間反
応した後、ＤＮＳ溶液１mLを加えて沸騰湯浴中で５分間
熱処理した後、氷水中で冷却し、脱イオン水４mLを加え
た。攪拌した後、Ｕ２０００スペクトロフォトメーター
（日立製作所製）を用いて５３５nmでの吸光度を測定し
た。酵素１単位は１分間に１μmolのグルコースを遊離
する酵素量とした。[Cellulase activity measuring method (3,5-dinitrosalicylic acid (DNS) method)] A final concentration of 1% CMC (manufactured by Nippon Paper) and 0.1 M glycine-sodium hydroxide (pH
9) 0.1 mL of the enzyme solution diluted to an appropriate concentration was added to 0.9 mL of the substrate solution containing the buffer solution, and reacted at 40 ° C. for 20 minutes. Then, 1 mL of the DNS solution was added, and the mixture was added for 5 minutes in a boiling water bath. After heat treatment, the mixture was cooled in ice water and 4 mL of deionized water was added. After stirring, the absorbance at 535 nm was measured using a U2000 spectrophotometer (manufactured by Hitachi, Ltd.). One unit of the enzyme was the amount of the enzyme that released 1 μmol of glucose per minute.
【００２１】[0021]
【発明の効果】本発明の耐熱性アルカリセルラーゼ遺伝
子を用いて、衣料用洗剤等に配合可能な耐熱性アルカリ
セルラーゼを大量に生産することが可能である。The heat-resistant alkaline cellulase gene of the present invention can be used to produce a large amount of heat-resistant alkaline cellulase that can be blended in detergents for clothing and the like.
【００２２】[0022]
【配列表】 SEQUENCE LISTING <110> KAO CORPORATION <120> Gene for Heat Resistant Alkaline Cellulase <130> P00181101 <160> 4 <210> 1 <211> 824 <212> PRT <213> Bacillus sp. <400> 1 Met Met Leu Arg Lys Lys Thr Lys Gln Leu Ile Ser Ser Ile Leu Ile 5 10 15 Leu Val Leu Leu Leu Ser Leu Phe Pro Ala Ala Leu Ala Ala Glu Gly 20 25 30 Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn Asp Asn Val 35 40 45 Lys Arg Pro Ser Glu Ala Gly Ala Leu Gln Leu Gln Glu Val Asp Gly 50 55 60 Gln Met Thr Leu Val Asp Gln His Gly Glu Lys Ile Gln Leu Arg Gly 65 70 75 80 Met Ser Thr His Gly Leu Gln Trp Phe Pro Glu Ile Leu Asn Asp Asn 85 90 95 Ala Tyr Lys Ala Leu Ser Asn Asp Trp Asp Ser Asn Met Ile Arg Leu 100 105 110 Ala Met Tyr Val Gly Glu Asn Gly Tyr Ala Thr Asn Pro Glu Leu Ile 115 120 125 Lys Gln Arg Val Ile Asp Gly Ile Glu Leu Ala Ile Glu Asn Asp Met 130 135 140 Tyr Val Ile Val Asp Trp His Val His Ala Pro Gly Asp Pro Arg Asp 145 150 155 160 Pro Val Tyr Ala Gly Ala Lys Asp Phe Phe Arg Glu Ile Ala Ala Leu 165 170 175 Tyr Pro Asn Asn Pro His Ile Ile Tyr Glu Leu Ala Asn Glu Pro Ser 180 185 190 Ser Asn Asn Asn Gly Gly Ala Gly Ile Pro Asn Asn Glu Glu Gly Trp 195 200 205 Lys Ala Val Lys Glu Tyr Ala Asp Pro Ile Val Glu Met Leu Arg Lys 210 215 220 Ser Gly Asn Ala Asp Asp Asn Ile Ile Ile Val Gly Ser Pro Asn Trp 225 230 235 240 Ser Gln Arg Pro Asp Leu Ala Ala Asp Asn Pro Ile Asp Asp His His 245 250 255 Thr Met Tyr Thr Val His Phe Tyr Thr Gly Ser His Ala Ala Ser Thr 260 265 270 Glu Ser Tyr Pro Ser Glu Thr Pro Asn Ser Glu Arg Gly Asn Val Met 275 280 285 Ser Asn Thr Arg Tyr Ala Leu Glu Asn Gly Val Ala Val Phe Ala Thr 290 295 300 Glu Trp Gly Thr Ser Gln Ala Ser Gly Asp Gly Gly Pro Tyr Phe Asp 305 310 315 320 Glu Ala Asp Val Trp Ile Glu Phe Leu Asn Glu Asn Asn Ile Ser Trp 325 330 335 Ala Asn Trp Ser Leu Thr Asn Lys Asn Glu Val Ser Gly Ala Phe Thr 340 345 350 Pro Phe Glu Leu Gly Lys Ser Asn Ala Thr Asn Leu Asp Pro Gly Pro 355 360 365 Asp His Val Trp Ala Pro Glu Glu Leu Ser Leu Ser Gly Glu Tyr Val 370 375 380 Arg Ala Arg Ile Lys Gly Val Asn Tyr Glu Pro Ile Asp Arg Thr Lys 385 390 395 400 Tyr Thr Lys Val Leu Trp Asp Phe Asn Asp Gly Thr Lys Gln Gly Phe 405 410 415 Gly Val Asn Ser Asp Ser Pro Asn Lys Glu Leu Ile Ala Val Asp Asn 420 425 430 Glu Asn Asn Thr Leu Lys Val Ser Gly Leu Asp Val Ser Asn Asp Val 435 440 445 Ser Asp Gly Asn Phe Trp Ala Asn Ala Arg Leu Ser Ala Asn Gly Trp 450 455 460 