JPH05219948A - Cyclodextrin glucanotransferase, its production and gene coding the same enzyme - Google Patents
Cyclodextrin glucanotransferase, its production and gene coding the same enzymeInfo
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- JPH05219948A JPH05219948A JP4799392A JP4799392A JPH05219948A JP H05219948 A JPH05219948 A JP H05219948A JP 4799392 A JP4799392 A JP 4799392A JP 4799392 A JP4799392 A JP 4799392A JP H05219948 A JPH05219948 A JP H05219948A
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- cgtase
- asp
- ser
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Abstract
Description
【0001】[0001]
【産業上の利用分野】本発明は、サイクロデキストリン
グルカノトランスフェラーゼ、その製造法、およびその
サイクロデキストリングルカノトランスフェラーゼをコ
ードする遺伝子に関する。TECHNICAL FIELD The present invention relates to a cyclodextrin glucanotransferase, a method for producing the same, and a gene encoding the cyclodextrin glucanotransferase.
【0002】[0002]
【従来の技術】サイクロデキストリン(以下、CDとい
う。)はグルコース分子が環状に連なった非還元性のマ
ルトオリゴ糖で、グルコース分子の数により、α(6
個)、β(7個)およびγ(8個)の3種が存在する。
CDはバチルス属(Bacillus)をはじめとする多くの微
生物が生産するCD生産酵素によりデンプンから生産さ
れる。BACKGROUND OF THE INVENTION Cyclodextrin (hereinafter referred to as CD) is a non-reducing maltooligosaccharide in which glucose molecules are linked in a ring, and α (6
There are three types, i.e.), β (7) and γ (8).
CD is produced from starch by a CD-producing enzyme produced by many microorganisms including Bacillus .
【0003】CDは、その分子構造により各種有機化合
物を分子空洞内に取り込んで優れた物性を持つ安定した
包接化合物を形成することが知られており、そのような
特性から、揮発性物質の安定化、酸化性・光分解性物質
の保護、溶解度や色、味などの性質の変化、水不溶性物
質の乳化などに広く用いられている物質であり、食品、
医薬品、香料などをはじめとする種々の分野でのCDの
需要が急速に高まりつつある。特に、γ−CDは、その
溶解度や生分解性において優れている他、分子内空洞が
最も大きいために多種類の物質を包接できる点で利用価
値が大きいが、3種のCDのうちでは最も生産量が少な
い。Due to its molecular structure, CD is known to take in various organic compounds into the molecular cavity to form a stable clathrate compound having excellent physical properties. It is a substance that is widely used for stabilization, protection of oxidative and photodegradable substances, changes in properties such as solubility, color and taste, and emulsification of water-insoluble substances.
Demand for CDs in various fields such as pharmaceuticals and fragrances is rapidly increasing. In particular, γ-CD is excellent in solubility and biodegradability, and has a great utility value in that it can enclose many kinds of substances because it has the largest intramolecular cavity. The smallest production volume.
【0004】サイクロデキストリングルカノトランスフ
ェラーゼ(以下、CGTaseという。)は、デンプン
を基質として直鎖状のデキストリンを生成すると同時
に、転移反応により上記3種のCDの混合物を生成する
転移酵素であり、同一の酵素分子が多種類の反応を触媒
する代表的な多機能酵素である。CGTaseは、表1
に示すようにバチルス属をはじめとする多くの微生物か
ら産生されることが知られており、これらは反応初期に
αまたはβ−CDを主として生成するが、反応の進行と
ともに主生産物以外のCDも生成してくる。Cyclodextrin glucanotransferase (hereinafter referred to as CGTase) is a transferase that produces linear dextrin from starch as a substrate and at the same time produces a mixture of the above-mentioned three types of CD by the same reaction. The enzyme molecule is a typical multifunctional enzyme that catalyzes many kinds of reactions. CGTase is shown in Table 1.
It is known that it is produced from many microorganisms including Bacillus as shown in Fig. 2, and these mainly produce α or β-CD in the early stage of the reaction, but as the reaction proceeds, CD other than the main product is produced. Will also be generated.
【0005】[0005]
【表1】 [Table 1]
【0006】CGTaseは酵素反応の主産物の相違に
より、α−CD生産型、β−CD生産型の2グループに
大別される。γ−CD生産型酵素についてはバチルス・
スブチリス(Bacillus subtilis) No.313 が生産する
CGTaseが知られているだけであり、通常γ−CD
はデンプンからα−CD、あるいはβ−CDを生産する
ときの副産物として少量生産されるにすぎない。バチル
ス・スブチリス(Bacillus subtilis) No.313 CGT
aseの場合でも、デンプンからのγ−CDの生産量は
対糖収率で2〜3%にすぎない状態であり、この値はα
−CD生産型やβ−CD生産型の酵素が副産物として生
産するγ−CDの量以下である。CGTases are roughly classified into two groups, α-CD producing type and β-CD producing type, depending on the difference in the main products of the enzymatic reaction. For the γ-CD producing enzyme, Bacillus
Only CGTase produced by Bacillus subtilis No. 313 is known, and usually γ-CD
Is only produced in a small amount as a by-product when producing α-CD or β-CD from starch. Bacillus subtilis No.313 CGT
Also in the case of asase, the production amount of γ-CD from starch is only 2-3% in terms of sugar yield, and this value is α
The amount is equal to or less than the amount of γ-CD produced as a by-product by the CD-producing or β-CD-producing enzyme.
【0007】一方、好アルカリ性細菌の1種であるバチ
ルス・オーベンシス(Bacillus ohbensis) C-1400 株
より生産されるCGTaseは、デンプンを基質として
β−CDを生産し、実際に工業的レベルでβ−CDの生
産に利用されている酵素である。そのアミノ酸配列およ
びそのDNA配列は本発明者らによって解明されてお
り、その遺伝子を大腸菌等に導入してそれらの発現系を
用いることによる大量生産も可能になった(特開平3-21
6191号)。この酵素は、通常の反応条件下ではαーCD
をほとんど生産しないことや、他のβ−CGTaseに
比べて比較的大量にγ−CDを生産するという特徴を持
っているが、β−CDとγ−CDとの混合物を分離する
際、HPLCを用いなければならず、コストの面で大き
な問題となっている。On the other hand, CGTase produced from Bacillus ohbensis C-1400 strain, which is one of the alkalophilic bacteria, produces β-CD using starch as a substrate, and actually produces β-CD at an industrial level. It is an enzyme used for the production of CD. The amino acid sequence and the DNA sequence thereof have been elucidated by the present inventors, and it became possible to mass-produce them by introducing the gene into Escherichia coli or the like and using their expression system (Japanese Patent Laid-Open No. 3-21).
No. 6191). This enzyme is α-CD under normal reaction conditions.
Is characterized in that γ-CD is produced in a relatively large amount as compared with other β-CGTases, but when separating a mixture of β-CD and γ-CD, HPLC is used. It has to be used, which is a big problem in terms of cost.
【0008】このように、γ−CDはその用途が最も多
いにもかかわらず、生産量が少なく高価であるのが現状
である。一方、遺伝子操作技術の普及とともに、CGT
ase遺伝子のクローン化が続々となされており、これ
までのCGTaseの構造と機能の相関の研究ととも
に、遺伝子を改変することにより酵素蛋白質を改良する
試みも行われている。例えば、木村らは、デンプンから
β−CDを主に生産する好アルカリ性バチルス(Bacill
us) 1011 CGTaseのC末端側の10個あるいは1
3個のアミノ酸を欠失させると、β−CD以外に相当量
のグルコース、マルトオリゴ糖およびα−CDを生産す
るようになることを示した(Biochemical and Biophysi
cal Research Communications, 161, 1273(1989)および
日本農芸化学大会講演要旨, 65, 286(1991) 参照)。し
かし、γ−CDを大量に得ることを目的として行なわれ
た報告例は今だにない。As described above, γ-CD is currently in a small amount of production and expensive although it has the most applications. On the other hand, with the spread of gene manipulation technology, CGT
The asase gene has been cloned one after another, and attempts have been made to improve the enzyme protein by modifying the gene, as well as studies on the correlation between the structure and function of CGTase to date. For example, Kimura et al. Reported that the alkalophilic Bacillus ( Bacill) mainly produces β-CD from starch.
us ) 1011 10 or 1 at the C-terminal side of CGTase
It was shown that deletion of 3 amino acids leads to the production of a considerable amount of glucose, maltooligosaccharides and α-CD in addition to β-CD (Biochemical and Biophysi).
cal Research Communications, 161, 1273 (1989) and Abstracts of the Japan Agricultural Chemistry Conference, 65, 286 (1991)). However, there are no reports to date for the purpose of obtaining a large amount of γ-CD.
【0009】[0009]
【発明が解決しようとする課題】従って、本発明の課題
はバチルス・オーベンシス(Bacillus ohbensis) C-1
400 株より生産されるCGTaseの遺伝子操作技術に
よってγ−CD生産性に優れたCGTase、その製造
法、およびそのCGTaseをコードする遺伝子を提供
することにある。SUMMARY OF THE INVENTION Therefore, the object of the present invention is to provide Bacillus ohbensis C-1.
It is intended to provide a CGTase excellent in γ-CD productivity, a method for producing the same, and a gene encoding the CGTase by gene manipulation technology of the CGTase produced from 400 strains.
【0010】[0010]
【課題を解決するための手段】本発明者らは、バチルス
・オーベンシス(Bacillus ohbensis) C-1400 株より
生産されるCGTaseが、通常の反応条件下ではαー
CDをほとんど生産しないことや、他のβ−CGTas
eに比べて比較的大量にγ−CDを生産するという特徴
を持つことに着目し、α−CDを主に生産するCGTa
seとβ−CDを主に生産するCGTaseの構造を比
較したところ、酵素特異性に関与すると考えられるいく
つかの領域において、大きく異なる点が存在することを
確認した。また、バチルス・オーベンシス(Bacillus
ohbensis) C-1400 株より生産されるCGTaseの特
定部位を他のアミノ酸に改変することにより、デンプン
から従来よりも高い収率でγ−CDを生産できることを
見出し、本発明に至った。The inventors of the present invention have found that CGTase produced from Bacillus ohbensis C-1400 strain produces almost no α-CD under normal reaction conditions. Β-CGTas
Focusing on the characteristic that γ-CD is produced in a relatively large amount compared to e, CGTa which mainly produces α-CD
Comparing the structures of CGTase that mainly produces se and β-CD, it was confirmed that there are large differences in some regions that are considered to be involved in enzyme specificity. Also, Bacillus orvensis ( Bacillus
The present inventors have found that γ-CD can be produced from starch in a higher yield than before by modifying a specific site of CGTase produced by the strain Ohbensis C-1400 with another amino acid.
【0011】すなわち、本発明は 1)バチルス・オーベンシス・エスピー・ノブ・C−1
400(Bacillus ohbensis sp. nov. C-1400 )由来の
配列番号1のアミノ酸配列において、188番目のチロ
シンがアラニン、セリンまたはトリプトファンに改変さ
れたサイクロデキストリングルカノトランスフェラー
ゼ、 2)バチルス・オーベンシス・エスピー・ノブ・C−1
400(Bacillus ohbensis sp. nov. C-1400 )のDN
Aにおける配列番号1の塩基配列を含む遺伝子におい
て、1756〜1758番目“TAT”が“GCT”、
“GCC”、“GCA”、GCG”、“TCT”、“T
CC”、TCA”、“TCG”、“AGT”、“AG
C”または“TGG”に改変されたサイクロデキストリ
ングルカノトランスフェラーゼ遺伝子、および 3)前記2に記載の遺伝子を導入した形質転換微生物を
培養し、培養液からサイクロデキストリングルカノトラ
ンスフェラーゼを取得することを特徴とするサイクロデ
キストリングルカノトランスフェラーゼの製造方法を提
供したものである。That is, the present invention is: 1) Bacillus obensis SP Knob C-1
In the amino acid sequence of SEQ ID NO: 1 derived from 400 (Bacillus ohbensis sp. Nov. C-1400), the cyclodextrin glucanotransferase in which the tyrosine at 188 is modified to alanine, serine or tryptophan, 2) Bacillus obensis sp. Knob C-1
DN of 400 (Bacillus ohbensis sp. Nov. C-1400)
In the gene containing the nucleotide sequence of SEQ ID NO: 1 in A, 1756 to 1758th "TAT" is "GCT",
"GCC", "GCA", GCG "," TCT "," T "
CC ”, TCA”, “TCG”, “AGT”, “AG
A cyclodextrin glucanotransferase gene modified to C "or" TGG ", and 3) culturing a transformed microorganism into which the gene described in 2 above is introduced, and obtaining a cyclodextrin glucanotransferase from the culture solution. The present invention provides a method for producing cyclodextrin glucanotransferase.
