JPS61187786A - Heat-resistant d-amino acid transaminase - Google Patents
Heat-resistant d-amino acid transaminaseInfo
- Publication number
- JPS61187786A JPS61187786A JP2673585A JP2673585A JPS61187786A JP S61187786 A JPS61187786 A JP S61187786A JP 2673585 A JP2673585 A JP 2673585A JP 2673585 A JP2673585 A JP 2673585A JP S61187786 A JPS61187786 A JP S61187786A
- Authority
- JP
- Japan
- Prior art keywords
- amino acid
- resistant
- acid transaminase
- heat
- bacillus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は新規な耐熱性Ｄ−アミノ酸トランスアミナーゼ
に関するものである。DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a novel thermostable D-amino acid transaminase.
（従来技術）
従来、Ｄ−アミノ酸トランスアミナーピはバチルススフ
エリカス（Ｂａｃｉｌｌｕｓ ５ｐｈａｒｉｃｕｓ）、
バチルスサブチルス（Ｂａｃｉｌｌｕｓ ５ｕｂｔｉｌ
ｌｉｓ）等により産生されるものが知られているが、い
ずれも常温では活性を示すが、５０℃以上では失活する
ものであった。しかし、産業上の酵素利用の観点からは
反応速度が速く、反応液が雑菌により汚染されにくい条
件下で使用可能な酵素を用いる事が要望される。(Prior Art) Conventionally, D-amino acid transaminarpi was obtained from Bacillus 5pharicus,
Bacillus subtilis
lis), etc., all of which exhibit activity at room temperature, but become inactive at temperatures above 50°C. However, from the viewpoint of industrial enzyme utilization, it is desired to use an enzyme that has a fast reaction rate and can be used under conditions where the reaction solution is unlikely to be contaminated by germs.
この様な条件を満たすには耐熱性酵素を用いるのがよく
、種々の耐熱性酵素の検索が行なわれている。To satisfy these conditions, it is best to use thermostable enzymes, and various thermostable enzymes are being searched for.
しかしながら、これまでのところ耐熱性のＤ−アミノ酸
トランスアミナーピは見いだされていない。However, no heat-stable D-amino acid transamino acids have been found so far.
（発明の目的）
本発明は各種の土壌等自然界の試料より、耐熱性かつ高
活性なり一アミノ酸トランスアミナーゼを産生ずる好熱
性細菌を探索し、もって新規な耐熱性Ｄ−アミノ酸トラ
ンスアミナーゼを得ることを目的とする。(Objective of the invention) The purpose of the present invention is to search for thermophilic bacteria that produce heat-resistant and highly active single-amino acid transaminases from various samples in the natural world such as soil, and thereby obtain a novel thermostable D-amino acid transaminase. shall be.
（発明の構成）
本発明者らは耐熱性Ｄ−アミノ酸トランスアミナーゼを
検索するため、多くの土壌よりＤ−アミノ酸トランスア
ミナーゼ活性を有する微生物の探索を行ない、目的とす
る酵素活性を有する中程度好熱性細菌を分離しバチルス
ＹＭ−１と命名した。(Structure of the Invention) In order to search for thermostable D-amino acid transaminase, the present inventors searched for microorganisms that have D-amino acid transaminase activity from many soils, and found that moderately thermophilic bacteria that have the desired enzyme activity. was isolated and named Bacillus YM-1.
分離した中程度好熱菌バチルスＹＭ−１は従来知られて
いる菌とは異なりバチルス属に属する新菌株として、工
業技術院微生物工業技術研究所に微工研菌寄第８０５７
号（ＦＥＲＭＰ−８０５７）として寄託されている。The isolated moderate thermophilic bacterium Bacillus YM-1 was submitted to the Institute of Microbiology, Agency of Industrial Science and Technology as a new strain belonging to the genus Bacillus, unlike previously known bacteria.
No. (FERMP-8057).
本発明者らはバチルスＹＭ−１（以下ＹＭ−１と示す。The present inventors produced Bacillus YM-1 (hereinafter referred to as YM-1).
）を培養し、Ｄ−アミノ酸トランスアミナーゼを抽出、
分離、精製することにより本発明を完成するに至った。) and extract D-amino acid transaminase.
Through separation and purification, the present invention was completed.