Gly Lys Ser Val Asp Ile Leu Gly Ala Glu Lys Leu Thr Met Asp Val 465 470 475 480 Ile Val Asp Glu Pro Thr Thr Val Ala Ile Ala Ala Ile Pro Gln Ser 485 490 495 Ser Lys Ser Gly Trp Ala Asn Pro Glu Arg Ala Val Arg Val Asn Ala 500 505 510 Glu Asp Phe Val Gln Gln Thr Asp Gly Lys Tyr Lys Ala Gly Leu Thr 515 520 525 Ile Thr Gly Glu Asp Ala Pro Asn Leu Lys Asn Ile Ala Phe His Glu 530 535 540 Glu Asp Asn Asn Met Asn Asn Ile Ile Leu Phe Val Gly Thr Asp Ala 545 550 555 560 Ala Asp Val Ile Tyr Leu Asp Asn Ile Lys Val Ile Gly Thr Glu Val 565 570 575 Glu Ile Pro Val Val His Asp Pro Lys Gly Glu Ala Val Leu Pro Ser 580 585 590 Val Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp Ala Gly Glu Ser 595 600 605 Gly Val Lys Thr Ala Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala 610 615 620 Leu Ser Trp Glu Phe Gly Tyr Pro Glu Val Lys Pro Ser Asp Asn Trp 625 630 635 640 Ala Thr Ala Pro Arg Leu Asp Phe Trp Lys Ser Asp Leu Val Arg Gly 645 650 655 Glu Asn Asp Tyr Val Ala Phe Asp Phe Tyr Leu Asp Pro Val Arg Ala 660 665 670 Thr Glu Gly Ala Met Asn Ile Asn Leu Val Phe Gln Pro Pro Thr Asn 675 680 685 Gly Tyr Trp Val Gln Ala Pro Lys Thr Tyr Thr Ile Asn Phe Asp Glu 690 695 700 Leu Glu Glu Ala Asn Gln Val Asn Gly Leu Tyr His Tyr Glu Val Lys 705 710 715 720 Ile Asn Val Arg Asp Ile Thr Asn Ile Gln Asp Asp Thr Leu Leu Arg 725 730 735 Asn Met Met Ile Ile Phe Ala Asp Val Glu Ser Asp Phe Ala Gly Arg 740 745 750 Val Phe Val Asp Asn Val Arg Phe Glu Gly Ala Ala Thr Thr Glu Pro 755 760 765 Val Glu Pro Glu Pro Val Asp Pro Gly Glu Glu Thr Pro Pro Val Asp 770 775 780 Glu Lys Glu Ala Lys Lys Glu Gln Lys Glu Ala Glu Lys Glu Glu Lys 785 790 795 800 Glu Ala Val Lys Glu Glu Lys Lys Glu Ala Lys Glu Glu Lys Lys Ala 805 810 815 Val Lys Asn Glu Ala Lys Lys Lys 820 [Sequence List] SEQUENCE LISTING <110> KAO CORPORATION <120> Gene for Heat Resistant Alkaline Cellulase <130> P00181101 <160> 4 <210> 1 <211> 824 <212> PRT <213> Bacillus sp. <400> 1 Met Met Leu Arg Lys Lys Thr Lys Gln Leu Ile Ser Ser Ile Leu Ile 5 10 15 Leu Val Leu Leu Leu Ser Leu Phe Pro Ala Ala Leu Ala Ala Glu Gly 20 25 30 Asn Thr Arg Glu Asp Asn Phe Lys His Leu Leu Gly Asn Asp Asn Val 35 40 45 Lys Arg Pro Ser Glu Ala Gly Ala Leu Gln Leu Gln Glu Val Asp Gly 50 55 60 Gln Met Thr Leu Val Asp Gln His Gly Glu Lys Ile Gln Leu Arg Gly 65 70 75 80 Met Ser Thr His Gly Leu Gln Trp Phe Pro Glu Ile Leu Asn Asp Asn 85 90 95 Ala Tyr Lys Ala Leu Ser Asn Asp Trp Asp Ser Asn Met Ile Arg Leu 100 105 110 Ala Met Tyr Val Gly Glu Asn Gly Tyr Ala Thr Asn Pro Glu Leu Ile 115 120 125 Lys Gln Arg Val Ile Asp Gly Ile Glu Leu Ala Ile Glu Asn Asp Met 130 135 140 Tyr Val Ile Val Asp Trp His Val His Ala Pro Gly Asp Pro Arg Asp 145 150 155 160 Pro Val Tyr Ala Gly Ala Lys Asp Phe Phe Arg Glu Ile Ala Ala Leu 165 1 70 175 Tyr Pro Asn Asn Pro His Ile Ile Tyr Glu Leu Ala Asn Glu Pro Ser 180 185 190 Ser Asn Asn Asn Gly Gly Ala Gly Ile Pro Asn Asn Glu Glu Gly Trp 195 200 205 Lys Ala Val Lys Glu Tyr Ala Asp Pro Ile Val Glu Met Leu Arg Lys 210 215 220 Ser Gly Asn Ala Asp Asp Asn Ile Ile Ile Val Gly Ser Pro Asn Trp 225 230 235 240 Ser Gln Arg Pro Asp Leu Ala Ala Asp Asn Pro Ile Asp Asp His His 245 250 255 Thr Met Tyr Thr Val His Phe Tyr Thr Gly Ser His Ala Ala Ser Thr 260 265 270 Glu Ser Tyr Pro Ser Glu Thr Pro Asn Ser Glu Arg Gly Asn Val Met 275 280 285 285 Ser Asn Thr Arg Tyr Ala Leu Glu Asn Gly Val Ala Val Phe Ala Thr 290 295 300 Glu Trp Gly Thr Ser Gln Ala Ser Gly Asp Gly Gly Pro Tyr Phe Asp 305 310 315 320 Glu Ala Asp Val Trp Ile Glu Phe Leu Asn Glu Asn Asn Ile Ser Trp 325 330 335 Ala Asn Trp Ser Leu Thr Asn Lys Asn Glu Val Ser Gly Ala Phe Thr 340 345 350 Pro Phe Glu