JP2011041550A - Preservation agent, activity activation agent and deactivation preventing agent for amidase - Google Patents
Preservation agent, activity activation agent and deactivation preventing agent for amidase Download PDFInfo
- Publication number
- JP2011041550A JP2011041550A JP2009193689A JP2009193689A JP2011041550A JP 2011041550 A JP2011041550 A JP 2011041550A JP 2009193689 A JP2009193689 A JP 2009193689A JP 2009193689 A JP2009193689 A JP 2009193689A JP 2011041550 A JP2011041550 A JP 2011041550A
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- JP
- Japan
- Prior art keywords
- amidase
- activity
- manganese
- cobalt
- nickel
- 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.)
- Pending
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- 108700023418 Amidases Proteins 0.000 title claims abstract description 125
- 102000005922 amidase Human genes 0.000 title claims abstract description 105
- 230000000694 effects Effects 0.000 title claims abstract description 65
- 230000009849 deactivation Effects 0.000 title claims abstract description 13
- 230000003405 preventing effect Effects 0.000 title claims description 7
- 230000004913 activation Effects 0.000 title abstract description 5
- 238000004321 preservation Methods 0.000 title description 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims abstract description 64
- 150000003839 salts Chemical class 0.000 claims abstract description 43
- 229910017052 cobalt Inorganic materials 0.000 claims abstract description 33
- 239000010941 cobalt Substances 0.000 claims abstract description 33
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims abstract description 33
- 229910052759 nickel Inorganic materials 0.000 claims abstract description 32
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229910052748 manganese Inorganic materials 0.000 claims abstract description 25
- 239000011572 manganese Substances 0.000 claims abstract description 25
- 239000003755 preservative agent Substances 0.000 claims abstract description 8
- 230000002335 preservative effect Effects 0.000 claims abstract description 7
- 239000012190 activator Substances 0.000 claims abstract description 6
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- 238000000034 method Methods 0.000 claims description 41
- 238000010438 heat treatment Methods 0.000 claims description 11
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims description 8
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- 229910021380 Manganese Chloride Inorganic materials 0.000 description 7
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Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
【課題】α−アミノ酸やα−ヒドロキシ酸等を製造するために用いるアミダーゼの保存剤、活性賦活剤及び失活予防剤を提供する。
【解決手段】Thermus sp.属、Thermus sp.0-3-1株、由来耐熱性アミダーゼ遺伝子を大腸菌JM109株にクローニングしてアミダーゼを生産することにより、アミダーゼ酵素溶液を調製した。該酵素溶液にニッケル、コバルト及びマンガン並びにそれらの塩からなる群より選ばれる少なくとも１種を共存させることにより、アミダーゼ活性を安定化し、保存、活性賦活及び失活予防に有効である。
【選択図】なしThe present invention provides a preservative, activity activator and deactivation preventive agent for amidase used for producing α-amino acid, α-hydroxy acid and the like.
An amidase enzyme solution was prepared by cloning a thermostable amidase gene derived from Thermus sp. Genus, Thermus sp. 0-3-1 strain, and Escherichia coli JM109 strain to produce amidase. By allowing the enzyme solution to coexist with at least one selected from the group consisting of nickel, cobalt, manganese and salts thereof, the amidase activity is stabilized and effective for storage, activation of activity and prevention of deactivation.
[Selection figure] None
Description
本発明は、アミダーゼの保存剤、活性賦活剤及び失活予防剤、アミダーゼの保存用キット、活性賦活用キット及び失活予防用キット、並びにそれらの使用方法に関する。 The present invention relates to an amidase preservative, an activity activator and an inactivation prevention agent, an amidase storage kit, an activity utilization kit and an inactivation prevention kit, and methods for using them.