【0012】以下、本発明を詳細に説明する。本発明者
らは、まずα−CD生産型の代表的酵素としてバチルス
・マセランス(Bacillus macerans) CGTase、
β−CD生産型の代表的酵素としてバチルス・ステアロ
テルモフィルス(Bacillus stearothermophilus) C
GTaseを用いて、バチルス・オーベンシス(Bacill
us ohbensis) C-1400 CGTaseと比較した。The present invention will be described in detail below. The present inventors first of all, as a representative enzyme of α-CD producing type, Bacillus macerans CGTase,
Bacillus stearothermophilus C as a representative enzyme of β-CD producing type
Using GTase, Bacillus orvensis ( Bacill
us ohbensis ) C-1400 CGTase.
【0013】バチルス・ステアロテルモフィルス(Baci
llus stearothermophilus) CGTaseは、β−C
D生産型の代表的酵素として知られており、X線結晶解
析による立体構造が解明されている(Kubota,M., Mikam
i,B., Tsujisaka,Y., & Morita,Y.,: J.Biochem., 104,
12(1988) および久保田倫夫,松浦良樹,堺修造,勝部
幸輝:日本農芸化学大会講演要旨,65, 671(1991) 参
照)数少ないCGTaseのひとつである。その立体構
造における活性中心近傍には、生成するCDの種類の決
定に関与すると考えられる構造上の特徴が認められる。
特に、生成したCDの空洞部にはフェニルアラニン19
1が存在すると推定され、この部位のアミノ酸残基が空
間的に大きいものになると、小さなCDの生成が困難に
なるものと考えられる。バチルス・ステアロテルモフィ
ルス(Bacillus stearothermophilus)CGTaseの
フェニルアラニン191の相当部としては、バチルス・
マセランス(Bacillus macerans) CGTaseでは
チロシン195、バチルス・オーベンシス(Bacillus
ohbensis) C-1400 CGTaseではチロシン188
残基であることがわかった。そこで、バチルス・オーベ
ンシス(Bacillus ohbensis) C-1400 CGTase
におけるチロシン188残基を、バチルス・ステアロテ
ルモフィルス(Bacillus stearothermophilus)型のフ
ェニルアラニンより空間的に大きいトリプトファンの
他、ヒスチジン、アラニンおよびセリンへと置換した場
合の酵素特性を調べたところ、アラニンおよびセリン置
換体ではβ−CDとγ−CDがほぼ同率で生成し、また
トリプトファンへ置換したときはγ−CDの生産量が対
糖収率で15%になることが確認され、この知見に基づ
き本発明に至ったものである。Bacillus stearothermophilus ( Baci
llus stearothermophilus ) CGTase is β-C
It is known as a representative D-producing enzyme, and its three-dimensional structure has been elucidated by X-ray crystallography (Kubota, M., Mikam
i, B., Tsujisaka, Y., & Morita, Y.,: J.Biochem., 104,
12 (1988) and Nobuo Kubota, Yoshiki Matsuura, Shuzo Sakai, Koki Katsube: Abstracts of the Japan Agricultural Chemistry Conference, 65 , 671 (1991)) It is one of the few CGTases. In the vicinity of the active center in the three-dimensional structure, structural features considered to be involved in determining the type of CD to be produced are recognized.
In particular, phenylalanine 19 is present in the cavity of the generated CD.
1 is presumed to exist, and if the amino acid residue at this site becomes spatially large, it may be difficult to generate a small CD. The equivalent portion of phenylalanine 191 of Bacillus stearothermophilus CGTase is Bacillus stearothermophilus.
Macerans (Bacillus macerans) CGTase in tyrosine 195, Bacillus Obenshisu (Bacillus
ohbensis ) Tyrosine 188 in C-1400 CGTase
It was found to be a residue. So, Bacillus ohbensis C-1400 CGTase
In addition to tryptophan, which is spatially larger than Bacillus stearothermophilus phenylalanine, the tyrosine 188 residue in Escherichia coli was replaced with histidine, alanine, and serine, and the enzymatic properties were found to be alanine and serine. It was confirmed that β-CD and γ-CD were produced at almost the same rate in the substitution product, and that the yield of γ-CD was 15% in terms of sugar yield when substituted with tryptophan. It was the invention.
【0014】本発明において用いる、アミノ酸あるいは
塩基の置換を起こさせる方法としては、目的とするアミ
ノ酸または塩基の置換を起こさせることができるもので
あれば如何なる方法を用いてもよい。高頻度で変異を起
こさせる方法の一つに部位特異的変異法が挙げられる。
これは、2本鎖DNAプラスミドに組み込んだ遺伝子を
鋳型として、目的変異部位に置換塩基を有するオリゴヌ
クレオチドを変異源として用いる方法(M.J.Zoller, M.S
mith, "Method in Enzymology", vol.100, R.Wu, L.Gro
ssmann, K.Moldave ed., P468, Academic Press (198
3))で、Kunkel法(Kunkel,T.A. (1985) Proc. Natl. Ac
ad. Sci. USA.,82, 488.) やGrapped duplex法(Karmer,
W. et al.(1984)Nucl. AcidsRes.,12, 9441) などいく
つかの改良法があるが、そのいずれを用いて改変を行な
ってもよい。Any method may be used as the method for causing the substitution of the amino acid or the base used in the present invention, as long as it can cause the substitution of the target amino acid or the base. Site-specific mutagenesis is one of the methods for causing mutations at a high frequency.
This is a method of using a gene incorporated in a double-stranded DNA plasmid as a template and an oligonucleotide having a substitution base at a target mutation site as a mutation source (MJZoller, MS
mith, "Method in Enzymology", vol.100, R.Wu, L.Gro
ssmann, K. Moldave ed., P468, Academic Press (198
3)), the Kunkel method (Kunkel, TA (1985) Proc. Natl. Ac
ad. Sci. USA., 82 , 488.) and Grapped duplex method (Karmer,
There are several improved methods such as W. et al. (1984) Nucl. Acids Res., 12 , 9441), and any of them may be used for modification.
【0015】また、アミノ酸の置換を起こさせる方法に
は、部位特異的変異法に限らず、ポリメラーゼチェーン
リアクション(PCR)を用いる方法(Mullis,K., Falo
ona,F.,Scharf, S.,Saiki, R/.Horn,G., and Erlich,
H.,(1986) Cold Spring Harber Symp. 51:263) や、カ
セットミュータジェネシス法(J.H.Richards, Nature, 3
23, 187 (1986)) などがあり、これらの方法を用いるこ
とも可能である。The method of causing amino acid substitution is not limited to the site-directed mutagenesis method, but a method using polymerase chain reaction (PCR) (Mullis, K., Falo
ona, F., Scharf, S., Saiki, R / .Horn, G., and Erlich,
H., (1986) Cold Spring Harber Symp. 51: 263) and cassette mutagenesis method (JHRichards, Nature, 3
23 , 187 (1986)), and these methods can also be used.
【0016】本発明において用いる、変異の鋳型として
の野生型CGTase遺伝子は、バチルス・オーベンシ
ス・エスピー・ノブ・C−1400(Bacillus ohbensi
s sp. nov. C-1400 )よりクローンしたDNA断片であ
る。クローニングは、公知の方法、例えば、特開平3-21
6191号に示された方法で行なうことができるが、その他
の方法を用いても構わない。このDNA断片中には、少
なくともプレプロ体部分を含む構造遺伝子がコードされ
ている部分が含まれていれば、そのDNAの長さや両端
の制限酵素切断部位には特に制限はなく如何なるもので
あってもよい。また、実際に変異操作を行なう際には、
必ずしもDNA断片の全てを用いる必要はなく、変異を
行なう部分を含んだ一部のDNA断片のみを変異操作に
用い、その後他の部分と結合し、その後の操作に用いて
もよい。本発明における改変したCGTaseは、変異
を起こさせた遺伝子を微生物中に導入し、その微生物を
培地中に培養することにより生産させ、培養液からCG
Taseを回収することによって取得できる。The wild-type CGTase gene used as a mutation template in the present invention is Bacillus obensis sp. Nob C-1400 (Bacillus ohbensi).
s sp. nov. C-1400). Cloning is a known method, for example, JP-A-3-21.
It can be carried out by the method shown in No. 6191, but other methods may be used. As long as this DNA fragment contains at least a portion encoding a structural gene containing a prepro body portion, there is no particular limitation on the length of the DNA or the restriction enzyme cleavage sites at both ends, and any DNA fragment can be used. Good. Also, when actually performing the mutation operation,
It is not always necessary to use all of the DNA fragments, and only a part of the DNA fragments containing the part to be mutated may be used for the mutation operation, then linked to other parts, and used for the subsequent operation. The modified CGTase according to the present invention is produced by introducing a mutated gene into a microorganism and culturing the microorganism in a medium to produce CG from the culture solution.
It can be obtained by collecting Tase.
【0017】この際、用いるDNAとしては、バチルス
・オーベンシス・エスピー・ノブ・C−1400(Baci
llus ohbensis sp. nov. C-1400 )由来の発現生産に関
与する部分を全て含んでいるCGTase遺伝子を用い
てもよいし、CGTaseの構造遺伝子部分に別のプロ
モーター領域や、シグナル配列、ターミネーター領域等
を結合したDNA断片を用いることも可能である。この
際用いるプロモーター領域や、シグナル配列、ターミネ
ーター領域等は、遺伝子を導入する宿主菌中でCGTa
seを発現、生産することができればどのようなものを
用いてもよい。At this time, the DNA to be used is Bacillus obensis SP Knob C-1400 (Baci
llus ohbensis sp. nov. C-1400) -derived CGTase gene containing all the parts involved in expression and production may be used, or another promoter region, signal sequence, terminator region or the like may be used in the structural gene part of CGTase. It is also possible to use a DNA fragment in which The promoter region, signal sequence, terminator region, etc. used at this time are CGTa in the host strain into which the gene is introduced.
Any substance may be used as long as it can express and produce se.
【0018】宿主菌としては、改変したCGTase遺
伝子を導入でき、発現・生産可能なものであればよい
が、生産した改変CGTaseを菌体外に分泌すること
から、工業的生産にはバチルス属細菌を用いることが望
ましい。さらに、バチルス・スブチリス(Bacillus su
btilis)207−21(Otozaki,K., et. al., J. Gen.
Appl. Microbial.,30,15(1984) )あるいはその変異株
を用いると、より好適に実施できる。As the host bacterium, any bacterium capable of introducing the modified CGTase gene and capable of expressing and producing can be used. However, since the produced modified CGTase is secreted out of the cells, a bacterium belonging to the genus Bacillus is used for industrial production. Is preferred. In addition, Bacillus su
btilis ) 207-21 (Otozaki, K., et. al., J. Gen.
Appl. Microbial., 30 , 15 (1984)) or a mutant strain thereof can be more preferably used.
【0019】宿主菌の形質転換は通常の方法で可能であ
る。例えば、バチルス属細菌では、コンピーテントセル
法(J.Spizizen, Proc. Natl. Sci. USA,44, 1072 (195
8))、プロトプラスト法(Chang,S., S.N.Cohen, Molec.