Leu Gly Lys Ser Asn Ala Thr Asn Leu Asp Pro Gly Pro 355 360 365 Asp His Val Trp Ala Pro Glu Glu Leu Ser Leu Ser Gly Glu Tyr Val 370 375 Three 80 Arg Ala Arg Ile Lys Gly Val Asn Tyr Glu Pro Ile Asp Arg Thr Lys 385 390 395 400 Tyr Thr Lys Val Leu Trp Asp Phe Asn Asp Gly Thr Lys Gln Gly Phe 405 410 415 Gly Val Asn Ser Asp Ser Pro Asn Lys Glu Leu Ile Ala Val Asp Asn 420 425 430 Glu Asn Asn Thr Leu Lys Val Ser Gly Leu Asp Val Ser Asn Asp Val 435 440 445 Ser Asp Gly Asn Phe Trp Ala Asn Ala Arg Leu Ser Ala Asn Gly Trp 450 455 460 Gly Lys Ser Val Asp Ile Leu Gly Ala Glu Lys Leu Thr Met Asp Val 465 470 475 475 480 Ile Val Asp Glu Pro Thr Thr Val Ala Ile Ala Ala Ile Pro Gln Ser 485 490 495 Ser Lys Ser Gly Trp Ala Asn Pro Glu Arg Ala Val Arg Val Asn Ala 500 505 510 Glu Asp Phe Val Gln Gln Thr Asp Gly Lys Tyr Lys Ala Gly Leu Thr 515 520 525 Ile Thr Gly Glu Asp Ala Pro Asn Leu Lys Asn Ile Ala Phe His Glu 530 535 540 Glu Asp Asn Asn Met Asn Asn Ile Ile Leu Phe Val Gly Thr Asp Ala 545 550 555 560 Ala Asp Val Ile Tyr Leu Asp Asn Ile Lys Val Ile Gly Thr Glu Val 565 570 575 Glu Ile Pro Val Val His Asp Pro Lys Gly Glu Ala Val Leu Pro Ser 580 585 Five 90 Val Phe Glu Asp Gly Thr Arg Gln Gly Trp Asp Trp Ala Gly Glu Ser 595 600 605 Gly Val Lys Thr Ala Leu Thr Ile Glu Glu Ala Asn Gly Ser Asn Ala 610 615 620 620 Leu Ser Trp Glu Phe Gly Tyr Pro Glu Val Lys Pro Ser Asp Asn Trp 625 630 635 640 Ala Thr Ala Pro Arg Leu Asp Phe Trp Lys Ser Asp Leu Val Arg Gly 645 650 655 Glu Asn Asp Tyr Val Ala Phe Asp Phe Tyr Leu Asp Pro Val Arg Ala 660 665 670 Thr Glu Gly Ala Met Asn Ile Asn Leu Val Phe Gln Pro Pro Thr Asn 675 680 685 Gly Tyr Trp Val Gln Ala Pro Lys Thr Tyr Thr Ile Asn Phe Asp Glu 690 695 700 Leu Glu Glu Ala Asn Gln Val Asn Gly Leu Tyr His Tyr Glu Val Lys 705 710 715 720 Ile Asn Val Arg Asp Ile Thr Asn Ile Gln Asp Asp Thr Leu Leu Arg 725 730 735 Asn Met Met Ile Ile Phe Ala Asp Val Glu Ser Asp Phe Ala Gly Arg 740 745 750 Val Phe Val Asp Asn Val Arg Phe Glu Gly Ala Ala Thr Thr Glu Pro 755 760 765 765 Val Glu Pro Glu Pro Val Asp Pro Gly Glu Glu Thr Pro Pro Val Asp 770 775 780 Glu Lys Glu Ala Lys Lys Glu Gln Lys Glu Ala Glu Lys Glu Glu Lys 785 790 795 8 00 Glu Ala Val Lys Glu Glu Lys Lys Glu Ala Lys Glu Glu Lys Lys Ala 805 810 815 Val Lys Asn Glu Ala Lys Lys Lys 820
【００２３】 <210> 2 <211> 3189 <212> DNA <213> Bacillus sp. <400> 2 gatttgccga tgcaacaggc ttatatttag aggaaatttc tttttaaatt gaatacggaa 60 taaaatcagg taaacaggtc ctgattttat ttttttgagt tttttagaga actgaagatt 120 gaaataaaag tagaagacaa aggacataag aaaattgcat tagttttaat tatagaaaac 180 gcctttttat aattatttat acctagaacg aaaatactgt ttcgaaagcg gtttactata 240 aaaccttata ttccggctct tttttaaaac agggggtaaa aattcactct agtattctaa 300 tttcaacatg ctataataaa tttgtaagac gcaatatgca tctctttttt tacgatatat 360 gtaagcggtt aaccttgtgc tatatgccga tttaggaagg ggggtagatt gagtcaagta 420 gtaataatat agataactta taagttgttg agaagcagga gagcatctgg gttactcaca 480 agttttttta aaactttaac gaaagcactt tcggtaatgc ttatgaattt agctatttga 540 ttcaattact ttaaaaatat ttaggaggta at atg atg tta aga aag aaa aca 593 Met Met Leu Arg Lys Lys Thr 5 aag cag ttg att tct tcc att ctt att tta gtt tta ctt cta tct tta 641 Lys Gln Leu Ile Ser Ser Ile Leu Ile Leu Val Leu Leu Leu Ser Leu 10 15 20 ttt ccg gca gct ctt gca gca gaa gga aac act cgt gaa gac aat ttt 689 Phe Pro Ala Ala Leu Ala Ala Glu Gly Asn Thr Arg Glu Asp Asn Phe 25 30 35 aaa cat tta tta ggt aat gac aat gtt aaa cgc cct tct gag gct ggc 737 Lys His Leu Leu Gly Asn Asp Asn Val Lys Arg Pro Ser Glu Ala Gly 40 45 50 55 gca tta caa tta caa gaa gtc gat gga caa atg aca tta gta gat caa 785 Ala Leu Gln Leu Gln Glu Val Asp Gly Gln Met Thr Leu Val Asp Gln 