JPH0675505B2 - Method for producing D-threonine aldolase - Google Patents
Method for producing D-threonine aldolaseInfo
- Publication number
- JPH0675505B2 JPH0675505B2 JP10137988A JP10137988A JPH0675505B2 JP H0675505 B2 JPH0675505 B2 JP H0675505B2 JP 10137988 A JP10137988 A JP 10137988A JP 10137988 A JP10137988 A JP 10137988A JP H0675505 B2 JPH0675505 B2 JP H0675505B2
- Authority
- JP
- Japan
- Prior art keywords
- threonine
- threonine aldolase
- producing
- aldolase
- enzyme
- 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.)
- Expired - Fee Related
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、微生物を用いてＤ−スレオニンアルドラーゼ
を製造する方法に関する。Ｄ−スレオニンアルドラーゼ
はＤ−スレオニンをグリシンとアセトアルデヒドに分解
する酵素でDL−スレオニンの光学分割、Ｄ−スレオニン
の定量分析及び逆反応を利用してグリシンとアルデヒド
類よりＤ−β−ハイドロキシアミノ酸類等に用いること
が出来る。TECHNICAL FIELD The present invention relates to a method for producing D-threonine aldolase using a microorganism. D-threonine aldolase is an enzyme that decomposes D-threonine into glycine and acetaldehyde, and uses optical resolution of DL-threonine, quantitative analysis of D-threonine, and reverse reaction to produce D-β-hydroxyamino acids from glycine and aldehydes. Can be used for
〔従来の技術及び発明が解決しようとする課題〕 本発明者らはＤ−スレオニンを分解する酵素を広く検索
した結果、先にアリスロバクター属、シュードモナス属
およびアルカリゲネス属に属する特定の微生物がＤ−ス
レオニンをグリシンとアセトアルデヒドに分解する新規
な酵素Ｄ−スレオニンアルドラーゼを産生することを見
出している（特開昭58−116680号公報）。それ以外には
未だ本酵素活性を示す微生物は知られていなかった。[Prior Art and Problems to be Solved by the Invention] As a result of a wide search of enzymes degrading D-threonine, the present inventors have found that specific microorganisms belonging to the genus Aithrobacter, Pseudomonas and Alcaligenes It has been found that it produces a novel enzyme D-threonine aldolase that decomposes threonine into glycine and acetaldehyde (Japanese Patent Laid-Open No. 58-116680). Other than that, no microorganism has been known that exhibits this enzyme activity.
今回、更にバクテリヤ、カビ及び酵母につき広範囲にわ
たりＤ−スレオニンアルドラーゼ活性を有する微生物を
検索した結果、新たにキサントモナス（Xanthomonas）
属に属する微生物もＤ−スレオニンアルドラーゼを産生
することを見い出し本発明を完成するに到った。This time, as a result of further searching for a microorganism having a D-threonine aldolase activity in a wide range of bacteria, molds and yeasts, Xanthomonas (Xanthomonas) was newly found.
It has been found that microorganisms belonging to the genus also produce D-threonine aldolase, and completed the present invention.
本発明において使用される微生物としてはキサントモナ
ス属に属しＤ−スレオニンアルドラーゼ産生能を有する
ものであり具体例としてはキサントモナス オリザエ
（Xanthomonas oryzae IAM1657）を挙げることが出来
る。The microorganism used in the present invention belongs to the genus Xanthomonas and has the ability to produce D-threonine aldolase. Specific examples thereof include Xanthomonas oryzae IAM1657.