光学活性なα−アミノ酸やα−ヒドロキシ酸は、各種工業製品等の中間体、食品添加物及び医薬品等として重要である。微生物あるいは微生物由来の酵素をα−アミノ酸アミド及びα−ヒドロキシ酸アミドの加水分解触媒として用いることにより、光学活性なα−アミノ酸（特許文献１及び２参照）やα−ヒドロキシ酸（特許文献３参照）を製造できることが知られている。近年、アミダーゼ遺伝子が導入された組換え体を用いた反応も報告されてきている（特許文献４及び５並びに非特許文献１参照）。 Optically active α-amino acids and α-hydroxy acids are important as intermediates for various industrial products, food additives, pharmaceuticals, and the like. By using microorganisms or microorganism-derived enzymes as hydrolysis catalysts for α-amino acid amides and α-hydroxy acid amides, optically active α-amino acids (see Patent Literatures 1 and 2) and α-hydroxy acids (see Patent Literature 3). It is known that can be produced. In recent years, reactions using recombinants into which amidase genes have been introduced have also been reported (see Patent Documents 4 and 5 and Non-Patent Document 1).
このような加水分解反応において、アミダーゼ活性を効率的に発現させるため、培養液中や反応液中に亜鉛塩などの金属塩を添加する試みがなされている。
例えば、Enterobacter Cloacae N-7901株由来のアミダーゼについて、培養液中に亜鉛塩を添加する試みがなされている（特許文献４参照）。また、Ochrobactrum anthropi NCIMB40321株由来のアミダーゼについて、反応液中への亜鉛塩の添加効果、及び培養液中への亜鉛塩の添加効果が示されている（特許文献５参照）。さらに、同菌株ついて、亜鉛、マグネシウム、マンガンの金属塩の反応液中への添加効果が示されている（非特許文献１参照）。
In such a hydrolysis reaction, an attempt has been made to add a metal salt such as a zinc salt to a culture solution or a reaction solution in order to efficiently express amidase activity.
For example, with respect to amidase derived from Enterobacter Cloacae N-7901, an attempt has been made to add a zinc salt to the culture solution (see Patent Document 4). In addition, with respect to amidase derived from Ochrobactrum anthropi NCIMB40321 strain, the effect of adding zinc salt to the reaction solution and the effect of adding zinc salt to the culture solution are shown (see Patent Document 5). Furthermore, the effect of adding zinc, magnesium, and manganese metal salts to the reaction solution is shown for this strain (see Non-Patent Document 1).
一方、上述のような、アミダーゼによる加水分解反応時の反応促進効果を目的とした金属塩の添加ではく、アミダーゼの保存性や活性の向上を目的として金属塩を利用する方法として、各種亜鉛塩を用いる方法が知られている（特許文献６参照）。 On the other hand, as a method of using a metal salt for the purpose of improving the preservability and activity of amidase instead of adding a metal salt for the purpose of promoting the reaction during the hydrolysis reaction with amidase as described above, various zinc salts can be used. A method using this is known (see Patent Document 6).
このような状況下において、アミダーゼの保存性や活性の向上及び失活防止をより効果的に実現することができる新たな金属塩の利用が求められていた。 Under such circumstances, there has been a demand for the use of a new metal salt that can more effectively realize storage stability and activity of amidase and prevent deactivation.
本発明者は、上記課題を解決するため鋭意研究を行った結果、ニッケル、コバルト若しくはマンガン又はそれらの塩を酵素液に添加する又は添加して加熱処理すると、効果的にアミダーゼを保存し、活性を賦活し、失活を予防し得ること見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventor effectively preserves amidase and adds activity when nickel, cobalt, manganese or a salt thereof is added to an enzyme solution or heat treatment is performed. Has been found to be capable of preventing deactivation and has led to the completion of the present invention.