60 65 70 cat gga gaa aaa att caa tta cgt gga atg agt aca cac gga tta cag 833 His Gly Glu Lys Ile Gln Leu Arg Gly Met Ser Thr His Gly Leu Gln 75 80 85 tgg ttt cct gag atc ttg aat gat aac gca tac aaa gct ctt tct aac 881 Trp Phe Pro Glu Ile Leu Asn Asp Asn Ala Tyr Lys Ala Leu Ser Asn 90 95 100 gat tgg gat tcc aat atg att cgt ctt gct atg tat gta ggt gaa aat 929 Asp Trp Asp Ser Asn Met Ile Arg Leu Ala Met Tyr Val Gly Glu Asn 105 110 115 ggg tac gct aca aac cct gag tta atc aaa caa aga gtg att gat gga 977 Gly Tyr Ala Thr Asn Pro Glu Leu Ile Lys Gln Arg Val Ile Asp Gly 120 125 130 135 att gag tta gcg att gaa aat gac atg tat gtt att gtt gac tgg cat 1025 Ile Glu Leu Ala Ile Glu Asn Asp Met Tyr Val Ile Val Asp Trp His 140 145 150 gtt cat gcg cca ggt gat cct aga gat cct gtt tat gca ggt gct aaa 1073 Val His Ala Pro Gly Asp Pro Arg Asp Pro Val Tyr Ala Gly Ala Lys 155 160 165 gat ttc ttt aga gaa att gca gct tta tac cct aat aat cca cac att 1121 Asp Phe Phe Arg Glu Ile Ala Ala Leu Tyr Pro Asn Asn Pro His Ile 170 175 180 att tat gag tta gcg aat gag ccg agt agt aat aat aat ggt gga gca 1169 Ile Tyr Glu Leu Ala Asn Glu Pro Ser Ser Asn Asn Asn Gly Gly Ala 185 190 195 ggg att ccg aat aac gaa gaa ggt tgg aaa gcg gta aaa gaa tat gct 1217 Gly Ile Pro Asn Asn Glu Glu Gly Trp Lys Ala Val Lys Glu Tyr Ala 200 205 210 215 gat cca att gta gaa atg tta cgt aaa agc ggt aat gca gat gac aac 1265 Asp Pro Ile Val Glu Met Leu Arg Lys Ser Gly Asn Ala Asp Asp Asn 220 225 230 att atc att gtt ggt agt cca aac tgg agt cag cgt ccg gac tta gca 1313 Ile Ile Ile Val Gly Ser Pro Asn Trp Ser Gln Arg Pro Asp Leu Ala 235 240 245 gct gat aat cca att gat gat cac cat aca atg tat act gtt cac ttc 1361 Ala Asp Asn Pro Ile Asp Asp His His Thr Met Tyr Thr Val His Phe 250 255 260 tac act ggt tca cat gct gct tca act gaa agc tat ccg tct gaa act 1409 Tyr Thr Gly Ser His Ala Ala Ser Thr Glu Ser Tyr Pro Ser Glu Thr 265 270 275 cct aac tct gaa aga gga aac gta atg agt aac act cgt tat gcg tta 1457 Pro Asn Ser Glu Arg Gly Asn Val Met Ser Asn Thr Arg Tyr Ala Leu 280 285 290 295 gaa aac gga gta gcg gta ttt gca aca gag tgg gga acg agt caa gct 1505 Glu Asn Gly Val Ala Val Phe Ala Thr Glu Trp Gly Thr Ser Gln Ala 300 305 310 agt gga gac ggt ggt cct tac ttt gat gaa gca gat gta tgg att gaa 1553 Ser Gly Asp Gly Gly Pro Tyr Phe Asp Glu Ala Asp Val Trp Ile Glu 315 320 325 ttt tta aat gaa aac aac att agc tgg gct aac tgg tct tta acg aat 1601 Phe Leu Asn Glu Asn Asn Ile Ser Trp Ala Asn Trp Ser Leu Thr Asn 330 335 340 aaa aat gaa gta tct ggt gca ttt aca cca ttc gag tta ggt aag tct 1649 Lys Asn Glu Val Ser Gly Ala Phe Thr Pro Phe Glu Leu Gly Lys Ser 345 350 355 aac gca acc aat ctt gac cca ggt cca gat cat gtg tgg gca cca gaa 1697 Asn Ala Thr Asn Leu Asp Pro Gly Pro Asp His Val Trp Ala Pro Glu 360 365 370 375 gaa tta agt ctt tct gga gaa tat gta cgt gct cgt att aaa ggt gtg 1745 Glu Leu Ser Leu Ser Gly Glu Tyr Val Arg Ala Arg Ile Lys Gly Val 380 385 390 aac tat gag cca atc gac cgt aca aaa tac acg aaa gta ctt tgg gac 1793 Asn Tyr Glu Pro Ile Asp Arg Thr Lys Tyr Thr Lys Val Leu Trp Asp 395 400 405 ttt aat gat gga acg aag caa gga ttt gga gtg aat tcg gat tct cca 1841 Phe Asn Asp Gly Thr Lys Gln Gly Phe Gly Val Asn Ser Asp Ser Pro 410 415 420 aat aaa gaa ctt att gca gtt gat aat gaa aac aac act ttg aaa gtt 1889 Asn Lys Glu Leu Ile Ala Val Asp Asn Glu Asn Asn Thr Leu Lys Val 425 430 435 tcg gga tta gat gta agt aac gat gtt tca gat ggc aac ttc tgg gct 1937 Ser Gly Leu Asp Val Ser Asn Asp Val Ser Asp Gly Asn Phe Trp Ala 440 445 450 455 aat gct cgt ctt tct gcc aac ggt tgg gga aaa agt gtt gat att tta 1985 Asn Ala Arg Leu Ser Ala Asn Gly Trp Gly Lys Ser