このような微生物を培養してＤ−スレオニンアルドラー
ゼを生成せしめるには特に困難はなく、通常の細菌類の
培養に準じて行なえばよい。すなわち、培地について炭
素源としてはグルコース、グリセロール、糖蜜等の糖類
あるいは酢酸、リンゴ酸等の有機酸など、窒素源として
は硫酸アンモニウム、塩化アンモニウム、尿素など、有
機栄養源として酵母エキス、ペプトン、肉エキス、コー
ンスティープリカーなど、そして無機イオンとしてマグ
ネシウム、鉄、マンガン、カリウム、リン酸塩などを含
むものを用いる。培地中にＤ−スレオニンを0.05〜0.5
％程度添加すると酵素の産生量が増加する場合もある。There is no particular difficulty in culturing such a microorganism to produce D-threonine aldolase, and it may be carried out in accordance with ordinary culturing of bacteria. That is, regarding the medium, sugars such as glucose, glycerol, molasses or organic acids such as acetic acid and malic acid are used as the carbon source, ammonium sulfate, ammonium chloride, urea and the like are used as the nitrogen source, and yeast extract, peptone and meat extract are used as the organic nutrient sources. , Corn steep liquor, etc., and those containing magnesium, iron, manganese, potassium, phosphate, etc. as inorganic ions are used. D-threonine was added to the medium at 0.05 to 0.5.
% Addition may increase the enzyme production.
培養方法も常法によればよく、例えば培地のpHを４〜10
として菌を接種後、20〜60℃で１〜３日間好気的に培養
すればよい。The culture method may be a conventional method, for example, the pH of the medium is 4 to 10
Then, after inoculating the bacteria, it may be aerobically cultured at 20 to 60 ° C. for 1 to 3 days.
このようにして得られるＤ−スレオニンアルドラーゼ
は、主に菌体内に生成蓄積される。そこで、Ｄ−スレオ
ニンアルドラーゼを酵素源として使用する場合には、培
養物あるいはそれから分離した菌体をそのままあるいは
乾燥して用いてもよいが、Ｄ−スレオニンアルドラーゼ
をより精製した形で使用する必要がある場合には、まず
菌体を機械的方法、酵素処理する方法、自己溶解法など
公知の方法によって破壊して粗抽出液を得る。それか
ら、この粗抽出液を硫安沈殿、アセトン又はエタノール
などによる溶媒沈殿、DEAE−セファロース、DEAE−セフ
ァデックス、リン酸カルシウムゲル等の種々のイオン交
換体や吸着剤を用いたクロマトグラフィーなどを適宜組
合せて精製することによって高純度の酵素標品を得るこ
とができる。本酵素の活性発現には、補酵素としてピリ
ドキサール−５′−リン酸を必要とするため、反応時に
は通常10-3〜10-5Ｍで存在させる。The D-threonine aldolase thus obtained is mainly produced and accumulated in the cells. Therefore, when D-threonine aldolase is used as an enzyme source, the culture or the bacterial cells separated therefrom may be used as it is or after drying, but it is necessary to use D-threonine aldolase in a more purified form. In some cases, first, the bacterial cells are disrupted by a known method such as a mechanical method, an enzyme treatment method or an autolysis method to obtain a crude extract. Then, the crude extract was purified by appropriately combining ammonium sulfate precipitation, solvent precipitation with acetone or ethanol, DEAE-Sepharose, DEAE-Sephadex, various ion exchangers such as calcium phosphate gel and chromatography using an adsorbent. By doing so, a highly pure enzyme preparation can be obtained. Since pyridoxal-5'-phosphate is required as a coenzyme for activity expression of this enzyme, it is usually present at 10 -3 to 10 -5 M during the reaction.
本酵素は、例えばDL−スレオニンを原料としてＬ−スレ
オニンを製造する方法に用いれば光学分割工程を簡略化
しうるが、そのほかＤ−スレオニンの定量分析などにも
有効である。The present enzyme can simplify the optical resolution process if used in a method for producing L-threonine from DL-threonine as a raw material, but is also effective for quantitative analysis of D-threonine.