（１）ニッケル、コバルト及びマンガン並びにそれらの塩からなる群より選ばれる少なくとも１種を含む、アミダーゼの保存剤、活性賦活剤又は失活予防剤。
（２）ニッケル、コバルト及びマンガン並びにそれらの塩からなる群より選ばれる少なくとも１種を含む、アミダーゼ保存用、アミダーゼ活性賦活用又はアミダーゼ失活予防用キット。
（３）アミダーゼを、ニッケル、コバルト及びマンガン並びにそれらの塩からなる群より選ばれる少なくとも１種を用いて処理することを含む、アミダーゼの保存方法、活性賦活方法又は失活予防方法。
上記(3)の方法は、例えば、前記処理に際し保温又は加熱することを含んでいてもよい。
(1) An amidase preservative, activity activator or deactivation preventive comprising at least one selected from the group consisting of nickel, cobalt and manganese and salts thereof.
(2) A kit for preserving amidase, utilizing amidase activity or preventing amidase inactivation, comprising at least one selected from the group consisting of nickel, cobalt and manganese and salts thereof.
(3) A method for preserving amidase, a method for activating activation, or a method for preventing deactivation, comprising treating amidase with at least one selected from the group consisting of nickel, cobalt and manganese and salts thereof.
The method (3) may include, for example, heat retention or heating during the treatment.
本発明により、ニッケル、コバルト若しくはマンガン又はそれらの塩を含む、アミダーゼの保存剤、活性賦活剤及び失活防止剤や、アミダーゼの保存用キット、活性賦活用キット及び失活防止用キット、並びにそれらの使用方法を提供することができる。
本発明の保存剤等は、アミダーゼ活性の低下を抑制して、アミダーゼ酵素液を安定的に保存し、酵素活性を賦活し、使用することができる。従って、本発明の保存剤等は極めて有用なものである。
According to the present invention, amidase preservatives, activity activators and deactivation inhibitors, amidase preservation kits, activity activation kits and deactivation prevention kits, and nickel, cobalt or manganese, or salts thereof, and the like How to use can be provided.
The preservative and the like of the present invention can be used by suppressing the decrease in amidase activity, stably storing the amidase enzyme solution, activating the enzyme activity. Therefore, the preservative and the like of the present invention are extremely useful.
以下、本発明を詳細に説明する。本発明の範囲はこれらの説明に拘束されることはなく、以下の例示以外についても、本発明の趣旨を損なわない範囲で適宜変更し実施することができる。なお、本明細書において引用された全ての刊行物、例えば先行技術文献、及び公開公報、特許公報その他の特許文献は、参照として本明細書に組み込まれる。 Hereinafter, the present invention will be described in detail. The scope of the present invention is not limited to these descriptions, and other than the following examples, the scope of the present invention can be appropriately changed and implemented without departing from the spirit of the present invention. It should be noted that all publications cited in the present specification, for example, prior art documents, and publications, patent publications, and other patent documents are incorporated herein by reference.
本発明は、ニッケル、コバルト若しくはマンガン又はそれらの塩を含む、アミダーゼの保存剤、活性賦活剤及び失活予防剤、アミダーゼの保存用キット、活性賦活用キット及び失活予防用キット、並びにそれらの使用方法である。
アミダーゼ酵素液に、ニッケル、コバルト若しくはマンガン又はそれらの塩を添加する、あるいは添加して熱処理する（ニッケル、コバルト若しくはマンガン又はそれらの塩の存在下で熱処理する）ことにより、それらを添加しない又は添加しないで熱処理した場合に比べて、活性が数倍から数十倍も向上することが示された。本発明は、このような知見に基づいて完成されたものである。
The present invention relates to a preservative for amidase, an activity activator and a deactivation preventive agent, a kit for preserving amidase, a kit for activation utilization and a kit for deactivation prevention, including nickel, cobalt, manganese or a salt thereof, and How to use.
Do not add or add nickel, cobalt or manganese or their salts to amidase enzyme solution, or add and heat-treat them (heat treatment in the presence of nickel, cobalt or manganese or their salts) It was shown that the activity was improved several times to several tens of times compared with the case where the heat treatment was performed without the heat treatment. The present invention has been completed based on such findings.