Val Asp Ile Leu 460 465 470 ggt gct gag aag ctt aca atg gat gtt att gtt gat gaa cca acg acg 2033 Gly Ala Glu Lys Leu Thr Met Asp Val Ile Val Asp Glu Pro Thr Thr 475 480 485 gta gct att gcg gcg att cca caa agt agt aaa agt gga tgg gca aat 2081 Val Ala Ile Ala Ala Ile Pro Gln Ser Ser Lys Ser Gly Trp Ala Asn 490 495 500 cca gag cgt gct gtt cga gtg aac gcg gaa gat ttt gtc cag caa acg 2129 Pro Glu Arg Ala Val Arg Val Asn Ala Glu Asp Phe Val Gln Gln Thr 505 510 515 gac ggt aag tat aaa gct gga tta aca att aca gga gaa gat gct cct 2177 Asp Gly Lys Tyr Lys Ala Gly Leu Thr Ile Thr Gly Glu Asp Ala Pro 520 525 530 535 aac cta aaa aat atc gct ttt cat gaa gaa gat aac aat atg aac aac 2225 Asn Leu Lys Asn Ile Ala Phe His Glu Glu Asp Asn Asn Met Asn Asn 540 545 550 atc att ctg ttc gtg gga act gat gca gct gac gtt att tac tta gat 2273 Ile Ile Leu Phe Val Gly Thr Asp Ala Ala Asp Val Ile Tyr Leu Asp 555 560 565 aac att aaa gta att gga aca gaa gtt gaa att cca gtt gtt cat gat 2321 Asn Ile Lys Val Ile Gly Thr Glu Val Glu Ile Pro Val Val His Asp 570 575 580 cca aaa gga gaa gct gtt ctt cct tct gtt ttt gaa gac ggt aca cgt 2369 Pro Lys Gly Glu Ala Val Leu Pro Ser Val Phe Glu Asp Gly Thr Arg 585 590 595 caa ggt tgg gac tgg gct gga gag tct ggt gtg aaa aca gct tta aca 2417 Gln Gly Trp Asp Trp Ala Gly Glu Ser Gly Val Lys Thr Ala Leu Thr 600 605 610 615 att gaa gaa gca aac ggt tct aac gcg tta tca tgg gaa ttt gga tat 2465 Ile Glu Glu Ala Asn Gly Ser Asn Ala Leu Ser Trp Glu Phe Gly Tyr 620 625 630 cca gaa gta aaa cct agt gat aac tgg gca aca gct cca cgt tta gat 2513 Pro Glu Val Lys Pro Ser Asp Asn Trp Ala Thr Ala Pro Arg Leu Asp 635 640 645 ttc tgg aaa tct gac ttg gtt cgc ggt gag aat gat tat gta gct ttt 2561 Phe Trp Lys Ser Asp Leu Val Arg Gly Glu Asn Asp Tyr Val Ala Phe 650 655 660 gat ttc tat cta gat cca gtt cgt gca aca gaa ggc gca atg aat atc 2609 Asp Phe Tyr Leu Asp Pro Val Arg Ala Thr Glu Gly Ala Met Asn Ile 665 670 675 aat tta gta ttc cag cca cct act aac ggg tat tgg gta caa gca cca 2657 Asn Leu Val Phe Gln Pro Pro Thr Asn Gly Tyr Trp Val Gln Ala Pro 680 685 690 695 aaa acg tat acg att aac ttt gat gaa tta gag gaa gcg aat caa gta 2705 Lys Thr Tyr Thr Ile Asn Phe Asp Glu Leu Glu Glu Ala Asn Gln Val 700 705 710 aat ggt tta tat cac tat gaa gtg aaa att aac gta aga gat att aca 2753 Asn Gly Leu Tyr His Tyr Glu Val Lys Ile Asn Val Arg Asp Ile Thr 715 720 725 aac att caa gat gac acg tta cta cgt aac atg atg atc att ttt gca 2801 Asn Ile Gln Asp Asp Thr Leu Leu Arg Asn Met Met Ile Ile Phe Ala 730 735 740 gat gta gaa agt gac ttt gca ggg aga gtc ttt gta gat aat gtt cgt 2849 Asp Val Glu Ser Asp Phe Ala Gly Arg Val Phe Val Asp Asn Val Arg 745 750 755 ttt gag ggg gct gct act act gag ccg gtt gaa cca gag cca gtt gat 2897 Phe Glu Gly Ala Ala Thr Thr Glu Pro Val Glu Pro Glu Pro Val Asp 760 765 770 775 cct ggc gaa gag acg cca cct gtc gat gag aag gaa gcg aaa aaa gaa 2945 Pro Gly Glu Glu Thr Pro Pro Val Asp Glu Lys Glu Ala Lys Lys Glu 780 785 790 caa aaa gaa gca gag aaa gaa gag aaa gaa gca gta aaa gaa gaa aag 2993 Gln Lys Glu Ala Glu Lys Glu Glu Lys Glu Ala Val Lys Glu Glu Lys 795 800 805 aaa gaa gct aaa gaa gaa aag aaa gca gtc aaa aat gag gct aag aaa 3041 Lys Glu Ala Lys Glu Glu Lys Lys Ala Val Lys Asn Glu Ala Lys Lys 810 815 820 aaa taatctatta aactagttat agggttatct aaaggtctga tgtagatctt ttagat 3100 Lys aacctttttc ttgcataact ggacacagag ttgttattaa agaaagtaag gtgtttaaac 3160 gataataaaa gggtagcgat tttaaattg 3189<210> 2 <211> 3189 <212> DNA <213> Bacillus sp. ttcgaaagcg gtttactata 240 aaaccttata ttccggctct tttttaaaac agggggtaaa aattcactct agtattctaa 300 tttcaacatg ctataataaa tttgtaagac gcaatatgca tctctttttt