Gen. Genet. 168, 111 (1979)) 等で実施できるが、こ
れらの方法に限られない。導入した変異CGTase遺
伝子は、プラスミド等を用いて、染色体外に存在、複製
する方法で安定に維持させてもよいし、染色体中に組み
込む(インテグレーション)方法でもよい。染色体外に
存在、複製する方法の場合には、宿主菌中で複製可能な
ベクターを用いる。例えば、バチルス属細菌を宿主とし
て用いる場合、pUB110、pUP110、pC19
4、pE194、pTP5、pSD3501など公知の
枯草菌用プラスミドや、上記の枯草菌プラスミドと大腸
菌プラスミドとのシャトルベクターなどを用いることが
できるが、宿主菌内で複製可能であれば、これに限られ
ない。Transformation of the host bacterium can be performed by a usual method. For example, in the case of Bacillus bacteria, the competent cell method (J. Spizizen, Proc. Natl. Sci. USA, 44 , 1072 (195
8)), protoplast method (Chang, S., SNCohen, Molec.
Gen. Genet. 168 , 111 (1979)), etc., but not limited to these methods. The introduced mutant CGTase gene may be stably maintained by a method of existing and replicating extrachromosomally using a plasmid or the like, or may be incorporated into a chromosome (integration). In the case of the method of existing and replicating extrachromosomally, a vector capable of replicating in the host bacterium is used. For example, when Bacillus bacterium is used as a host, pUB110, pUP110, pC19
4, known plasmids for Bacillus subtilis such as pE194, pTP5, pSD3501 and shuttle vectors of the above Bacillus subtilis plasmid and E. coli plasmid can be used. Absent.
【0020】一方、染色体中に組み込む方法では、ベク
ターを用いないか、あるいは宿主菌内で複製しないもの
を用いることが望ましく、例えば、大腸菌プラスミドp
BR322、pUC18、pHSG396、pSD31
65、pACYC184、等を用いることができる。形
質転換株の選択は、導入遺伝子に結合した遺伝子マーカ
ーの発現等により行なうことができる。On the other hand, in the method of integrating into the chromosome, it is desirable to use no vector or one that does not replicate in the host bacterium. For example, E. coli plasmid p
BR322, pUC18, pHSG396, pSD31
65, pACYC184, etc. can be used. The transformant can be selected by the expression of a gene marker linked to the transgene.
【0021】上記のようにして得た形質転換株を培養す
る培地としては特に制限はなく、該形質転換株が生育す
るものであればどのような培地でも用いることができ
る。培養液からのCGTaseの分離精製は、一般的な
蛋白質の分離精製法に従って行なうことができる。例え
ば、コーンスターチのようなデンプンを添加して吸着・
沈殿させてから緩衝液に酵素を溶出させることにより可
能であるが、これに限らず他の方法でも可能である。The medium for culturing the transformant obtained as described above is not particularly limited, and any medium can be used as long as the transformant can grow. Separation and purification of CGTase from the culture solution can be performed according to a general method for separating and purifying proteins. For example, adding starch such as corn starch
This can be done by precipitating and then eluting the enzyme in a buffer solution, but the method is not limited to this, and other methods are also possible.
【0022】本発明においては、CGTaseの活性は
以下に示すようにblue value法により測定し
た。すなわち、可溶性デンプン(potato starch,シグマ
社製)を、50mM Na2 CO3 −NaHCO3 緩衝
液(pH10.0)に、可溶性デンプン(potato starch,シ
グマ社製)が0.025 %になるように溶かしたものを基質
溶液とした。基質溶液2.5 mlに対して酵素溶液0.2 m
lを加え、40℃で10分間反応させた後に、0.6 N
HCl 2mlを加えて反応を停止させ、0.1 % KI
−0.01%I2 1mlを加え、660nmでの吸光度を測
定した。酵素1単位は上記の条件下、660nmでの吸
光度を1.0 減少させる量とした。In the present invention, the activity of CGTase was measured by the blue value method as shown below. That is, soluble starch (potato starch, manufactured by Sigma) was dissolved in 50 mM Na 2 CO 3 -NaHCO 3 buffer solution (pH 10.0) so that soluble starch (potato starch, manufactured by Sigma) was 0.025%. The thing was used as the substrate solution. 0.2 m of enzyme solution for 2.5 ml of substrate solution
1 was added and reacted at 40 ° C. for 10 minutes, then 0.6 N
The reaction was stopped by adding 2 ml of HCl, and 0.1% KI
The -0.01% I 2 1 ml was added, and the absorbance was measured at 660 nm. One unit of enzyme was used as an amount that reduces the absorbance at 660 nm by 1.0 under the above conditions.
【0023】[0023]
【実施例】以下に本発明について代表的な例を示し、さ
らに具体的に説明する。ただし、これらは単なる例示で
あり、本発明はこれらのみに限られるものではない。 実施例1:改変CGTase遺伝子の構築 改変CGTase遺伝子を図1(1) 〜(5) に示すように
構築した。The present invention will be described in more detail below with reference to typical examples. However, these are merely examples, and the present invention is not limited to these. Example 1: Construction of modified CGTase gene A modified CGTase gene was constructed as shown in FIGS. 1 (1) to (5).
【0024】バチルス・オーベンシス・エスピー・ノブ
・C−1400(Bacillus ohbensis sp. nov. C-1400
)からクローニングされた野生型CGTaseの遺伝
子をSphIとHindIII で部分消化することにより
約4.8 KbのpUC19CGT5A断片を切り出し、こ
れをプラスミドpUP110BSCsacQ(Uozumi,
T., et. al., Agric. Biol. Chem.,55(9),2367-2374(19
91))と混合し、T4ファージDNAリガーゼ処理して
連結した。このプラスミドをpUP110Ce−CGT
とした。さらに、CGTase遺伝子の発現量を増加さ
せるために、バチルス・スブチリス セルラーゼ遺伝子
の発現量を上昇させることが知られているバチルス・ス
ブチリスsacQ遺伝子を、プラスミド上のBamHI
−SphIの間に組み込んだ。こうして得られたプラス
ミドをpUP110CeCGTsacQとする。このD
NAを用い、大腸菌MV1184に形質転換を行ない、
アンピシリンに耐性の形質転換体を選択した。この形質
転換体より、常法によりプラスミドDNAを抽出、精製
し、分析したところ、野生型プラスミドDNApUP1
10CeCGTsacQを保有していた。Bacillus ohbensis sp. Nov. C-1400
The fragment of the wild type CGTase gene cloned from (1) was partially digested with SphI and HindIII to excise a pUC19CGT5A fragment of about 4.8 Kb, which was excised from the plasmid pUP110BSCsacQ (Uozumi,
T., et.al., Agric. Biol. Chem., 55 (9), 2367-2374 (19
91)), mixed with T4 phage DNA ligase and ligated. This plasmid was designated as pUP110Ce-CGT.
And Furthermore, in order to increase the expression level of the CGTase gene, the Bacillus subtilis sacQ gene, which is known to increase the expression level of the Bacillus subtilis cellulase gene, was added to BamHI on a plasmid.
-Incorporated between SphI. The plasmid thus obtained is designated as pUP110CeCGTsacQ. This D
E. coli MV1184 was transformed with NA,
Transformants resistant to ampicillin were selected. From this transformant, plasmid DNA was extracted by a conventional method, purified, and analyzed to find that wild-type plasmid DNA pUP1
Had 10CeCGTsacQ.
【0025】部位特異的変異は、以下のようにKunk
el法に従って行なった。プラスミドpUP110Ce
CGTsacQを大腸菌CJ236に形質転換し、プラ
スミドpUP110CeCGTsacQをもつ大腸菌C
J236を得る。この大腸菌から、Messing らの方法(V
ieira,J. and Messing,J. (1987) Methodin Enzymolog
y, 153, 3)に従い1本鎖DNAを調整しDNA中のデオ
キシチアミン(dT)の一部がデオキシウラシル(d
U)に置き代わった1本鎖DNAを得る。配列番号2〜
6に示したオリゴヌクレオチドを各々化学合成し、T4
ポリヌクレオチドキナーゼにより5′末端をリン酸化す
る。The site-directed mutation is Kunk as follows.
It was performed according to the EL method. Plasmid pUP110Ce
CGTsacQ was transformed into Escherichia coli CJ236 and E. coli C harboring the plasmid pUP110CeCGTsacQ
Get J236. From this E. coli, the method of Messing et al.
ieira, J. and Messing, J. (1987) Methodin Enzymolog
Single-stranded DNA was prepared according to y, 153 , 3), and a part of deoxythiamine (dT) in the DNA was deoxyuracil (dT).
A single-stranded DNA replacing U) is obtained. Sequence number 2
Each of the oligonucleotides shown in 6 was chemically synthesized, and T4
The 5'end is phosphorylated by polynucleotide kinase.
【0026】このオリゴヌクレオチドを用いて上記の1
本鎖DNAをアニーリングバッファー(200mM T
ris−HCl,pH 8.0,100mM MgCl2 ,
500mM NaCl,10mM DTT)中で混合
し、65℃で15分間静置後、さらに37℃で15分間
静置し、オリゴヌクレオチドを変異の目的部位にアニー
リングさせた。次に、このDNA混合液に 2.5倍量のエ
クステンションバッファー(50mMTris−HC
l,pH 8.0,60mM 酢酸アンモニウム,5mM
MgCl2 ,5mM DTT,1mM NAD, 0.5m
M dNTP(A,C,G,T))を加え、さらにE.
coli DNAリガーゼとT4DNAポリメラーゼを
加えて25℃で2時間静置して相補鎖の合成を行なっ
た。その後、このDNAを大腸菌BMH71−18 m
utSに常法に従い形質転換しアンピシリン耐性の形質
転換株を選択した。得られた形質転換株のいくつかか
ら、常法に従い1本鎖DNAを調製し、Sangerら
によるジデオキシ法により塩基配列を決定し、チロシン
188をコードする遺伝子の塩基である“TAT”が
“TGG”、“GCT”、“TCT”、“TTT”およ
び“CAT”に改変されているプラスミドDNApUP
110CeCGTsacQ−Y188W、pUP110
CeCGTsacQ−Y188A、pUP110CeC
GTsacQ−Y188S、pUP110CeCGTs
acQ−Y188FおよびpUP110CeCGTsa
cQ−Y188Hを取得した。Using this oligonucleotide, the above 1
Annealing buffer (200 mM T
ris-HCl, pH 8.0, 100 mM MgCl 2 ,
The mixture was mixed in 500 mM NaCl, 10 mM DTT), left standing at 65 ° C. for 15 minutes, and further left at 37 ° C. for 15 minutes to anneal the oligonucleotide to the target site of mutation. Next, add 2.5 times as much extension buffer (50 mM Tris-HC
1, pH 8.0, 60 mM ammonium acetate, 5 mM
MgCl 2 , 5 mM DTT, 1 mM NAD, 0.5 m
MdNTP (A, C, G, T)) was added, and E.
E. coli DNA ligase and T4 DNA polymerase were added and the mixture was allowed to stand at 25 ° C. for 2 hours to synthesize a complementary strand. Then, this DNA was used for Escherichia coli BMH71-18 m
utS was transformed by a conventional method to select an ampicillin-resistant transformant. Single-stranded DNA was prepared from some of the obtained transformants according to a conventional method, and the nucleotide sequence was determined by the dideoxy method by Sanger et al., And the base "TAT" of the gene encoding tyrosine 188 was "TGG.""PCT,""GCT","TCT","TTT" and "CAT"
110CeCGTsacQ-Y188W, pUP110
CeCGTsacQ-Y188A, pUP110CeC
GTsacQ-Y188S, pUP110CeCGTs
acQ-Y188F and pUP110CeCGTsa
cQ-Y188H was obtained.