１．アミダーゼの調製
本発明において、アミダーゼは、好熱菌由来、常温菌由来、好冷菌由来及び低温菌由来のいずれのものでもよいが、中でも好熱菌由来のものが好ましい。
ここで、「好熱菌」とは、一般には５５℃以上で生育可能な細菌をいう（極限環境微生物ハンドブック、大島泰郎監修、株式会社サイエンスフォーラム）。好熱菌の例としては、限定されるわけではないが、サーマス（Thermus）属に属する細菌等（例えば、Thermus sp.、Thermus thermophilus、Thermus aquaticus等）を挙げることができる。
また、一般に「好冷菌」とは最適生育温度が１５℃以下であって生育限界温度が２０℃前後の菌を、「低温菌」とは最適生育温度が１５℃以下であって生育限界温度が２０℃以上のものをいう。
「常温菌」とは、上記好熱菌、好冷菌及び低温菌の定義に当てはまらないものを言い、従って、最適生育温度が１５〜４５℃であって５５℃以上で生育不可能な細菌を指す。現在単離されている微生物の多くは常温菌であり、常温菌の例としては、限定されるわけではないが、エンテロバクター（Enterobacter属）に属する細菌、エッシェリヒア（Escherichia）属に属する細菌、クレブシエラ（Krebsiella）属に属する細菌、オクロバクトラム（Ochrobactrum）属に属する細菌、ロドコッカス（Rhodococcus）属に属する細菌、マイコバクテリウム（Mycobacterium）属に属する細菌等を挙げることができる。
1. Preparation of amidase In the present invention, the amidase may be derived from a thermophilic bacterium, a thermophilic bacterium, a psychrophilic bacterium, or a psychrophilic bacterium.
Here, “thermophilic bacteria” generally refers to bacteria that can grow at 55 ° C. or higher (Extreme Environmental Microorganism Handbook, supervised by Yasuo Oshima, Science Forum Inc.). Examples of thermophilic bacteria include, but are not limited to, bacteria belonging to the genus Thermus (for example, Thermus sp., Thermus thermophilus, Thermus aquaticus, etc.).
Further, in general, “thermophilic bacteria” means bacteria having an optimum growth temperature of 15 ° C. or less and a growth limit temperature of around 20 ° C., and “pyrophilic bacteria” means an optimum growth temperature of 15 ° C. or less and a growth limit temperature. Means 20 ° C. or higher.
The term “thermophilic bacteria” refers to those that do not fall within the above definition of thermophilic bacteria, psychrophilic bacteria, and thermophilic bacteria. Therefore, bacteria having an optimum growth temperature of 15 to 45 ° C. and unable to grow at 55 ° C. or more are referred to. Point to. Many of the microorganisms currently isolated are psychrophilic bacteria. Examples of psychrophilic bacteria include, but are not limited to, bacteria belonging to the genus Enterobacter, bacteria belonging to the genus Escherichia, and Klebsiella. Examples include bacteria belonging to the genus (Krebsiella), bacteria belonging to the genus Ochrobactrum, bacteria belonging to the genus Rhodococcus, bacteria belonging to the genus Mycobacterium, and the like.
本発明において、アミダーゼは、例えば、配列番号２で示されるアミノ酸配列を含むタンパク質であって、かつアミダーゼ活性を有するタンパク質を含む。 In the present invention, an amidase includes, for example, a protein comprising the amino acid sequence represented by SEQ ID NO: 2 and having amidase activity.
配列番号２で示されるアミノ酸配列からなるアミダーゼは、サーマス・エスピー（Thermus sp.）株由来のアミダーゼであり、配列番号１で示されるアミダーゼ遺伝子の塩基配列によりコードされる。 The amidase having the amino acid sequence represented by SEQ ID NO: 2 is an amidase derived from the Thermus sp. Strain, and is encoded by the base sequence of the amidase gene represented by SEQ ID NO: 1.