tacgatatat 360 gtaagcggtt aaccttgtgc tatatgccga tttaggaagg ggggtagatt gagtcaagta 420 gtaataatat agataactta taagttgttg agaagcagga gagcatctgg gttactcaca 480 agttttttta aaactttaac gaaagcactt tcggtaatgc ttatgaattt agctatttga 540 ttcaattact ttaaaaatat ttaggaggta at atg atg tta aga aag aaa aca 593 Met Met Leu Arg Lys Lys Thr 5 aag cag ttg att tct tcc att ctt att tta gtt tta ctt cta tct tta 641 Lys Gln Leu Ile Ser Ser Ile Leu Ile Leu Val Leu Leu Leu Ser Leu 10 15 20 ttt ccg gca gct ctt gca gca gaa gga aac act cgt gaa gac a at ttt 689 Phe Pro Ala Ala Leu Ala Ala Glu Gly Asn Thr Arg Glu Asp Asn Phe 25 30 35 aaa cat tta tta ggt aat gac aat gtt aaa cgc cct tct gag gct ggc 737 Lys His Leu Leu Gly Asn Asp Asn Val Lys Arg Pro Ser Glu Ala Gly 40 45 50 55 gca tta caa tta caa gaa gtc gat gga caa atg aca tta gta gat caa 785 Ala Leu Gln Leu Gln Glu Val Asp Gly Gln Met Thr Leu Val Asp Gln 60 65 70 cat gga gaa aaa att caa tta cgt gga atg agt aca cac gga tta cag 833 His Gly Glu Lys Ile Gln Leu Arg Gly Met Ser Thr His Gly Leu Gln 75 80 85 tgg ttt cct gag atc ttg aat gat aac gca tac aaa gct ctt tct aac 881 Trp Phe Pro Glu Ile Leu Asn Asp Asn Ala Tyr Lys Ala Leu Ser Asn 90 95 100 gat tgg gat tcc aat atg att cgt ctt gct atg tat gta ggt gaa aat 929 Asp Trp Asp Ser Asn Met Ile Arg Leu Ala Met Tyr Val Gly Glu Asn 105 110 115 ggg tac gct aca aac cct gag tta atc aaa caa aga gtg att gat gga 977 Gly Tyr Ala Thr Asn Pro Glu Leu Ile Lys Gln Arg Val Ile Asp Gly 120 125 130 135 att gag tta gcg att gaa aat gac atg tat gtt att gtt gac tgg cat 1025 Ile Glu Leu Ala Ile Glu Asn Asp Met Tyr Val Ile Val Asp Trp His 140 145 150 gtt cat gcg cca ggt gat cct aga gat cct gtt tat gca ggt gct aaa 1073 Val His Ala Pro Gly Asp Pro Arg Asp Pro Val Tyr Ala Gly Ala Lys 155 160 165 gat ttc ttt aga gaa att gca gct tta tac cct aat aat cca cac att 1121 Asp Phe Phe Arg Glu Ile Ala Ala Leu Tyr Pro Asn Asn Pro His Ile 170 175 180 att tat gag tta gcgat gag ccg agt agt aat aat aat ggt gga gca 1169 Ile Tyr Glu Leu Ala Asn Glu Pro Ser Ser Asn Asn Asn Gly Gly Ala 185 190 195 ggg att ccg aat aac gaa gaa ggt tgg aaa gcg gta aaa gaa tat gle Pro 1217 G Asn Asn Glu Glu Gly Trp Lys Ala Val Lys Glu Tyr Ala 200 205 210 215 gat cca att gta gaa atg tta cgt aaa agc ggt aat gca gat gac aac 1265 Asp Pro Ile Val Glu Met Leu Arg Lys Ser Gly Asn Ala Asp Asp Asn 220 225 230 att atc att gtt ggt agt cca aac tgg agt cag cgt ccg gac tta gca 1313 Ile Ile Ile Val Gly Ser Pro Asn Trp Ser Gln Arg Pro Asp Leu Ala 235 240 245 gct gat aat cca att gat gat cac cat a ca atg tat act gtt cac ttc 1361 Ala Asp Asn Pro Ile Asp Asp His His Thr Met Tyr Thr Val His Phe 250 255 260 tac act ggt tca cat gct gct tca act gaa agc tat ccg tct gaa act 1409 Tyr Thr Gly Ser His Ala Ala Ser Thr Glu Ser Tyr Pro Ser Glu Thr 265 270 275 cct aac tct gaa aga gga aac gta atg agt aac act cgt tat gcg tta 1457 Pro Asn Ser Glu Arg Gly Asn Val Met Ser Asn Thr Arg Tyr Ala Leu 280 285 290 290 295 gaa aac gga gta gcg gta ttt gca aca gag tgg gga acg agt caa gct 1505 Glu Asn Gly Val Ala Val Phe Ala Thr Glu Trp Gly Thr Ser Gln Ala 300 305 310 agt gga gac ggt ggt cct tac ttt gat gaa gca gat gta tgg att gaa 1553 Ser Gly Asp Gly Gly Pro Tyr Phe Asp Glu Ala Asp Val Trp Ile Glu 315 320 325 ttt tta aat gaa aac aac att agc tgg gct aac tgg tct tta acg aat 1601 Phe Leu Asn Glu Asn Asn Ile Ser Trp Ala Asn Trp Ser Leu Thr Asn 330 335 340 aaa aat gaa gta tct ggt gca ttt aca cca ttc gag tta ggt aag tct 1649 Lys Asn Glu Val Ser Gly Ala Phe Thr Pro Phe Glu Leu Gly Lys Ser 345 350 355 aac gca acc aat ct t gac cca ggt cca gat cat gtg tgg gca cca gaa 1697 Asn Ala Thr Asn Leu Asp Pro Gly Pro Asp His Val Trp