【0027】実施例2:バチルス・スブチリス(Bacill
us subtilis)207−21による改変CGTaseの
生産 実施例1で得られた野生型プラスミドDNApUP11
0CeCGTsacQおよび改変プラスミドDNApU
P110CeCGTsacQ−Y188W、pUP11
0CeCGTsacQ−Y188A、pUP110Ce
CGTsacQ−Y188S、pUP110CeCGT
sacQ−Y188F、およびpUP110CeCGT
sacQ−Y188Hを用い、バチルス・スブチリス
(Bacillussubtilis)207−21をプロトプラスト法
により形質転換し、カナマイシン耐性の形質転換体を選
択した。これらの形質転換体より、常法によりプラスミ
ドDNAを抽出、精製し、分析したところ、いずれも、
野生型プラスミドDNApUP110CeCGTsac
Qおよび改変プラスミドDNApUP110CeCGT
sacQ−Y188W、pUP110CeCGTsac
Q−Y188A、pUP110CeCGTsacQ−Y
188S、pUP110CeCGTsacQ−Y188
F、およびpUP110CeCGTsacQ−Y188
Hを保有していた。該形質転換株を、終濃度で10μg
/mlのカナマイシンを含有するL−broth10m
l中で37℃、36時間振盪培養し、培地中に改変酵素
を生産させた。この培養液を、4℃、15000rpmで遠心分
離し、菌体を除去して、上清をとり、粗酵素液を調製し
た。なお、プラスミドDNApUP110CeCGTs
acQ−Y188Wを組入れた微生物であるバチルス・
スブチリス(Bacillus subtilis)207−21(pU
P110CeCGTasesacQ(Y188W))は
微生物工業技術研究所に受託番号微工研菌寄第12720 号
(FERM P−12720 )で1992年1月22日に寄託さ
れている。[0027] Example 2: Bacillus subtilis (Bacill
production of modified CGTase by us subtilis ) 207-21 The wild-type plasmid DNA pUP11 obtained in Example 1
0CeCGTsacQ and modified plasmid DNApU
P110CeCGTsacQ-Y188W, pUP11
0CeCGTsacQ-Y188A, pUP110Ce
CGTsacQ-Y188S, pUP110CeCGT
sacQ-Y188F, and pUP110CeCGT
using sacQ-Y188H, Bacillus subtilis and (Bacillus subtilis) 207-21 was transformed by the protoplast method, and transformants were selected for kanamycin resistance. From these transformants, plasmid DNA was extracted, purified, and analyzed by a conventional method.
Wild-type plasmid DNA pUP110CeCGTsac
Q and modified plasmid DNA pUP110CeCGT
sacQ-Y188W, pUP110CeCGTsac
Q-Y188A, pUP110CeCGTsacQ-Y
188S, pUP110CeCGTsacQ-Y188
F, and pUP110CeCGTsacQ-Y188
Had H. 10 μg of the transformant at a final concentration
L-broth containing 100 mg / ml kanamycin
The cells were cultivated with shaking at 37 ° C. for 36 hours to produce the modified enzyme in the medium. This culture solution was centrifuged at 4 ° C. and 15000 rpm to remove bacterial cells, and the supernatant was taken to prepare a crude enzyme solution. In addition, plasmid DNApUP110CeCGTs
Bacillus which is a microorganism incorporating acQ-Y188W
Subtilis (Bacillus subtilis) 207-21 (pU
P110CeCGTasesacQ (Y188W)) has been deposited at the Institute of Microbial Science and Technology on Jan. 22, 1992 under the deposit number, Microindustrial Research Institute No. 12720 (FERM P-12720).
【0028】実施例3:改変CGTaseの精製 実施例2により得られた各種粗酵素液に対して、デンプ
ンを2%になるように加え、4℃で1時間撹拌して酵素
を吸着させた。遠心分離(4℃、7000rpm )にデンプン
を集め、50mMリン酸ナトリウム緩衝液(pH 6.8)
で洗浄後、同じ緩衝液に懸濁し、40℃で2時間撹拌す
ることにより酵素を溶出させた。遠心分離によりデンプ
ンを除去し、その上清を精製酵素標品とした。必要によ
りセントリプレップ−30(アミコン社製)を用いて限
外ろ過を行ない、酵素溶液の濃縮を行なう。なお、精製
過程はSDS−PAGEにより確認した。Example 3: Purification of modified CGTase Starch was added to the various crude enzyme solutions obtained in Example 2 in an amount of 2% to stir at 4 ° C. for 1 hour to adsorb the enzyme. Collect starch by centrifugation (4 ℃, 7000rpm), 50mM sodium phosphate buffer (pH 6.8)
After washing with, the cells were suspended in the same buffer and stirred at 40 ° C. for 2 hours to elute the enzyme. The starch was removed by centrifugation, and the supernatant was used as a purified enzyme preparation. If necessary, Centriprep-30 (manufactured by Amicon) is used for ultrafiltration to concentrate the enzyme solution. The purification process was confirmed by SDS-PAGE.
【0029】実施例4:改変CGTaseの酵素特性 実施例3により得られた酵素液を用いて、各変異酵素の
特性を野生型酵素と比較することによって調べた。 (1)分子量の測定 実施例3で得られた各種変異酵素の分子量をSDS−P
AGEにより調べ、野生型のCGTaseと比較したと
ころ、分子量はすべて約80,000ダルトンであることがわ
かった。SDS−PAGEによるゲル上のバンドの様子
を図2に示す。Example 4: Enzymatic properties of modified CGTase Using the enzyme solution obtained in Example 3, the properties of each mutant enzyme were examined by comparing with the wild-type enzyme. (1) Measurement of molecular weight The molecular weights of various mutant enzymes obtained in Example 3 were measured by SDS-P.
When examined by AGE and compared with wild type CGTase, it was found that the molecular weights were all about 80,000 daltons. The appearance of bands on the gel by SDS-PAGE is shown in FIG.
【0030】(2)至適pHの測定 可溶性デンプンを基質として、40℃で10分間反応さ
せ、各種変異酵素のpHによる活性を調べた。最大活性
に対する相対活性の測定結果を図3に示す。野生型CG
TaseがpH5.0 とpH10.0の2つの活性領域を有す
るのに対して、アラニン、ヒスチジン、セリン置換体変
異CGTaseでは、その至適pHがpH6.0 付近に変
化していた。(2) Measurement of optimum pH The soluble starch as a substrate was reacted at 40 ° C. for 10 minutes, and the activity of various mutant enzymes depending on pH was examined. The measurement results of relative activity with respect to maximum activity are shown in FIG. Wild type CG
While Tase has two active regions of pH 5.0 and pH 10.0, the optimum pH of alanine-, histidine-, and serine-substitute mutant CGTases was changed to around pH 6.0.
【0031】(3)酵素反応生産物の分析 デンプンを基質として、各種変異酵素の加水分解能を経
時的に調べた。生産物であるCDの分析はHPLCによ
り行なった。100mM McIlvaine緩衝液
(pH6.0 )に可溶性デンプンが6%になるように溶か
した基質溶液2.5 mlに対して、15単位の酵素溶液を
各々0.5 ml加え、50℃で1〜24時間反応させた後
に、10分間の煮沸で酵素反応を停止し、分析に用い
た。NH2 −1251−N(4.6 ×250mm、センシュー
科学社製)カラムに反応液20μlを添加し、65%ア
セトニトリルを用いて、1.5 ml/minの流速で溶出
させ、屈折率検出機(ERC−7510、Erma Opt
ical Works社製)で生産物であるCDを検出
した。(3) Analysis of enzymatic reaction products Using starch as a substrate, the hydrolyzing ability of various mutant enzymes was examined with time. The product CD was analyzed by HPLC. 0.5 ml each of 15 units of enzyme solution was added to 2.5 ml of a substrate solution prepared by dissolving 6% soluble starch in 100 mM McIlvaine buffer (pH 6.0) and reacted at 50 ° C. for 1 to 24 hours. After that, the enzymatic reaction was stopped by boiling for 10 minutes and used for analysis. 20 μl of the reaction solution was added to an NH 2 1251-N (4.6 × 250 mm, Senshu Scientific Co., Ltd.) column, which was eluted with 65% acetonitrile at a flow rate of 1.5 ml / min, and a refractive index detector (ERC-7510) was used. , Erma Opt
The product, CD, was detected by using ICAL WORKS.
【0032】各変異酵素の反応生産物の経時的変化を図
4に示す。図4からもわかるように、野生型CGTas
eにおけるチロシン188置換体変異酵素において、ア
ラニン、セリンおよびトリプトファン置換体変異酵素
は、野生型CGTaseと比べてβ−CDに対するγ−
CDの相対生産量が増大していることがわかる。特に、
アラニン、セリン置換体変異酵素ではβ−CDとγ−C
Dがほぼ同率で生成された。また、トリプトファン置換
体変異酵素はγ−CDの生産量が対糖収率で15%に達
した。The time-dependent changes in the reaction products of each mutant enzyme are shown in FIG. As can be seen from FIG. 4, wild-type CGTas
In the tyrosine 188 substitution mutant enzyme in e, the alanine, serine and tryptophan substitution mutant enzyme has a γ-to β-CD compared to the wild-type CGTase.
It can be seen that the relative production of CD is increasing. In particular,
Alanine and serine substitution mutant enzymes have β-CD and γ-C
D was produced at approximately the same rate. In addition, the tryptophan-substituted mutant enzyme produced a γ-CD production amount of 15% in terms of sugar yield.
【0033】[0033]
【発明の効果】バチルス・オーベンシス・エスピー・ノ
ブ・C−1400(Bacillus ohbensis sp. nov. C-140
0 )由来の配列番号1のアミノ酸配列において、188
番目のチロシンを他のアミノ酸に置換することにより、
産生される酵素の性質や、その酵素と基質とも相互作用
に大きな影響を及ぼし、チロシン188残基をより空間
的に大きな他のアミノ酸であるトリプトファンの他、ア
ラニン、セリンに置換した場合、その置換変異CGTa
seのデンプンから生産するCDは、野生型CGTas
eと比べてβ−CDに対するγ−CDの相対生産量が増
大する。アラニン、セリン置換体変異酵素ではβ−CD
とγ−CDがほぼ同率で生成され、トリプトファン置換
体変異酵素はγ−CDの生産量が対糖収率で15%に達
したことから、公知である分野への応用に限らず、従来
困難であったγ−CDの大量生産に利用できる。EFFECT OF THE INVENTION Bacillus ohbensis sp. Nov. C-1400 (Bacillus ohbensis sp. Nov. C-140)
0) in the amino acid sequence of SEQ ID NO: 1
By substituting the tyrosine at the other with another amino acid,
When the tyrosine 188 residue is replaced with another spatially larger amino acid, tryptophan, alanine, or serine, it has a great influence on the properties of the produced enzyme and its interaction with the substrate. Mutant CGTa
The CD produced from the starch of Se is wild-type CGTas.
The relative production amount of γ-CD with respect to β-CD is increased as compared with e. Β-CD for alanine and serine substitution mutant enzymes
And γ-CD were produced at almost the same rate, and the tryptophan-substituted mutant enzyme produced γ-CD at a yield of 15% in terms of sugar yield. The above can be used for mass production of γ-CD.