また、本発明で用いられるアミダーゼには、配列番号２で示されるアミノ酸配列と６５％以上、好ましくは７０％以上、より好ましくは７５％以上、さらに好ましくは８５％以上、最も好ましくは９０％、９５％、９７％、９８％又は９９％の相同性（同一性）を有するアミノ酸配列を含み、かつアミダーゼ活性を有するタンパク質も含まれる。 The amidase used in the present invention is 65% or more, preferably 70% or more, more preferably 75% or more, still more preferably 85% or more, most preferably 90%, with the amino acid sequence represented by SEQ ID NO: 2. Also included are proteins that contain amino acid sequences with 95%, 97%, 98% or 99% homology (identity) and have amidase activity.
さらに、本発明におけるアミダーゼには、配列番号２で示されるアミノ酸配列において、１又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列を含み、かつ、アミダーゼ活性を有するタンパク質も含まれる。
配列番号２で示されるアミノ酸配列において、１個又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列としては、例えば、(i) 配列番号２で示されるアミノ酸配列において、１〜１０個（例えば、１〜５個、好ましくは１〜３個、さらに好ましくは１〜２個）のアミノ酸が欠失したアミノ酸配列、（ii）配列番号２で示されるアミノ酸配列の１〜１０個（例えば、１〜５個、好ましくは１〜３個、さらに好ましくは１〜２個）のアミノ酸が他のアミノ酸で置換されたアミノ酸配列、（iii）配列番号２で示されるアミノ酸配列に１〜１０個（例えば、１〜５個、好ましくは１〜３個、さらに好ましくは１〜２個）のアミノ酸が付加したアミノ酸配列、（iv）上記（i）〜（iii）を組み合わせたアミノ酸配列が挙げられる。
Furthermore, the amidase in the present invention includes an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 2, and a protein having amidase activity.
Examples of the amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 include (i) 1-10 amino acids in the amino acid sequence shown in SEQ ID NO: 2 (For example, 1 to 5, preferably 1 to 3, more preferably 1 to 2) amino acid sequences from which amino acids have been deleted, (ii) 1 to 10 amino acid sequences represented by SEQ ID NO: 2 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2 amino acid sequences substituted with other amino acids, (iii) 1 to 10 amino acid sequences represented by SEQ ID NO: 2 (For example, an amino acid sequence to which 1 to 5, preferably 1 to 3, more preferably 1 to 2 amino acids are added), and (iv) an amino acid sequence in which the above (i) to (iii) are combined. .
本発明で用いられるアミダーゼは、酸アミド基を加水分解してカルボン酸とアミン又はアンモニアを生ずる反応（ＲＣＯＮＨＲ’＋Ｈ2Ｏ→ＲＣＯＯＨ＋ＮＨ2Ｒ’）を触媒する加水分解酵素であり、好ましくはα−アミノ酸アミド又はα−ヒドロキシ酸アミドを立体選択的に加水分解する活性を有する酵素である。したがって、本発明において、「アミダーゼ活性」とは、酸アミド基を加水分解してカルボン酸とアミン又はアンモニアとを生ずる反応を触媒する活性、好ましくはα−アミノ酸アミド又はα−ヒドロキシ酸アミドを立体選択的に加水分解して、Ｌ−α−アミノ酸又はＬ−α−ヒドロキシ酸と、アンモニアとを生ずる反応を触媒する活性を意味する。アミダーゼ活性の測定方法については、後述する。 The amidase used in the present invention is a hydrolase that catalyzes a reaction (RCONHR ′ + H 2 O → RCOOH + NH 2 R ′) that hydrolyzes an acid amide group to generate a carboxylic acid and an amine or ammonia, preferably α− It is an enzyme having an activity of stereoselectively hydrolyzing amino acid amide or α-hydroxy acid amide. Therefore, in the present invention, “amidase activity” means an activity that catalyzes a reaction that hydrolyzes an acid amide group to produce a carboxylic acid and an amine or ammonia, preferably an α-amino acid amide or an α-hydroxy acid amide. It means the activity of catalyzing the reaction that selectively hydrolyzes to produce L-α-amino acid or L-α-hydroxy acid and ammonia. A method for measuring amidase activity will be described later.