即ち、本発明は、バチルス属に属する耐熱性Ｄ−アミノ
酸トランスアミナーゼ産生菌が産生ずる耐熱性Ｄ−アミ
ノ酸トランスアミナーゼである。That is, the present invention is a thermostable D-amino acid transaminase produced by a thermostable D-amino acid transaminase-producing bacterium belonging to the genus Bacillus.
以下本発明の内容をＹＭ−１の産生ずる耐熱性Ｄ−アミ
ノ酸トランスアミナーゼについて詳述するが、本発明は
これに限定されるものではない。The present invention will be described in detail below regarding the heat-stable D-amino acid transaminase produced by YM-1, but the present invention is not limited thereto.
本発明の耐熱性Ｄ−アミノ酸トランスアミナーゼは、Ｙ
Ｍ−１株を５７℃で５時間通気培養し、培養液中の菌体
を集菌し、超音波により細胞を破砕して得た菌体抽出液
をＤＥＡＥ−トヨバール、ヒドロキシアパタイト、ブチ
ルトヨパールなどの各種りａマドグラフィにより分離、
精製することにより、電気泳動的にまた超遠心分析的に
均一なものとして得ることができる。The thermostable D-amino acid transaminase of the present invention is Y
Strain M-1 was aerated and cultured at 57°C for 5 hours, the cells in the culture solution were collected, and the cells were disrupted by ultrasonic waves. Separation by various kinds of atomography, such as
By purification, it can be obtained as homogeneous electrophoretically and ultracentrifugally.
一方、従来からＤ−アミノ酸トランスアミナーゼの産生
の知られているバチルススフエリカスからも、同様の方
法により同酵素を抽出、分離、精製し、両者の酵素学的
な比較を行った。On the other hand, the same enzyme was extracted, separated, and purified from Bacillus sphaericus, which has been known to produce D-amino acid transaminase, by the same method, and enzymological comparisons were made between the two.
酵素のアミノ酸組成について比較した結果を第１表に示
す。アミノ酸組成は両者は全く異なり、セリン、グリシ
ン、アラニン、シスチン、ロイシン、トリプトファン等
の含有量に著しい差異があり、従って酵素蛋白質の立体
構造が異なり、両酵素の耐熱性あるいは基質特異性に差
があるものと考えられる。Table 1 shows the results of comparing the amino acid compositions of the enzymes. The amino acid composition of the two enzymes is completely different, with significant differences in the content of serine, glycine, alanine, cystine, leucine, tryptophan, etc. Therefore, the three-dimensional structure of the enzyme protein is different, and there is a difference in the thermostability or substrate specificity of the two enzymes. It is thought that there is.
第１表 酵素のアミノ酸組成 さらに、両酵素の性質の比較を第２表に示す。Table 1 Amino acid composition of enzymes Furthermore, a comparison of the properties of both enzymes is shown in Table 2.
ＹＭ−１の産生ずる酵素は従来から知られているものに
比べて、至適Ｅ）Ｈが低く、至適温度は１５℃も高く、
かつ熱安定性が２０℃も高い、耐熱性酵素である。The enzyme used to produce YM-1 has a lower optimum E)H and a 15°C higher optimum temperature than those previously known.
It is also a thermostable enzyme with a high thermostability of 20°C.
基質特異性について、従来から知られているバチルスス
フエリス及びバチールスサブチルスの産生する酵素とＹ
Ｍ−１の酵素を比較した結果を第３表に示す。表に示さ
れている様に基質に対する相対活性は従来から知られて
いるものと著しく異なっている。Regarding substrate specificity, the enzymes produced by Bacillus sphaeris and Bacillus subtilis and Y
Table 3 shows the results of comparing the M-1 enzymes. As shown in the table, the relative activity towards the substrate is significantly different from what has been previously known.
以上、示した結果から、ＹＭ−１の産生ずるＤ−アミノ
酸トランスアミナーゼは従来から知られているものと各
種の性質が異なり、さらに耐熱性のよいものであり、全
く新規のものである。From the results shown above, the D-amino acid transaminase produced by YM-1 has various properties different from those conventionally known, and has good heat resistance, and is completely new.
（発明の効果）
本発明は、これまで見出されていなかった耐熱性Ｄ−ア
ミノ酸トランスアミナービを供給することにより、反応
液の雑菌馬乗、反応速度等の面で産業上有利な酵素反応
プロセスの設定を可能にした。(Effects of the Invention) The present invention provides an enzyme reaction process that is industrially advantageous in terms of eliminating bacteria in the reaction solution, reaction rate, etc. by supplying a heat-stable D-amino acid transamino acid that has not been discovered so far. settings.