Ala Pro Glu 360 365 370 375 gaa tta agt ctt tct gga gaa tat gta cgt gct cgt att aaa ggt gtg 1745 Glu Leu Ser Leu Ser Gly Glu Tyr Val Arg Ala Arg Ile Lys Gly Val 380 385 390 aac tat gag cca atc gac cgt aca aaa tac acg aaa gta ctt tgg gac 1793 Asn Tyr Glu Pro Ile Asp Arg Thr Lys Tyr Thr Lys Val Leu Trp Asp 395 400 405 ttt aat gat gga acg aag caa gga ttt gga gtg aat tcg gat tct cca 1841 Phe Asn Asp Gly Thr Lys Gln Gly Phe Gly Val Asn Ser Asp Ser Pro 410 415 420 aat aaa gaa ctt att gca gtt gat aat gaa aac aac act ttg aaa gtt 1889 Asn Lys Glu Leu Ile Ala Val Asp Asn Glu Asn Asn Thr Leu Lys Val 425 430 435 tcg gga tta gat gta agt aac gat gtt tca gat ggc aac ttc tgg gct 1937 Ser Gly Leu Asp Val Asn Asp Val Ser Asp Gly Asn Phe Trp Ala 440 445 450 455 aat gct cgt ctt tct gcc aac ggt tgg gga aaa agt gtt gat att tta 1985 Asn Ala Arg Leu Ser Ala Asn Gly Trp Gly Lys Ser Val Asp Ile Leu 460 465 470 ggt gct gag aag ctt aca atg gat gtt att gtt gat gaa cca acg acg 2033 Gly Ala Glu Lys Leu Thr Met Asp Val Ile Val Asp Glu Pro Thr Thr 475 480 485 gta gct att gcg gcg att cca caa agt agt agt gga tgg gca aat 2081 Val Ala Ile Ala Ala Ile Pro Gln Ser Ser Lys Ser Gly Trp Ala Asn 490 495 500 cca gag cgt gct gtt cga gtg aac gcg gaa gat ttt gtc cag caa acg 2129 Pro Glu Arg Ala Val Arg Val Asn Ala Glu Asp Phe Val Gln Gln Thr 505 510 515 gac ggt aag tat aaa gct gga tta aca att aca gga gaa gat gct cct 2177 Asp Gly Lys Tyr Lys Ala Gly Leu Thr Ile Thr Gly Glu Asp Ala Pro 520 525 530 530 535 aac cta aaa aat atc gct ttt cat gaa gaa gat aac aat atg aac aac 2225 Asn Leu Lys Asn Ile Ala Phe His Glu Glu Asp Asn Asn Met Asn Asn 540 545 550 atc att ctg ttc gtg gga act gat gca gct gac gtt att tac tta gat Ile Leu Phe Val Gly Thr Asp Ala Ala Asp Val Ile Tyr Leu Asp 555 560 565 aac att aaa gta att gga aca gaa gtt gaa att cca gtt gtt cat gat 2321 Asn Ile Lys Val Ile Gly Thr Glu Val Glu Ile Pro Val Val His Asp 570 575 580 cca aaa gga gaa gct gtt ctt cct tct gtt ttt gaa gac ggt aca cgt 2369 Pro Lys Gly Glu Ala Val Leu Pro Ser Val Phe Glu Asp Gly Thr Arg 585 590 595 595 caa ggt tgg gac tgg gct gga gag tct ggt gtg aaa aca gct tta aca 2417 Gln Gly Trp Asp Trp Ala Gly Glu Ser Gly Val Lys Thr Ala Leu Thr 600 605 610 615 att gaa gaa gca aac ggt tct aac gcg tta tca tgg gaa ttt gga tat 2465 Ale Glu Asn Gly Ser Asn Ala Leu Ser Trp Glu Phe Gly Tyr 620 625 630 cca gaa gta aaa cct agt gat aac tgg gca aca gct cca cgt tta gat 2513 Pro Glu Val Lys Pro Ser Asp Asn Trp Ala Thr Ala Pro Arg Leu Asp 635 640 645 ttc tgg aaa tct gac ttg gtt cgc ggt gag aat gat tat gta gct ttt 2561 Phe Trp Lys Ser Asp Leu Val Arg Gly Glu Asn Asp Tyr Val Ala Phe 650 655 660 gat ttc tat cta gat cca gtt cgt gca acaga atg aat atc 2609 Asp Phe Tyr Leu Asp Pro Val Arg Ala Thr Glu Gly Ala Met Asn Ile 665 670 675 aat tta gta ttc cag cca cct act aac ggg tat tgg gta caa gca cca 2657 Asn Leu Val Phe Gln Pro Pro Thr A sn Gly Tyr Trp Val Gln Ala Pro 680 685 690 695 aaa acg tat acg att aac ttt gat gaa tta gag gaa gcg aat caa gta 2705 Lys Thr Tyr Thr Ile Asn Phe Asp Glu Leu Glu Glu Ala Asn Gln Val 700 705 710 aat ggt tta tat cac tat gaa gtg aaa att aac gta aga gat att aca 2753 Asn Gly Leu Tyr His Tyr Glu Val Lys Ile Asn Val Arg Asp Ile Thr 715 720 725 aac att caa gat gac acg tta cta cgt aac atg atg atc att ttt gca 2801 Asn Ile Gln Asp Asp Thr Leu Leu Arg Asn Met Met Ile Ile Phe Ala 730 735 740 gat gta gaa agt gac ttt gca ggg aga gtc ttt gta gat aat gtt cgt 2849 Asp Val Glu Ser Asp Phe Ala Gly Arg Val Phe Val Asp Asn Val Arg 745 750 755 ttt gag ggg gct gct act act gag ccg gtt gaa cca gag cca gtt gat 2897 Phe Glu Gly Ala Ala Thr Thr Glu Pro Val Glu Pro Glu