また、本発明で用いられるアミダーゼは、形質転換体、形質転換体懸濁液、形質転換体破砕物、粗精製アミダーゼ酵素液又はアミダーゼ酵素液を７０℃で１時間処理した後も、処理前の活性と比較して、例えば、７０％、好ましくは少なくとも８０％、より好ましくは少なくとも９０％、さらに好ましくは少なくとも９５％の活性を保持していることを特徴とする。 In addition, the amidase used in the present invention can be obtained by treating a transformant, a transformant suspension, a transformant crushed material, a crude purified amidase enzyme solution or an amidase enzyme solution at 70 ° C. for 1 hour. Compared to activity, for example, it is characterized by retaining 70%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 95% activity.
本発明のアミダーゼ遺伝子は、本発明で用いられるアミダーゼをコードする遺伝子である。本発明のアミダーゼ遺伝子は、例えば、配列番号１で示される塩基配列からなる遺伝子が挙げられるが、これに限定されるものではない。 The amidase gene of the present invention is a gene encoding the amidase used in the present invention. Examples of the amidase gene of the present invention include, but are not limited to, a gene consisting of the base sequence represented by SEQ ID NO: 1.
本発明のアミダーゼ遺伝子には、配列番号１で示される塩基配列に相補的な塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNAであって、かつアミダーゼ活性を有するタンパク質をコードする遺伝子も含まれる。
このような遺伝子の例として、配列番号１で示される塩基配列と６５％以上、好ましくは７０％以上、より好ましくは７５％以上、さらに好ましくは８５％以上、最も好ましくは９０％、９５％、９７％、９８％又は９９％の相同性（同一性）を有する塩基配列を含み、かつアミダーゼ活性を有するタンパク質をコードする遺伝子が挙げられる。
The amidase gene of the present invention includes a DNA that hybridizes under stringent conditions with a DNA consisting of a base sequence complementary to the base sequence represented by SEQ ID NO: 1 and encodes a protein having amidase activity Is also included.
Examples of such a gene include 65% or more, preferably 70% or more, more preferably 75% or more, still more preferably 85% or more, most preferably 90%, 95%, the nucleotide sequence represented by SEQ ID NO: 1. Examples thereof include genes encoding a protein having a base sequence having 97%, 98% or 99% homology (identity) and having amidase activity.
上記遺伝子は、配列番号１で示される塩基配列からなるDNAの一部をプライマーとして、公知の核酸増幅方法又はプライマーウォーキング法により取得することができる。また、本発明のアミダーゼ遺伝子は、配列番号１で示される塩基配列からなるDNAの一部をプローブとして、コロニーハイブリダーゼーション、プラークハイブリダイゼーション、サザンブロットなどの公知のハイブリダイゼーション法により取得することもできる。
上記方法には、アミダーゼ遺伝子を含むベクター、mRNA、total RNA、cDNA、ゲノムDNA、又はそれらのライブラリーを用いることができる。また、市販のライブラリーを使用しても良い。
本明細書において、ストリンジェントな条件は、ハイブリダイゼーション後の洗浄条件として、例えば、「2×SSC、0.1％SDS、42℃」、「1×SSC、0.1％SDS、37℃」、よりストリンジェントな条件としては、例えば、「1×SSC、0.1％SDS、65℃」、「0.5×SSC、0.1％SDS、50℃」等の条件を挙げることができる。
ハイブリダイゼーションは、公知の方法によって行うことができる。ハイブリダイゼーションの方法は、例えば、「Molecular Cloning, A Laboratory Manual 2nd ed.」（Cold Spring Harbor Laboratory Press（1989））、「Current Protocols in Molecular Biology」（John Wiley & Sons（1987-1997））等を参照することができる。
The gene can be obtained by a known nucleic acid amplification method or primer walking method using a part of the DNA consisting of the base sequence represented by SEQ ID NO: 1 as a primer. The amidase gene of the present invention can also be obtained by a known hybridization method such as colony hybridization, plaque hybridization, Southern blotting, etc. using a part of the DNA consisting of the base sequence represented by SEQ ID NO: 1 as a probe. it can.