又、本酵素反応が可逆反応であることを利用し、グリシ
ンと各種アルデヒド類よりＤ−β−ハイドロキシアミノ
酸類の合成に用いることも出来る。Further, by utilizing the fact that this enzymatic reaction is a reversible reaction, it can be used for the synthesis of D-β-hydroxyamino acids from glycine and various aldehydes.
以下、実施例を示す。なお、％は全て重量％である。 Examples will be shown below. All percentages are by weight.
実施例１ ポリペプトン0.5％、酵母エキス0.5％、KH2PO40.1％、M
gSO40.05％、Ｌ−グルタミン酸0.1％、およびＤ−スレ
オニン0.1％からなるpH8.0の培地を調製し、120℃で15
分間加熱殺菌した。この培地にキサントモナス オリザ
エ（Xanthomonas oryzae）IAM1657を接種し、30℃で20
時間培養した。Example 1 Polypeptone 0.5%, yeast extract 0.5%, KH 2 PO 4 0.1%, M
A medium of pH 8.0 consisting of 0.05% of gSO 4 , 0.1% of L-glutamic acid, and 0.1% of D-threonine was prepared, and the medium at 120 ° C.
Heat sterilized for minutes. This medium was inoculated with Xanthomonas oryzae IAM1657 and incubated at 30 ° C for 20
Incubated for hours.
培養終了後、培養液5mlから菌体を遠心分離し、生理食
塩水で１回洗浄後この湿菌体を0.1mMピリドキサール−
５′−リン酸及び50mMD−スレオニンを含むpH7.5の0.1M
トリス−塩酸緩衝液5ml中に懸濁し、35℃で分解反応を
行った。24時間後に遠心分離により得られた反応液上清
をHPLC分析した結果、グリシンが32mM生成しておりＤ−
スレオニンアルドラーゼ活性を有することを認めた。After culturing, the cells were centrifuged from 5 ml of the culture solution, washed once with physiological saline, and the wet cells were washed with 0.1 mM pyridoxal-
0.1 M pH 7.5 containing 5'-phosphate and 50 mM D-threonine
The suspension was suspended in 5 ml of Tris-hydrochloric acid buffer and the decomposition reaction was carried out at 35 ° C. As a result of HPLC analysis of the reaction solution supernatant obtained by centrifugation after 24 hours, glycine was produced at 32 mM and D-
It was confirmed to have threonine aldolase activity.
本発明によれば大量かつ安価に簡便な方法でＤ−スレオ
ニンアルドラーゼを製造することができる。According to the present invention, a large amount of D-threonine aldolase can be produced at low cost by a simple method.
Claims (1)
アルドラーゼを産生しうる微生物を栄養培地に培養し、
培養物からＤ−スレオニンアルドラーゼを採取すること
を特徴とするＤ−スレオニンアルドラーゼの製造法。1. A microorganism capable of producing D-threonine aldolase belonging to the genus Xanthomonas is cultured in a nutrient medium,
A method for producing D-threonine aldolase, which comprises collecting D-threonine aldolase from a culture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10137988A JPH0675505B2 (en) | 1988-04-26 | 1988-04-26 | Method for producing D-threonine aldolase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP10137988A JPH0675505B2 (en) | 1988-04-26 | 1988-04-26 | Method for producing D-threonine aldolase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH01273586A JPH01273586A (en) | 1989-11-01 |
| JPH0675505B2 true JPH0675505B2 (en) | 1994-09-28 |
Family
ID=14299157
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP10137988A Expired - Fee Related JPH0675505B2 (en) | 1988-04-26 | 1988-04-26 | Method for producing D-threonine aldolase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0675505B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| BRPI0607205A2 (en) | 2005-01-31 | 2011-07-19 | Koninkl Philips Electronics Nv | sensor device for detecting magnetic particles, and method for a biosensor process |
-
1988
- 1988-04-26 JP JP10137988A patent/JPH0675505B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH01273586A (en) | 1989-11-01 |
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Free format text: JAPANESE INTERMEDIATE CODE: R313531 |
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