Pro Val Asp 760 765 770 770 775 cct ggc gaa gag acg cca cct gtc gat gag aag gaa gcg aaa aaa gaa 2945 Pro Gly Glu Glu Thr Pro Pro Val Asp Glu Lys Glu Ala Lys Lys Glu 780 785 790 caa aaa gaa gca gag aaa gaa gag aaa gaa gca gta aaa gaa gaa Gag 2993 Gln u Ala Glu Lys Glu Glu Lys Glu Ala Val Lys Glu Glu Lys 795 800 805 aaa gaa gct aaa gaa gaa aag aaa gca gtc aaa aat gag gct aag aaa 3041 Lys Glu Ala Lys Glu Glu Lys Lys Ala Val Lys Asn Glu Ala Lys 810 815 820 aaa taatctatta aactagttat agggttatct aaaggtctga tgtagatctt ttagat 3100 Lys aacctttttc ttgcataact ggacacagag ttgttattaa agaaagtaag gtgtttaaac 3160 gataataaaa gggtagcgat tttaaattg 3189
【００２４】 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 gatgcaacag gcttatattt agag 24<210> 3 <211> 24 <212> DNA <213> Artificial Sequence <400> 3 gatgcaacag gcttatattt agag 24
【００２５】 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 aaattacttc atcattctat cac 23<210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> 4 aaattacttc atcattctat cac 23
───────────────────────────────────────────────────── 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 1/21 Ｃ１２Ｒ 1:19） (72)発明者 小林 徹 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 BA12 CA01 CA03 CA20 DA06 DA07 EA04 GA19 GA21 GA27 4B050 CC01 CC03 DD02 EE01 LL04 LL05 4B065 AA15Y AA19X AA26X AB01 AC14 AC15 BA02 BA10 BA23 BB01 BB16 BB23 BC01 BC03 BC26 BD50 CA31 CA57 ──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 7 Identification symbol FI Theme coat ゛ (Reference) (C12N 1/21 C12R 1:19) (72) Inventor Toru Kobayashi 2606 Kabane-cho, Kaigacho, Haga-gun, Tochigi Pref. In-house F-term (reference) 4B024 AA03 BA12 CA01 CA03 CA20 DA06 DA07 EA04 GA19 GA21 GA27 4B050 CC01 CC03 DD02 EE01 LL04 LL05 4B065 AA15Y AA19X AA26X AB01 AC14 AC15 BA02 BA10 BA23 BB01 BB16 CA50
Claims (5)
アミノ酸配列の１若しくは数個のアミノ酸が欠失、置換
或いは付加されたアミノ酸配列をコードする耐熱性アル
カリセルラーゼ遺伝子。1. A heat-resistant alkaline cellulase gene encoding the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence in which one or several amino acids of the amino acid sequence have been deleted, substituted or added.
列番号２の塩基番号５７３〜３０４４に示す塩基配列又
は該塩基配列の１若しくは数個の塩基が欠失、置換或い
は付加された塩基配列を有する請求項１記載の耐熱性ア
ルカリセルラーゼ遺伝子。2. The heat-resistant alkaline cellulase gene has a nucleotide sequence represented by nucleotide numbers 573 to 3044 of SEQ ID NO: 2 or a nucleotide sequence in which one or several nucleotides of the nucleotide sequence are deleted, substituted or added. Item 6. The thermostable alkaline cellulase gene according to Item 1.
なる塩基配列又は該塩基配列の１若しくは数個の塩基が
欠失、置換或いは付加された塩基配列を有するものであ
る請求項１又は２記載の耐熱性アルカリセルラーゼ遺伝
子。3. The method according to claim 1, which has a base sequence consisting of base numbers 1 to 3044 of SEQ ID NO: 2 or a base sequence in which one or several bases of the base sequence are deleted, substituted or added. The described thermostable alkaline cellulase gene.
組換えベクター。4. A recombinant vector comprising the gene according to claim 1, 2 or 3.
質転換体。A transformant comprising the recombinant vector according to claim 4.
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|---|---|---|---|
| JP11013049A JP2000210081A (en) | 1999-01-21 | 1999-01-21 | Heat-resistant alkali cellulase gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP11013049A JP2000210081A (en) | 1999-01-21 | 1999-01-21 | Heat-resistant alkali cellulase gene |
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| JP2000210081A true JP2000210081A (en) | 2000-08-02 |
Family
ID=11822279
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-
1999
- 1999-01-21 JP JP11013049A patent/JP2000210081A/en active Pending
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