【0034】[0034]
【0035】配列番号:1 配列の長さ:4785 配列の型:核酸 鎖の数:二本鎖 トポロジー:直鎖状 配列の種類:Genomic DNA 起源:バチルス・オーベンシス・エスピー・ノブ・C-14
00(Bacillus ohbensissp. nov. C-1400 ) 配列の特徴 特徴を表わす記号:mat peptide 存在位置:1195..3219 特徴を決定した方法:S 配列 GAGCTCGCCG CCTTCGTAAG CGCCTTCATT ATTTAAACCA ACATCCTCCG TATCATATTC 60 CCCTACATCA CCGCCAAAAA TATAGGCACC GTAATTTTTG AGGAAAGGAA AAGCAAAATA 120 GAGATCATTA GGTCTCATCA GAAATCCATA TTGATCAGCT GATGCATCCG TATTTTCTTC 180 AAGAATCGTT CTTAAATCTT CGATCGTCTC TGGTGCTTCT GGTACAATTT CTTTATTGTA 240 GAATATTCCA TATGTTTCGA TAACAGCTGG AACTCCATAA ATATCCGTTT CTCCTTCGAA 300 CTCATAGGTG ACCGCATCTA TAGAGGAACT AGCATAATTG CTTAACTCGT CATCTGATAG 360 AGTAAGAGGA TCAGCTAATC CTTGTGCCAC AATATTTCCG ATCTGGTCAT GTGGCTGGAA 420 AAATAAATCG GGACCATTCC CTTCTGGACC TGCTAATGAC AATTCCTGCA ATTGGTCCAT 480 CATCGGCTTT GTCTCAACCT TCACGTTAAT TCCCGTTTGC TCTGTATAAT CGTTGCTATT 540 TTTTCAATCG CCTCTAATTG CTCTTCTCTA TCATTTGCCC AGATGGGTTA ATTCTTCAGG 600 TTGATCCGCT TCCCCACCAT TTGTATCCGT GGTTACTGCC TCTTCTTCCC GTTCAGGTGC 660 ACATGCCGCT AAGAGAATAA TCATTAAAAA CATGGTAATC AGTGATAATA ATGAAAAACC 720 TTTTTTCATT TCATTTCCCC CCTGAAAAAA TTATATTAAT CTAGTAAATG TTGAGCTACC 780 TTTACATAAA GGAAAGTAAC GCAAACGTTT GCACTAGATT AGATACAACA ATCATACATG 840 GCAATTCAAT TTTTTGCAAT CGTTTACAGT AAAGAATTTT TTGAATTTTT ATTTAGTTAC 900 AATTACCTAT TTACAAATGA AATGTCTATG AGATAAAAAT AAATTAACAA CTATGAATGT 960 TCATATTCTC AATACGAGGG CAATGCTTTT GTTTTTTACA AACAACCCCG TCTTTTATTC 1020 CATTAAAAGG CGGGGTTATT TTATTGGATA AGGAGGGTTG TATTGCTTGT TCATACGATT 1080 CATTTTTAAT GTATAGGGGG AGTATTT TTG AAA AAT TTA ACG GTT TTA TTG AAA 1134 Met Lys Asn Leu Thr Val Leu Leu Lys -25 ACG ATT CCA TTA GCT CTG TTA CTT TTC ATC CTT CTT TCT TTA CCA ACA 1182 Thr Ile Pro Leu Ala Leu Leu Leu Phe Ile Leu Leu Ser Leu Pro Thr -20 -15 -10 -5 GCG GCT CAG GCT GAC GTT ACA AAT AAG GTT AAT TAT ACA AGA GAT GTT 1230 Ala Ala Gln Ala Asp Val Thr Asn Lys Val Asn Tyr Thr Arg Asp Val 1 5 10 ATT TAT CAA ATT GTG ACC GAC CGT TTT TCT GAT GGA GAT CCA TCC AAC 1278 Ile Tyr Gln Ile Val Thr Asp Arg Phe Ser Asp Gly Asp Pro Ser Asn 15 20 25 AAC CCA ACG GGG GCG ATT TAT AGT CAG GAT TGT AGC GAC CTT CAT AAA 1326 Asn Pro Thr Gly Ala Ile Tyr Ser Gln Asp Cys Ser Asp Leu His Lys 30 35 40 TAT TGT GGG GGC GAC TGG CAA GGA ATT ATC GAC AAA ATC AAT GAC GGG 1374 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly 45 50 55 60 TAT TTA ACC GAT TTA GGA ATT ACA GCG ATT TGG ATT TCA CAG CCT GTA 1422 Tyr Leu Thr Asp Leu Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val 65 70 75 GAA AAT GTC TAT GCT CTT CAT CCG AGT GGT TAT ACC TCT TAT CAC GGG 1470 Glu Asn Val Tyr Ala Leu His Pro Ser Gly Tyr Thr Ser Tyr His Gly 80 85 90 TAT TGG GCA AGA GAT TAT AAA AGA ACG AAC CCT TTT TAT GGC GAT TTC 1518 Tyr Trp Ala Arg Asp Tyr Lys Arg Thr Asn Pro Phe Tyr Gly Asp Phe 95 100 105 TCT GAC TTT GAC CGG TTA ATG GAT ACT GCA CAT AGT AAT GGC ATT AAG 1566 Ser Asp Phe Asp Arg Leu Met Asp Thr Ala His Ser Asn Gly Ile Lys 110 115 120 GTA ATC ATG GAC TTT ACG CCC AAC CAT TCA TCA CCA GCT CTT GAA ACC 1614 Val Ile Met Asp Phe Thr Pro Asn His Ser Ser Pro Ala Leu Glu Thr 125 130 135 140 GAT CCA AGC TAT GCC GAG AAC GGA GCG GTT TAT AAT GAT GGC GTA TTA 1662 Asp Pro Ser Tyr Ala Glu Asn Gly Ala Val Tyr Asn Asp Gly Val Leu 145 150 155 ATA GGC AAT TAT TCT AAC GAC CCT AAC AAC CTC TTT CAC CAT AAT GGT 1710 Ile Gly Asn Tyr Ser Asn Asp Pro Asn Asn Leu Phe His His Asn Gly 160 165 170 GGG ACA GAT TTT TCA TCC TAT GAA GAT AGC ATT TAT CGA AAT CTA TAT 1758 Gly Thr Asp Phe Ser Ser Tyr Glu Asp Ser Ile Tyr Arg Asn Leu Tyr 175 180 185 GAT TTA GCC GAC TAT GAT TTA AAC AAT ACT GTA ATG GAC CAA TAT TTA 1806 Asp Leu Ala Asp Tyr Asp Leu Asn Asn Thr Val Met Asp Gln Tyr Leu 190 195 200 AAA GAA AGT ATA AAG CTT TGG TTA GAT AAA GGA ATT GAT GGG ATT CGA 1854 Lys Glu Ser Ile Lys Leu Trp Leu Asp Lys Gly Ile Asp Gly Ile Arg 205 210 215 220 GTC GAT GCG GTC AAA CAT ATG TCC GAG GGC TGG CAA ACC TCC TTA ATG 1902 Val Asp Ala Val Lys His Met Ser Glu Gly Trp Gln Thr Ser Leu Met 225 230 235 AGT GAT ATT TAT GCA CAC GAG CCT GTC TTT ACA TTT GGC GAA TGG TTT 1950 Ser Asp Ile Tyr Ala His Glu Pro Val Phe Thr Phe Gly Glu Trp Phe 240 245 250 TTA GGA TCA GGG GAA GTC GAC CCA CAA AAT CAT CAC TTT GCC AAT GAA 1998 Leu Gly Ser Gly Glu Val Asp Pro Gln Asn His His Phe Ala Asn Glu 255 260 265 AGT GGT ATG AGC TTA CTA GAC TTT CAG TTT GGT CAA ACG ATT AGA GAT 2046 Ser Gly Met Ser Leu Leu Asp Phe Gln Phe Gly Gln Thr Ile Arg Asp 270 275 280 GTA TTA ATG GAC GGT AGC AGC AAT TGG TAT GAC TTT AAT GAG ATG ATT 2094 Val Leu Met Asp Gly Ser Ser Asn Trp Tyr Asp Phe Asn Glu Met Ile 285 290 295 300 GCA AGC ACC GAA GAG GAT TAT GAC GAA GTG ATT GAT CAG GTT ACG TTT 2142 Ala Ser Thr Glu Glu Asp Tyr Asp Glu Val Ile Asp Gln Val Thr Phe 305 310 315 ATT GAT AAT CAT GAC ATG AGC CGT TTT TCC TTT GAA CAA TCT TCA AAC 2190 Ile Asp Asn His Asp Met Ser Arg Phe Ser Phe Glu Gln Ser Ser Asn 320 325 330 CGC CAT ACA GAC ATT GCT TTA GCT GTC CTG TTA ACC TCT CGT GGA GTT 2238 Arg His Thr Asp Ile Ala Leu Ala Val Leu Leu Thr Ser Arg Gly Val 335 340 345 CCG ACT ATA TAT TAC GGT ACA GAG CAA TAT TTA ACA GGA GGC AAC GAC 2286 Pro Thr Ile Tyr Tyr Gly Thr Glu Gln Tyr Leu Thr Gly Gly Asn Asp 350 355 360 CCT GAA AAC CGC AAG CCG ATG AGC GAT TTT GAC CGT ACG ACA AAT TCT 2334 Pro Glu Asn Arg Lys Pro Met Ser Asp Phe Asp Arg Thr Thr Asn Ser 365 370 375 380 TAT CAA ATT ATT AGT ACG CTT GCT TCC TTA AGA CAA AAC AAC CCG GCA 2382 Tyr Gln Ile Ile Ser Thr Leu Ala Ser Leu Arg Gln Asn Asn Pro Ala 385 390 395 TTA GGC TAT GGA AAT ACA AGC GAA CGA TGG ATT AAC TCC GAC GTT TAT 2430 Leu Gly Tyr Gly Asn Thr Ser Glu Arg Trp Ile Asn Ser Asp Val Tyr 400 405 410 ATT TAT GAA CGA TCC TTC GGA GAC AGT GTC GTG CTT ACT GCT GTT AAC 2478 Ile Tyr Glu Arg Ser Phe Gly Asp Ser Val Val Leu Thr Ala Val Asn 415 420 425 AGT GGC GAT ACT AGT TAC ACG ATT AAT AAC TTA AAT ACC TCG TTA CCT 2526 Ser Gly Asp Thr Ser Tyr Thr Ile Asn Asn Leu Asn Thr Ser Leu Pro 430 435 440 CAA GGT CAA TAT ACA GAT GAG CTT CAG CAG CTT TTG GAT GGA AAT GAG 2574 Gln Gly Gln Tyr Thr Asp Glu Leu Gln Gln Leu Leu Asp Gly Asn Glu 445 450 455 460 ATT ACA GTC AAT AGC AAT GGA GCT GTA GAC TCG TTC CAG CTA AGT GCA 2622 Ile Thr Val Asn Ser Asn Gly Ala Val Asp Ser Phe Gln Leu Ser Ala 465 470 475 AAT GGA GTA TCC GTG TGG CAA ATA ACC GAA GAG CAT GCT TCT CCT CTA 2670 Asn Gly Val Ser Val Trp Gln Ile Thr Glu Glu His Ala Ser Pro Leu 480 485 490 ATC GGC CAT GTA GGA CCA ATG ATG GGT AAA CAC GGA AAT ACT GTC ACT 2718 Ile Gly His Val Gly Pro Met Met Gly Lys His Gly Asn Thr Val Thr 495 500 505 ATA ACG GGA GAA GGC TTT GGT GAC AAT GAG GGA AGT GTT CTC TTT GAT 2766 Ile Thr Gly Glu Gly Phe Gly Asp Asn Glu Gly Ser Val Leu Phe Asp 510 515 520 TCT GAT TTC TCT GAT GTC CTT TCT TGG TCT GAC ACG AAA ATA GAA GTA 2814 Ser Asp Phe Ser Asp Val Leu Ser Trp Ser Asp Thr Lys Ile Glu Val 525 530 535 540 AGC GTT CCG GAT GTA ACA GCT GGT CAC TAT GAT ATT AGC GTT GTA AAT 2862 Ser Val Pro Asp Val Thr Ala Gly His Tyr Asp Ile Ser Val Val Asn 545 550 555 GCT GGA GAC AGC CAA AGT CCA ACG TAT GAT AAG TTT GAA GTA TTA ACC 2910 Ala Gly Asp Ser Gln Ser Pro Thr Tyr Asp Lys Phe Glu Val Leu