JP2017529849A - ニトリルヒドラターゼ活性を有する微生物の培養方法 - Google Patents
ニトリルヒドラターゼ活性を有する微生物の培養方法 Download PDFInfo
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- JP2017529849A JP2017529849A JP2017516326A JP2017516326A JP2017529849A JP 2017529849 A JP2017529849 A JP 2017529849A JP 2017516326 A JP2017516326 A JP 2017516326A JP 2017516326 A JP2017516326 A JP 2017516326A JP 2017529849 A JP2017529849 A JP 2017529849A
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- Prior art keywords
- nitrile hydratase
- microorganism
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- saccharide
- acid
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
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Abstract
Description
(a)前記微生物と、糖類(i)および有機酸(ii)を含む水性組成物とを接触させるステップ;
(b)(a)の前記水性組成物中で前記微生物を培養するステップ
を含む前記方法に関する。
実施例1
ロドコッカス亜種(Rhodococcus ssp.)の培養
マイクロ発酵システム内で、異なるロドコッカス株(R.ロドクラウス(R.rhodochrous)「CIBA」(NCIMB 41164)およびR.ロドクラウス(R.rhodochrous)「J1」(FERM BP−1478))について、様々な糖/有機酸の比を評価した。
ニトリルヒドラターゼ活性の測定
実施例1の培養培地から試料を採取し、50mmol/lのKH2PO4緩衝液(pH7.0)で1:10(v/v)で希釈した。得られた溶液をさらに、50mmol/lのKH2PO4緩衝液(pH7.0)で1:20(v/v)で希釈し、培養培地に対して1:200の最終希釈率に達する。
KH2PO4緩衝液(pH7.0)875μlを、ピペットで2mlエッペンドルフチューブに移した。希釈した培養培地100μlを添加し、この混合物を、サーモミキサー中で25℃で500rpmで5分間プレインキュベートした。プレインキュベーション後、アクリロニトリル25μlの添加によって反応を開始した。この反応を、25℃、1,000rpmで10分間行った。
反応時間が10分間経過した後、この反応溶液300μlを、1.4%(m/v)塩酸300μlの入った1.5mlのエッペンドルフチューブに移すことによって、この反応をクエンチした。この混合物を短時間ボルテックス処理し、卓上遠心分離機で10,000rpmで1分間遠心分離して、細胞を分離させた。澄明な上澄み100μlと水900μlとを混合した。次いで、この混合物をHPLCで分析した。
・カラム:アクア(Aqua)5μ C18 125A、250×4.60mm(フェノメネックス(Phenomenex))
・オーブン温度:45℃
・注入量:1μl
・検出:UV 210nm
・流量:1.0ml/分
・溶媒A:25mmol/l KH2PO4 pH2.5
・溶媒B:アセトニトリル
・分離:10%B(イソクラティック)
・保持:アクリルアミド:約3.6分間
・HPLC値は、0.25〜1.25mmol/lであるべきである。
・c:生成物の物質量[mM](HPLC値)
・t:インキュベーション時間[分]、本試験では10分間
・k:希釈係数 −出発試料からHPLC試料への合計希釈
活性(kU/l)の計算
・活性(kU/l)=c/t×k
1活性単位[U]とは、反応時間1分間中にアクリルアミドが1μmol生成されることと定義される。
Claims (15)
- ニトリルヒドラターゼ産生微生物を培養するための方法であって、以下:
(a)前記微生物と、糖類(i)および有機酸(ii)を含む水性組成物とを接触させるステップ;および
(b)(a)の前記水性組成物中で前記微生物を培養するステップ
を含む、前記方法。 - ニトリルヒドラターゼ産生微生物を培養するための組成物であって、糖類(i)および有機酸(ii)を含む、前記組成物。
- ニトリルヒドラターゼ産生微生物を培養するための、糖類(i)と有機酸(ii)とを含む組成物の使用。
- 前記糖類(i)が単糖類である、請求項1に記載の方法、請求項2に記載の組成物または請求項3に記載の使用。
- 前記糖類(i)中に含まれる炭素原子数が、少なくとも5であり、好ましくは少なくとも6である、請求項1もしくは4に記載の方法、請求項2もしくは4に記載の組成物または請求項3もしくは4に記載の使用。
- 前記糖類(i)が、グルコース、フルクトース、リボースおよびマンノースからなる群から選択される、請求項1、4もしくは5に記載の方法、請求項2、4もしくは5に記載の組成物または請求項3から5までのいずれか1項に記載の使用。
- 前記有機酸(ii)が、3個以下のカルボキシル基、好ましくは2個以下のカルボキシル基、最も好ましくは1個以下のカルボキシル基を含む、請求項1もしくは4から6までのいずれか1項に記載の方法、請求項2もしくは4から6までのいずれか1項に記載の組成物または請求項3から6までのいずれか1項に記載の使用。
- 前記有機酸(ii)が、6個以下の炭素原子、好ましくは4個以下の炭素原子、最も好ましくは3個以下の炭素原子を含む、請求項1もしくは4から7までのいずれか1項に記載の方法、請求項2もしくは4から7までのいずれか1項に記載の組成物または請求項3から7までのいずれか1項に記載の使用。
- 前記有機酸(ii)が、乳酸、酒石酸、クエン酸、リンゴ酸およびコハク酸からなる群から選択される、請求項1もしくは4から8までのいずれか1項に記載の方法、請求項2もしくは4から8までのいずれか1項に記載の組成物または請求項3から8までのいずれか1項に記載の使用。
- 前記糖類(i)と前記有機酸(ii)との比が、重量で3:7〜7:3である、請求項1もしくは4から9までのいずれか1項に記載の方法、請求項2もしくは4から9までのいずれか1項に記載の組成物または請求項3から9までのいずれか1項に記載の使用。
- 前記微生物が、ロドコッカス・ロドクラウス(Rhodococcus rhodochrous)またはロドコッカス・ピリジノボランス(Rhodococcus pyridinovorans)の種に属する、請求項1もしくは4から10までのいずれか1項に記載の方法、請求項2もしくは4から10までのいずれか1項に記載の組成物または請求項4から10までのいずれか1項に記載の使用。
- 前記ニトリルヒドラターゼが、配列番号1および/または配列番号3に示されるヌクレオチド配列と少なくとも70%同一であるヌクレオチド配列を含むポリヌクレオチドによりコードされる、請求項1もしくは4から11までのいずれか1項に記載の方法、請求項2もしくは4から11までのいずれか1項に記載の組成物または請求項4から11までのいずれか1項に記載の使用。
- 前記ニトリルヒドラターゼが、配列番号2および/または配列番号4に示されるアミノ酸配列と少なくとも70%同一であるアミノ酸配列を有する、請求項1もしくは4から12までのいずれか1項に記載の方法、請求項2もしくは4から12までのいずれか1項に記載の組成物または請求項4から12までのいずれか1項に記載の使用。
- 請求項1または4から13までのいずれか1項に記載の培養方法によって得ることができる、微生物。
- 前記微生物が、培養後に乾燥される、請求項1もしくは4から13までのいずれか1項に記載の方法または請求項14に記載の微生物。
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