実施例
下記の培地５００ｍを用いてｐ）−１７，２゜５７℃で
６時間ＹＭ−１株を通気培養し、集菌後超音波破砕した
。Example YM-1 strain was aerated and cultured for 6 hours at -17.2°C and 57°C using 500ml of the following medium, and the cells were collected and disrupted by ultrasonication.
得られた菌体抽出液をＤＥＡＥ−１，＋パルをカラムと
して粗分離を行い、さらにヒドロキシアパタイト、ブチ
ルトヨバールのカラムを用いて精製し、Ｄ−アミノ酸ト
ランスアミナーゼを得た。The obtained bacterial cell extract was roughly separated using DEAE-1,+PAL as a column, and further purified using hydroxyapatite and butyltoyobal columns to obtain D-amino acid transaminase.
この酵素を用いて、５０℃、Ｉ）Ｈ８で反応を行った結
果、α−ケトグルタル酸とＤ−アラニンを基質にした時
の活性を１００とすると、Ｄ−アラニンの替りにＤ−グ
ルタミン、Ｄ−アスパラギン酸、Ｄ−ノルバリンを基質
としだ時の活性は、それぞれ、５３．５７および２９で
あった。その他同様の相対活性は先に第３表に示した通
りである。As a result of a reaction using this enzyme at 50°C and I) H8, assuming that the activity when α-ketoglutaric acid and D-alanine were used as substrates was 100, D-glutamine and D The activities when using -aspartic acid and D-norvaline as substrates were 53.57 and 29, respectively. Other similar relative activities are as shown in Table 3 above.
（培 地）
ペプトン １ ％酵母エキス
０．２５
グリセリン ０．２
ＮａＣＩ ｏ、２
に２ＨＰＯ４０，２
ＫＨ２ＰＯ４０，２
Ｍｇ５０ ・７ＨＯＯ，０１
ビオチン ４μｇ／ｊＮａＯＨにより
ｐＨ７，２に調整。(Medium) Peptone 1% yeast extract
0.25 Glycerin 0.2 NaCI o,2 to 2HPO40,2 KH2PO40,2 Mg50 ・7HOO,01 Biotin 4 μg/j Adjusted to pH 7.2 with NaOH.
Claims (1)
ーゼ産生菌が産生する耐熱性アミノ酸トランスアミナー
ゼ。A thermostable amino acid transaminase produced by a thermostable D-amino acid transaminase-producing bacterium belonging to the genus Bacillus.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2673585A JPS61187786A (en) | 1985-02-14 | 1985-02-14 | Heat-resistant d-amino acid transaminase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2673585A JPS61187786A (en) | 1985-02-14 | 1985-02-14 | Heat-resistant d-amino acid transaminase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS61187786A true JPS61187786A (en) | 1986-08-21 |
| JPH055472B2 JPH055472B2 (en) | 1993-01-22 |
Family
ID=12201564
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2673585A Granted JPS61187786A (en) | 1985-02-14 | 1985-02-14 | Heat-resistant d-amino acid transaminase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS61187786A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63207387A (en) * | 1987-02-24 | 1988-08-26 | Daicel Chem Ind Ltd | Dna sequence coding d-amino acid transaminase |
| US6413752B1 (en) | 1999-03-19 | 2002-07-02 | Sumitomo Chemical Company, Limited | Protein capable of catalyzing transamination stereoselectively, gene encoding said protein and use thereof |
-
1985
- 1985-02-14 JP JP2673585A patent/JPS61187786A/en active Granted
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS63207387A (en) * | 1987-02-24 | 1988-08-26 | Daicel Chem Ind Ltd | Dna sequence coding d-amino acid transaminase |
| US6413752B1 (en) | 1999-03-19 | 2002-07-02 | Sumitomo Chemical Company, Limited | Protein capable of catalyzing transamination stereoselectively, gene encoding said protein and use thereof |
| US6727083B2 (en) | 1999-03-19 | 2004-04-27 | Sumitomo Chemical Company, Limited | Protein capable of catalyzing transamination stereoselectively, gene encoding said protein and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH055472B2 (en) | 1993-01-22 |
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