Thr 560 565 570 GGT GAC CAA GTT AGT ATC CGT TTT GCT GTT AAT AAT GCG ACC ACT AGC 2958 Gly Asp Gln Val Ser Ile Arg Phe Ala Val Asn Asn Ala Thr Thr Ser 575 580 585 CTT GGT ACC AAT CTG TAT ATG GTT GGA AAT GTG AAT GAG CTT GGA AAT 3006 Leu Gly Thr Asn Leu Tyr Met Val Gly Asn Val Asn Glu Leu Gly Asn 590 595 600 TGG GAC CCT GAT CAA GCG ATT GGG CCG ATG TTT AAT CAA GTG ATG TAT 3054 Trp Asp Pro Asp Gln Ala Ile Gly Pro Met Phe Asn Gln Val Met Tyr 605 610 615 620 CAA TAC CCA ACC TGG TAC TAT GAT ATA AGT GTT CCT GCC GAG GAA AAT 3102 Gln Tyr Pro Thr Trp Tyr Tyr Asp Ile Ser Val Pro Ala Glu Glu Asn 625 630 635 CTT GAA TAT AAA TTT ATT AAA AAG GAC AGC AGC GGA AAC GTC GTT TGG 3150 Leu Glu Tyr Lys Phe Ile Lys Lys Asp Ser Ser Gly Asn Val Val Trp 640 645 650 GAA AGT GGA AAT AAC CAT ACG TAT ACA ACA CCT GCA ACT GGA ACC GAT 3198 Glu Ser Gly Asn Asn His Thr Tyr Thr Thr Pro Ala Thr Gly Thr Asp 655 660 665 ACA GTC CTC GTC GAC TGG CAA TAACACAAAA TACACATCAA CTTAAGGGCT 3249 Thr Val Leu Val Asp Trp Gln 670 675 ATGCATTGTG ATCATACACA ACAACCCCAT TTTCCAAGAT TGGAAATAGG GGTTGTTTTA 3309 TAATGAAAGG GGAATTTTCC TATGTTAAAA GAAGCGATTT ATCACCGACC AAAAAATAAT 3369 TATGCGTACG CCTATTCAAA GGATACGCTA CACATTCGTC TACGCACCAA AAAGAATGAT 3429 CTCACGCAAG TCGAGCTTCT CTATGCTGAT CCTTACAATT GGAATGAAGA TGGTTGGCTC 3489 TATGAGCAAA AAGCGATGCG TTTAGAGGCT TCTGACCACT TATTTGACTA TTGGATGATT 3549 GATGTTACCG TCCCATACCG TCGACTCCGC TATGGCTTTA AGCTTACAAG TGATGATGAA 3609 ACGTTATATT ATACGGAAAA AGGCTTTTAT GAAACAGCAC CTACCGATGA TACGGCTTAC 3669 TATTTTTGTT TTCCCTTTAT CAATCCTGTT GATATCTTTC AAGCTCCTGA ATGGGTCAAA 3729 AAGACCGTTT GGTATCAAAT CTTCCCGGAG CGTTTTGCCA ATGGTGACTC GAGCATTAAT 3789 CCAGCCTCAA CCCTCCCTTG GGGAAGTACA GAAGCTACAC CAACTAATTT TTTCGGTGGA 3849 GATTTCGAGG GAATACTTAA TCATTTAGAT TATTTAGTTG ATTAGGCATT AATGGCATTT 3909 ACTTTACTCG ATTTTTAAAG CTAAATCAAA CCATAAGTAC GATACGATTG ATTATATGGA 3969 AATGATCCAC AGTTTGGAGA TAAAGAAACC TTCCGGAAGC TCGTTAATGC CTGTCATGAA 4029 AAAGGGATAA AAATCATGCT TGATGCTGTA TTTAATCATA GTGGCTATTA TTTCGAAGCG 4089 TTTCAGGATG TCCTGAAGCA TCAGGAGCAA TCGAAGTATA AAGATTGGTT TCATATACGC 4149 GACTTTCCAG TCACTCCTGG TCCTAAGCCT AATTATGATA CATTTGGGTT TGTTGAATAT 4209 ATGCCGAAGT TAAATACGGA AAACCAAGAG GTGAAGGATT ATTTATTAAA AGTGGCGCGC 4269 TACTGGATCG AGGAATTTAA TATCGATGGC TGGCGTCTTG ATGTCGCCAA TGAAGTCGAC 4329 CATCAATTTT GGAGAGATTT TAGAAGAGAA GTAAAAGCGA TTAACCCCGA TGTTTATATC 4389 TTAGGTGAGA TATGGCATGA CTCGATGCCG TGGCTGCAGG GCGATCAGTT CGATGCCGTC 4449 ATGAATTATC CTTTTACGGT AGCGGCTCTC GATTATATTG CGAAGGATAA GATTAATGCC 4509 GAAGAGTTTG CTCATCAGCT GACCGATGTT CTTTGCTCGT ATCCAGCCAA TATTCATGAA 4569 GTGACCTTTA ACCTGCTTGG CAGTCATGAC ACTGCACGAG TCCTGACCGT ATGTAAGGAC 4629 AACAAAGAAA AAACGAAGCT ACTTTATTTA CTATTGTTAT CATCTAAAGG AAGTCCTTGC 4689 ATTTTTTATG GCGAGGAAAT CGGCATGGCC GGTGAAAACG ATCCAGGCTG CAGAGACTGC 4749 ATGATTTGGG AGGAAGACCA ACAAGACCTC GAATTC 4785SEQ ID NO: 1 Sequence Length: 4785 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Genomic DNA Origin: Bacillus obensis SP Knob C-14
00 (Bacillus ohbensissp. Nov. C-1400) Sequence features Characteristic symbols: mat peptide Location: 1195. . 3219 method to determine the characteristics: S sequence GAGCTCGCCG CCTTCGTAAG CGCCTTCATT ATTTAAACCA ACATCCTCCG TATCATATTC 60 CCCTACATCA CCGCCAAAAA TATAGGCACC GTAATTTTTG AGGAAAGGAA AAGCAAAATA 120 GAGATCATTA GGTCTCATCA GAAATCCATA TTGATCAGCT GATGCATCCG TATTTTCTTC 180 AAGAATCGTT CTTAAATCTT CGATCGTCTC TGGTGCTTCT GGTACAATTT CTTTATTGTA 240 GAATATTCCA TATGTTTCGA TAACAGCTGG AACTCCATAA ATATCCGTTT CTCCTTCGAA 300 CTCATAGGTG ACCGCATCTA TAGAGGAACT AGCATAATTG CTTAACTCGT CATCTGATAG 360 AGTAAGAGGA TCAGCTAATC CTTGTGCCAC AATATTTCCG ATCTGGTCAT GTGGCTGGAA 420 AAATAAATCG GGACCATTCC CTTCTGGACC TGCTAATGAC AATTCCTGCA ATTGGTCCAT 480 CATCGGCTTT GTCTCAACCT TCACGTTAAT TCCCGTTTGC TCTGTATAAT CGTTGCTATT 540 TTTTCAATCG CCTCTAATTG CTCTTCTCTA TCATTTGCCC AGATGGGTTA ATTCTTCAGG 600 TTGATCCGCT TCCCCACCAT TTGTATCCGT GGTTACTGCC TCTTCTTCCC GTTCAGGTGC 660 ACATGCCGCT AAGAGAATAA TCATTAAAAA CATGGTAATC AGTGATAATA ATGAAAAACC 720 TTTTTTCATT TCATTTCCCC CCTGAAAAAA TTATATTAAT CTAGTAAATG TTGAGCTACC 780 TTTACATAAA GGAAAGTAAC GCAAACGTTT GCACTAGATT A GATACAACA ATCATACATG 840 GCAATTCAAT TTTTTGCAAT CGTTTACAGT AAAGAATTTT TTGAATTTTT ATTTAGTTAC 900 AATTACCTAT TTACAAATGA AATGTCTATG AGATAAAAAT AAATTAACAA CTATGAATGT 960 TCATATTCTC AATACGAGGG CAATGCTTTT GTTTTTTACA AACAACCCCG TCTTTTATTC 1020 CATTAAAAGG CGGGGTTATT TTATTGGATA AGGAGGGTTG TATTGCTTGT TCATACGATT 1080 CATTTTTAAT GTATAGGGGG AGTATTT TTG AAA AAT TTA ACG GTT TTA TTG AAA 1134 Met Lys Asn Leu Thr Val Leu Leu Lys -25 ACG ATT CCA TTA GCT CTG TTA CTT TTC ATC CTT CTT TCT TTA CCA ACA 1182 Thr Ile Pro Leu Ala Leu Leu Leu Phe Ile Leu Leu Ser Leu Pro Thr -20 -15 -10 -5 GCG GCT CAG GCT GAC GTT ACA AAT AAG GTT AAT TAT ACA AGA GAT GTT 1230 Ala Ala Gln Ala Asp Val Thr Asn Lys Val Asn Tyr Thr Arg Asp Val 1 5 10 ATT TAT CAA ATT GTG ACC GAC CGT TTT TCT GAT GGA GAT CCA TCC AAC 1278 Ile Tyr Gln Ile Val Thr Asp Arg Phe Ser Asp Gly Asp Pro Ser Asn 15 20 25 AAC CCA ACG GGG GCG ATT TAT AGT CAG GAT TGT AGC GAC CTT CAT AAA 1326 Asn Pro Thr Gly Ala Ile Tyr Ser Gln Asp Cys Ser Asp Leu His Lys 30 35 40 TAT TGT GG G GGC GAC TGG CAA GGA ATT ATC GAC AAA ATC AAT GAC GGG 1374 Tyr Cys Gly Gly Asp Trp Gln Gly Ile Ile Asp Lys Ile Asn Asp Gly 45 50 55 60 TAT TTA ACC GAT TTA GGA ATT ACA GCG ATT TGG ATT TCA CAG CCT GTA 1422 Tyr Leu Thr Asp Leu Gly Ile Thr Ala Ile Trp Ile Ser Gln Pro Val 65 70 75 GAA AAT GTC TAT GCT CTT CAT CCG AGT GGT TAT ACC TCT TAT CAC GGG 1470 Glu Asn Val Tyr Ala Leu His Pro Ser Gly Tyr Thr Ser Tyr His Gly 80 85 90 TAT TGG GCA AGA GAT TAT AAA AGA ACG AAC CCT TTT TAT GGC GAT TTC 1518 Tyr Trp Ala Arg Asp Tyr Lys Arg Thr Asn Pro Phe Tyr Gly Asp Phe 95 100 105 TCT GAC TTT GAC CGG TTA ATG GAT ACT GCA CAT AGT AAT GGC ATT AAG 1566 Ser Asp Phe Asp Arg Leu Met Asp Thr Ala His Ser Asn Gly Ile Lys 110 115 120 GTA ATC ATG GAC TTT ACG CCC AAC CAT TCA TCA CCA GCT CTT GAA ACC 1614 Val Ile Met Asp Phe Thr Pro Asn His Ser Ser Pro Ala Leu Glu Thr 125 130 135 140 GAT CCA AGC TAT GCC GAG AAC GGA GCG GTT TAT AAT GAT GGC GTA TTA 1662 Asp Pro Ser Tyr Ala Glu Asn Gly Ala Val Tyr Asn Asp Gly Val Leu 145 150 15 5 ATA GGC AAT TAT TCT AAC GAC CCT AAC AAC CTC TTT CAC CAT AAT GGT 1710 Ile Gly Asn Tyr Ser Asn Asp Pro Asn Asn Leu Phe His His Asn Gly 160 165 170 GGG ACA GAT TTT TCA TCC TAT GAA GAT AGC ATT TAT CGA AAT CTA TAT 1758 Gly Thr Asp Phe Ser Ser Tyr Glu Asp Ser Ile Tyr Arg Asn Leu Tyr 175 180 185 GAT TTA GCC GAC TAT GAT TTA AAC AAT ACT GTA ATG GAC CAA TAT TTA 1806 Asp Leu Ala Asp Tyr Asp Leu Asn Asn Thr Val Met Asp Gln Tyr Leu 190 195 200 AAA GAA AGT ATA AAG CTT TGG TTA GAT AAA GGA ATT GAT GGG ATT CGA 1854 Lys Glu Ser Ile Lys Leu Trp Leu Asp Lys Gly Ile Asp Gly Ile Arg 205 210 215 220 GTC GAT GCG GTC AAA CAT ATG TCC GAG GGC TGG CAA ACC TCC TTA ATG 1902 Val Asp Ala Val Lys His Met Ser Glu Gly Trp Gln Thr Ser Leu Met 225 230 235 AGT GAT ATT TAT GCA CAC GAG CCT GTC TTT ACA TTT GGC GAA TGG TTT 1950 Ser Asp Ile Tyr Ala His Glu Pro Val Phe Thr Phe Gly Glu Trp Phe 240 245 250 TTA GGA TCA GGG GAA GTC GAC CCA CAA AAT CAT CAC TTT GCC AAT GAA 1998 Leu Gly Ser Gly Glu Val Asp Pro Gln Asn His His Phe Ala Asn Glu 255 260 265 AGT GGT ATG AGC TTA CTA GAC TTT CAG TTT GGT CAA ACG ATT AGA GAT 2046 Ser Gly Met Ser Leu Leu Asp Phe Gln Phe Gly Gln Thr Ile Arg Asp 270 275 280 GTA TTA ATG GAC GGT AGC AGC AAT TGG TAT GAC TTT AAT GAG ATG ATT 2094 Val Leu Met Asp Gly Ser Ser Asn Trp Tyr Asp Phe Asn Glu Met Ile 285 290 295 300 GCA AGC ACC GAA GAG GAT TAT GAC GAA GTG ATT GAT CAG GTT ACG TTT 2142 Ala Ser Thr Glu Glu Asp Tyr Asp Glu Val Ile Asp Gln Val Thr Phe 305 310 315 ATT GAT AAT CAT GAC ATG AGC CGT TTT TCC TTT GAA CAA TCT TCA AAC 2190 Ile Asp Asn His Asp Met Ser Arg Phe Ser Phe Glu Gln Ser Ser Asn 320 325 330 CGC CAT ACA GAC ATT GCT TTA GCT GTC CTG TTA ACC TCT CGT GGA GTT 2238 Arg His Thr Asp Ile Ala Leu Ala Val Leu Leu Thr Ser Arg Gly Val 335 340 345 CCG ACT ATA TAT TAC GGT ACA GAG CAA TAT TTA ACA GGA GGC AAC GAC 2286 Pro Thr Ile Tyr Tyr Gly Thr Glu Gln Tyr Leu Thr Gly Gly Asn Asp 350 355 360 CCT GAA AAC CGC AAG CCG ATG AGC GAT TTT GAC CGT ACG ACA AAT TCT 2334 Pro Glu Asn Arg Lys Pro Met Ser Asp Phe Asp Arg Thr Thr Asn Ser 365 370 375 380 TAT CAA ATT ATT AGT ACG CTT GCT TCC TTA AGA CAA AAC AAC CCG GCA 2382 Tyr Gln Ile Ile Ser Thr Leu Ala Ser Leu Arg Gln Asn Asn Pro Ala 385 390 395 TTA GGC TAT GGA AAT ACA AGC GAA CGA TGG ATT AAC TCC GAC GTT TAT 2430 Leu Gly Tyr Gly Asn Thr Ser Glu Arg Trp Ile Asn Ser Asp Val Tyr 400 405 410 ATT TAT GAA CGA TCC TTC GGA GAC AGT GTC GTG CTT ACT GCT GTT AAC 2478 Ile Tyr Glu Arg Ser Phe Gly Asp Ser Val Val Leu Thr Ala Val Asn 415 420 425 AGT GGC GAT ACT AGT TAC ACG ATT AAT AAC TTA AAT ACC TCG TTA CCT 2526 Ser Gly Asp Thr Ser Tyr Thr Ile Asn Asn Leu Asn Thr Ser Leu Pro 430 435 440 CAA GGT CAA TAT ACA GAT GAG CTT CAG CAG CTT TTG GAT GGA AAT GAG 2574 Gln Gly Gln Tyr Thr Asp Glu Leu Gln Gln Leu Leu Asp Gly Asn Glu 445 450 455 460 ATT ACA GTC AAT AGC AAT GGA GCT GTA GAC TCG TTC CAG CTA AGT GCA 2622 Ile Thr Val Asn Ser Asn Gly Ala Val Asp Ser Phe Gln Leu Ser Ala 465 470 475 AAT GGA GTA TCC GTG TGG CAA ATA ACC GAA GAG CAT GCT TCT CCT CTA 2670 Asn Gly Val S er Val Trp Gln Ile Thr Glu Glu His Ala Ser Pro Leu 480 485 490 ATC GGC CAT GTA GGA CCA ATG ATG GGT AAA CAC GGA AAT ACT GTC ACT 2718 Ile Gly His Val Gly Pro Met Met Gly Lys His Gly Asn Thr Val Thr 495 500 505 ATA ACG GGA GAA GGC TTT GGT GAC AAT GAG GGA AGT GTT CTC TTT GAT 2766 Ile Thr Gly Glu Gly Phe Gly Asp Asn Glu Gly Ser Val Leu Phe Asp 510 515 520 TCT GAT TTC TCT GAT GTC CTT TCT TGG TCT GAC ACG AAA ATA GAA GTA 2814 Ser Asp Phe Ser Asp Val Leu Ser Trp Ser Asp Thr Lys Ile Glu Val 525 530 535 540 AGC GTT CCG GAT GTA ACA GCT GGT CAC TAT GAT ATT AGC GTT GTA AAT 2862 Ser Val Pro Asp Val Thr Ala Gly His Tyr Asp Ile Ser Val Val Asn 545 550 555 GCT GGA GAC AGC CAA AGT CCA ACG TAT GAT AAG TTT GAA GTA TTA ACC 2910 Ala Gly Asp Ser Gln Ser Pro Thr Tyr Asp Lys Phe Glu Val Leu Thr 560 565 570 GGT GAC CAA GTT AGT ATC CGT TTT GCT GTT AAT AAT GCG ACC ACT AGC 2958 Gly Asp Gln Val Ser Ile Arg Phe Ala Val Asn Asn Ala Thr Thr Ser 575 580 585 CTT GGT ACC AAT CTG TAT ATG GTT GGA AAT GTG AAT GAG CTT GGA AAT 3006 Leu Gly Thr Asn Leu Tyr Met Val Gly Asn Val Asn Glu Leu Gly Asn 590 595 600 TGG GAC CCT GAT CAA GCG ATT GGG CCG ATG TTT AAT CAA GTG ATG TAT 3054 Trp Asp Pro Asp Gln Ala Ile Gly Pro Met Phe Asn Gln Val Met Tyr 605 610 615 620 CAA TAC CCA ACC TGG TAC TAT GAT ATA AGT GTT CCT GCC GAG GAA AAT 3102 Gln Tyr Pro Thr Trp Tyr Tyr Asp Ile Ser Val Pro Ala Glu Glu Asn 625 630 635 CTT GAA TAT AAA TTT ATT AAA AAG GAC AGC AGC GGA AAC GTC GTT TGG 3150 Leu Glu Tyr Lys Phe Ile Lys Lys Asp Ser Ser Gly Asn Val Val Trp 640 645 650 GAA AGT GGA AAT AAC CAT ACG TAT ACA ACA CCT GCA ACT GGA ACC GAT 3198 Glu Ser Gly Asn Asn His Thr Tyr Thr Thr Pro Ala Thr Gly Thr Asp 655 660 665 ACA GTC CTC GTC GAC TGG CAA TAACACAAAA TACACATCAA CTTAAGGGCT 3249 Thr Val Leu Val Asp Trp Gln 670 675 ATGCATTGTGAATCATATCATCGAG TATCATACACAGAAT TGGAATTAG 3G CCTATTCAAA GGATACGCTA CACATTCGTC TACGCACCAA AAAGAATGAT 3429 CTCACGCAAG TCGAGCTT CT CTATGCTGAT CCTTACAATT GGAATGAAGA TGGTTGGCTC 3489 TATGAGCAAA AAGCGATGCG TTTAGAGGCT TCTGACCACT TATTTGACTA TTGGATGATT 3549 GATGTTACCG TCCCATACCG TCGACTCCGC TATGGCTTTA AGCTTACAAG TGATGATGAA 3609 ACGTTATATT ATACGGAAAA AGGCTTTTAT GAAACAGCAC CTACCGATGA TACGGCTTAC 3669 TATTTTTGTT TTCCCTTTAT CAATCCTGTT GATATCTTTC AAGCTCCTGA ATGGGTCAAA 3729 AAGACCGTTT GGTATCAAAT CTTCCCGGAG CGTTTTGCCA ATGGTGACTC GAGCATTAAT 3789 CCAGCCTCAA CCCTCCCTTG GGGAAGTACA GAAGCTACAC CAACTAATTT TTTCGGTGGA 3849 GATTTCGAGG GAATACTTAA TCATTTAGAT TATTTAGTTG ATTAGGCATT AATGGCATTT 3909 ACTTTACTCG ATTTTTAAAG CTAAATCAAA CCATAAGTAC GATACGATTG ATTATATGGA 3969 AATGATCCAC AGTTTGGAGA TAAAGAAACC TTCCGGAAGC TCGTTAATGC CTGTCATGAA 4029 AAAGGGATAA AAATCATGCT TGATGCTGTA TTTAATCATA GTGGCTATTA TTTCGAAGCG 4089 TTTCAGGATG TCCTGAAGCA TCAGGAGCAA TCGAAGTATA AAGATTGGTT TCATATACGC 4149 GACTTTCCAG TCACTCCTGG TCCTAAGCCT AATTATGATA CATTTGGGTT TGTTGAATAT 4209 ATGCCGAAGT TAAATACGGA AAACCAAGAG GTGAAGGATT ATTTATTAAA AGTGGCGCGC 4269 TACTGGATCG AGGAATTTAA TAT CGATGGC TGGCGTCTTG ATGTCGCCAA TGAAGTCGAC 4329 CATCAATTTT GGAGAGATTT TAGAAGAGAA GTAAAAGCGA TTAACCCCGA TGTTTATATC 4389 TTAGGTGAGA TATGGCATGA CTCGATGCCG TGGCTGCAGG GCGATCAGTT CGATGCCGTC 4449 ATGAATTATC CTTTTACGGT AGCGGCTCTC GATTATATTG CGAAGGATAA GATTAATGCC 4509 GAAGAGTTTG CTCATCAGCT GACCGATGTT CTTTGCTCGT ATCCAGCCAA TATTCATGAA 4569 GTGACCTTTA ACCTGCTTGG CAGTCATGAC ACTGCACGAG TCCTGACCGT ATGTAAGGAC 4629 AACAAAGAAA AAACGAAGCT ACTTTATTTA CTATTGTTAT CATCTAAAGG AAGTCCTTGC 4689 ATTTTTTATG GCGAGGAAAT CGGCATGGCC GGTGAAAACG ATCCAGGCTG CAGAGACTGC 4749 ATGATTTGGG AGGAAGACCA ACAAGACCTC GAATTC 4785
【0036】配列番号:2 配列の長さ:24 配列の型:核酸 鎖の数:一本鎖 トポロジー:直鎖状 配列の種類:他の核酸 合成DNA 配列 TCGAAATCTA TGGGATTTAG CCGA 24SEQ ID NO: 2 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence TCGAAATCTA TGGGATTTAG CCGA 24
【0037】配列番号:3 配列の長さ:24 配列の型:核酸 鎖の数:一本鎖 トポロジー:直鎖状 配列の種類:他の核酸 合成DNA 配列 ATCGAAATCT AGCTGATTTA GCCG 24SEQ ID NO: 3 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence ATCGAAATCT AGCTGATTTA GCCG 24
【0038】配列番号:4 配列の長さ:19 配列の型:核酸 鎖の数:一本鎖 トポロジー:直鎖状 配列の種類:他の核酸 合成DNA 配列 GAAATCTATC TGATTTAGC 19SEQ ID NO: 4 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence GAAATCTATC TGATTTAGC 19
【0039】配列番号:5 配列の長さ:19 配列の型:核酸 鎖の数:一本鎖 トポロジー:直鎖状 配列の種類:他の核酸 合成DNA 配列 GAAATCTATT TGATTTAGC 19SEQ ID NO: 5 Sequence length: 19 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence GAAATCTATT TGATTTAGC 19
【0040】配列番号:6 配列の長さ:19 配列の型:核酸 鎖の数:一本鎖 トポロジー:直鎖状 配列の種類:他の核酸 合成DNA 配列 CGAAATCTAC ATGATTTAG 19SEQ ID NO: 6 Sequence Length: 19 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence CGAAATCTAC ATGATTTAG 19
【図1】(1) 〜(5) は本発明の改変CGTase遺伝子
の構築を示す。FIG. 1 (1) to (5) show the construction of the modified CGTase gene of the present invention.
【図2】野生型CGTaseおよび各種チロシン188
変異CGTaseのSDS−PAGEにおけるゲル上の
バンドを示す図である。FIG. 2 Wild type CGTase and various tyrosine 188
It is a figure which shows the band on the gel in SDS-PAGE of mutant CGTase.
【図3】野生型CGTaseおよび各種チロシン188
変異CGTaseのpHによる相対活性の変化を示すグ
ラフである。FIG. 3 Wild-type CGTase and various tyrosine 188
It is a graph which shows the change of relative activity of mutant CGTase with pH.
【図4】野生型CGTaseおよび各種チロシン188
変異CGTaseにおける、デンプンを基質とした場合
の反応生成物であるCDの経時的変化を示すグラフであ
る。FIG. 4 Wild-type CGTase and various tyrosine 188
It is a graph which shows the time-dependent change of CD which is a reaction product in the case of using starch as a substrate in mutant CGTase.
─────────────────────────────────────────────────────
─────────────────────────────────────────────────── ───
【手続補正書】[Procedure amendment]
【提出日】平成4年3月12日[Submission date] March 12, 1992
【手続補正1】[Procedure Amendment 1]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0024[Correction target item name] 0024
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0024】 バチルス・オーベンシス・エスピー・ノ
ブ・C−1400(Bacillus ohbensi
s sp.nov.C−1400)からクローニングさ
れた野生型CGTase遺伝子を保持するpUC19C
GT5A(Sin,K.−A.et.al.,App
l.Microbiol.Biotechnol.,3
5,600−605(1991))をSphIとHin
dIIIで部分消化することにより約2.5KbのDN
A断片を切り出し、これをプラスミドpUP110BS
CsacQ(Nakamura A.et.al.,A
gric.Biol.Chem.,55(9),236
7−2374(1991)のSphI・HindIII
消化物と混合し、T4ファージDNAリガーゼ処理して
連結した。このプラスミドをpUP110Ce−CGT
とした。さらに、CGTase遺伝子の発現量を増加さ
せるために、バチルス・スブチリス セルラーゼ遺伝子
の発現量を上昇させることが知られているsacQ遺伝
子を、プラスミド上のHindIII−SphIの間に
組み込んだ。こうして得られたプラスミドをpUP11
0CeCGTsacQとする。このDNAを用い、バチ
ルス・スブチリス(Bacillus subtili
s)207−21の形質転換を行ない、カナマイシンに
耐性の形質転換体を選択した。この形質転換体より、常
法によりプラスミドDNAを抽出、精製し、分析したと
ころ、野生型プラスミドDNApUP110CeCGT
sacQを保有していた。Bacillus obensis SP Knob C-1400 (Bacillus ohbensi
s sp. nov. P-1C19) carrying a wild-type CGTase gene cloned from C-1400)
GT5A (Sin, K.-A. et. Al., App
l. Microbiol. Biotechnol. , 3
5,600-605 (1991)) with SphI and Hin
Approximately 2.5 Kb DN by partial digestion with dIII
The A fragment was cut out and used as a plasmid pUP110BS.
CsacQ (Nakamura A. et. Al., A
gric. Biol. Chem. , 55 (9), 236
7-2374 (1991) SphI and HindIII
It was mixed with the digest, treated with T4 phage DNA ligase and ligated. This plasmid was designated as pUP110Ce-CGT.
And Furthermore, in order to increase the expression level of the CGTase gene, the sacQ gene known to increase the expression level of the Bacillus subtilis cellulase gene was incorporated between HindIII- SphI on the plasmid. The thus obtained plasmid was designated as pUP11.
0CeCGTsacQ. Using this DNA, bee
Bacillus subtilis
s) Transformation of 207-21 was performed, and transformants resistant to kanamycin were selected. From this transformant, plasmid DNA was extracted, purified, and analyzed by a conventional method. As a result, wild-type plasmid DNA pUP110CeCGT was obtained.
Had sacQ.
【手続補正2】[Procedure Amendment 2]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0028[Correction target item name] 0028
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0028】実施例3:改変CGTaseの精製 実施例2により得られた各種粗酵素液に対して、デンプ
ンを2%になるように加え、4℃で1時間撹拌して酵素
を吸着させた。遠心分離(4℃、7000rpm)によ
りデンプンを集め、50mMリン酸ナトリウム緩衝液
(pH 6.8)で洗浄後、同じ緩衝液に懸濁し、40
℃で2時間撹拌することにより酵素を溶出させた。遠心
分離によりデンプンを除去し、その上清を精製酵素標品
とした。必要によりセントリプレップ−30(アミコン
社製)を用いて限外ろ過を行ない、酵素溶液の濃縮を行
なう。なお、精製過程はSDS−PAGEにより確認し
た。Example 3: Purification of modified CGTase Starch was added to the various crude enzyme solutions obtained in Example 2 in an amount of 2% to stir at 4 ° C. for 1 hour to adsorb the enzyme. Centrifugation (4 ℃, 7000rpm) to
The starch was collected, washed with 50 mM sodium phosphate buffer (pH 6.8), suspended in the same buffer, and
The enzyme was eluted by stirring for 2 hours at ° C. The starch was removed by centrifugation, and the supernatant was used as a purified enzyme preparation. If necessary, Centriprep-30 (manufactured by Amicon) is used for ultrafiltration to concentrate the enzyme solution. The purification process was confirmed by SDS-PAGE.
【手続補正3】[Procedure 3]
【補正対象書類名】明細書[Document name to be amended] Statement
【補正対象項目名】0033[Correction target item name] 0033
【補正方法】変更[Correction method] Change
【補正内容】[Correction content]
【0033】[0033]
【発明の効果】 バチルス・オーベンシス・エスピー・
ノブ・C−1400(Bacillus ohbens
is sp.nov.C−1400)由来の配列番号1
のアミノ酸配列において、188番目のチロシンを他の
アミノ酸に置換することにより、産生される酵素の性質
や、その酵素と基質との相互作用に大きな影響を及ぼ
し、チロシン188残基をより空間的に大きな他のアミ
ノ酸であるトリプトファンの他、アラニン、セリンに置
換した場合、その置換変異CGTaseのデンプンから
生産するCDは、野生型CGTaseと比べてβ−CD
に対するγ−CDの相対生産量が増大する。アラニン、
セリン置換体変異酵素ではβ−CDとγ−CDがほぼ同
率で生成され、トリプトファン置換体変異酵素はγ−C
Dの生産量が対糖収率で15%に達したことから、公知
である分野への応用に限らず、従来困難であったγ−C
Dの大量生産に利用できる。[Effects of the Invention] Bacillus obensis SP
Knob C-1400 (Bacillus ohbens
is sp. nov. C-1400) -derived SEQ ID NO: 1
In the amino acid sequence of, the substitution of the 188th tyrosine with another amino acid has a great influence on the properties of the produced enzyme and the interaction between the enzyme and the substrate, and makes the tyrosine 188 residue more spatial. When substituted with alanine or serine in addition to tryptophan, which is another large amino acid, the CD produced from starch of the substitution mutant CGTase is β-CD as compared with wild-type CGTase.
Relative production of γ-CD with respect to Alanine,
The serine-substituting mutant enzyme produces β-CD and γ-CD at almost the same rate, and the tryptophan-substituting mutant enzyme produces γ-C.
Since the production amount of D reached 15% in terms of sugar yield, it was not limited to the application to a known field, and γ-C which had been difficult in the past.
It can be used for mass production of D.
Claims (3)
ブ・C−1400(Bacillus ohbensis sp. nov. C-140
0 )由来の配列番号1のアミノ酸配列において、188
番目のチロシンがアラニン、セリンまたはトリプトファ
ンに改変されたサイクロデキストリングルカノトランス
フェラーゼ。1. Bacillus ohbensis sp. Nov. C-140
0) in the amino acid sequence of SEQ ID NO: 1
Cyclodextrin glucanotransferase in which the second tyrosine is modified to alanine, serine or tryptophan.
ブ・C−1400(Bacillus ohbensis sp. nov. C-140
0 )のDNAにおける配列番号1の塩基配列を含む遺伝
子において、1756〜1758番目の“TAT”が
“GCT”、“GCC”、“GCA”、GCG”、“T
CT”、“TCC”、TCA”、“TCG”、“AG
T”、“AGC”または“TGG”に改変されたサイク
ロデキストリングルカノトランスフェラーゼ遺伝子。2. Bacillus ohbensis sp. Nov. C-140
In the gene containing the base sequence of SEQ ID NO: 1 in the DNA of (0), the 1756th to 1758th "TAT" s are "GCT", "GCC", "GCA", GCG ", and" T ".
CT "," TCC ", TCA", "TCG", "AG"
A cyclodextrin glucanotransferase gene modified to T "," AGC "or" TGG ".
転換微生物を培養し、培養液からサイクロデキストリン
グルカノトランスフェラーゼを取得することを特徴とす
るサイクロデキストリングルカノトランスフェラーゼの
製造方法。3. A method for producing a cyclodextrin glucanotransferase, which comprises culturing a transformed microorganism into which the gene according to claim 2 is introduced and obtaining cyclodextrin glucanotransferase from the culture solution.
Priority Applications (1)
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JP4799392A JP3155324B2 (en) | 1992-02-04 | 1992-02-04 | Cyclodextrin glucanotransferase, method for producing the same, and gene encoding the enzyme |
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---|---|---|---|
JP4799392A JP3155324B2 (en) | 1992-02-04 | 1992-02-04 | Cyclodextrin glucanotransferase, method for producing the same, and gene encoding the enzyme |
Publications (2)
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JPH05219948A true JPH05219948A (en) | 1993-08-31 |
JP3155324B2 JP3155324B2 (en) | 2001-04-09 |
Family
ID=12790848
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0630967A1 (en) * | 1993-06-24 | 1994-12-28 | Consortium für elektrochemische Industrie GmbH | Cyclodextrin glycosyltransferase for the production of gamma-cyclodextrin |
EP0802259A1 (en) * | 1996-04-18 | 1997-10-22 | Consortium für elektrochemische Industrie GmbH | Cyclodextrin glucosyltransferases for the production of gamma-cyclodextrin |
US6004790A (en) * | 1995-04-21 | 1999-12-21 | Novo Nordisk A/S | Cyclomaltodextrin glucanotransferase variants |
EP2031066A4 (en) * | 2006-06-08 | 2009-11-18 | Japan Maize Prod | Mutant and gene encoding the same |
CN112301011A (en) * | 2019-07-23 | 2021-02-02 | 波顿(上海)生物技术有限公司 | Glycosyltransferase variants and uses thereof |
-
1992
- 1992-02-04 JP JP4799392A patent/JP3155324B2/en not_active Expired - Fee Related
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0630967A1 (en) * | 1993-06-24 | 1994-12-28 | Consortium für elektrochemische Industrie GmbH | Cyclodextrin glycosyltransferase for the production of gamma-cyclodextrin |
JPH078275A (en) * | 1993-06-24 | 1995-01-13 | Consortium Elektrochem Ind Gmbh | Cyclodextringlycosyl transferase and method for production of said transferase and gamma-cyclodextrin |
US5474917A (en) * | 1993-06-24 | 1995-12-12 | Consortium Fur Elektrochemische Industrie Gmbh | Modified cyclodextrin glycosyltransferases for producing γ-cyclodextrins |
JP2635932B2 (en) * | 1993-06-24 | 1997-07-30 | コンゾルテイウム フユール エレクトロケミツシエ インヅストリー ゲゼルシヤフト ミツト ベシユレンクテル ハフツング | Cyclodextrin glycosyltransferase, method for producing the transferase, and method for producing γ-cyclodextrin |
US6004790A (en) * | 1995-04-21 | 1999-12-21 | Novo Nordisk A/S | Cyclomaltodextrin glucanotransferase variants |
EP0802259A1 (en) * | 1996-04-18 | 1997-10-22 | Consortium für elektrochemische Industrie GmbH | Cyclodextrin glucosyltransferases for the production of gamma-cyclodextrin |
EP2031066A4 (en) * | 2006-06-08 | 2009-11-18 | Japan Maize Prod | Mutant and gene encoding the same |
CN112301011A (en) * | 2019-07-23 | 2021-02-02 | 波顿(上海)生物技术有限公司 | Glycosyltransferase variants and uses thereof |
CN112301011B (en) * | 2019-07-23 | 2023-12-08 | 波顿(上海)生物技术有限公司 | Glycosyltransferase variants and uses thereof |
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