In the above method, a vector containing amidase gene, mRNA, total RNA, cDNA, genomic DNA, or a library thereof can be used. A commercially available library may be used.
In the present specification, stringent conditions include, for example, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.”, and more stringent as washing conditions after hybridization. Examples of such conditions include “1 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 50 ° C.”, and the like.
Hybridization can be performed by a known method. Hybridization methods include, for example, “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Laboratory Press (1989)), “Current Protocols in Molecular Biology” (John Wiley & Sons (1987-1997)), etc. You can refer to it.
さらに、本発明のアミダーゼ遺伝子には、配列番号１で示される塩基配列において、１又は数個の塩基配列が欠失、置換又は付加された塩基配列を含み、かつ、アミダーゼ活性を有するタンパク質をコードする遺伝子も含まれる。
配列番号１で示される塩基配列において、１個又は数個の塩基が欠失、置換又は付加された塩基配列としては、例えば、(i) 配列番号１で示される塩基配列において、１〜１０個（例えば、１〜５個、好ましくは１〜３個、さらに好ましくは１〜２個）の塩基が欠失した塩基配列、（ii）配列番号１で示される塩基配列の１〜１０個（例えば、１〜５個、好ましくは１〜３個、さらに好ましくは１〜２個）の塩基が他の塩基で置換された塩基配列、（iii）配列番号１で示される塩基配列に１〜１０個（例えば、１〜５個、好ましくは１〜３個、さらに好ましくは１〜２個）の塩基が付加した塩基配列、（iv）上記（i）〜（iii）を組み合わせた塩基配列が挙げられる。
Furthermore, the amidase gene of the present invention includes a base sequence in which one or several base sequences are deleted, substituted or added in the base sequence represented by SEQ ID NO: 1, and encodes a protein having amidase activity. Gene to be included.
Examples of the base sequence in which one or several bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 1 include (i) 1 to 10 in the base sequence shown in SEQ ID NO: 1. (For example, 1 to 5, preferably 1 to 3, more preferably 1 to 2) base sequences from which bases have been deleted, (ii) 1 to 10 base sequences represented by SEQ ID NO: 1 (for example, 1 to 5, preferably 1 to 3, more preferably 1 to 2) base sequence in which bases are substituted with other bases, and (iii) 1 to 10 base sequences represented by SEQ ID NO: 1. (For example, 1 to 5, preferably 1 to 3, more preferably 1 to 2) a base sequence added with a base, (iv) a base sequence combining (i) to (iii) above. .
配列番号１で示される塩基配列において１又は数個の核酸に欠失、置換又は付加などの変異の生じた塩基配列を含むDNAは、「Molecular Cloning, A Laboratory Manual 2nd ed.」(Cold Spring Harbor Press (1989))、「Current Protocols in Molecular Biology」(John Wiley & Sons (1987-1997))、Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92、Kunkel (1988) Method. Enzymol. 85: 2763-6等に記載の部位特異的変異誘発法等の方法に従って調製することができる。 A DNA containing a nucleotide sequence in which mutation such as deletion, substitution or addition has occurred in one or several nucleic acids in the nucleotide sequence shown in SEQ ID NO: 1 is “Molecular Cloning, A Laboratory Manual 2nd ed.” (Cold Spring Harbor Press (1989)), `` Current Protocols in Molecular Biology '' (John Wiley & Sons (1987-1997)), Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-92, Kunkel (1988) Method. Enzymol. 85: 2763-6 etc. and can be prepared according to a method such as site-directed mutagenesis.
また、DNAに変異を導入するには、Kunkel法やGapped duplex法等の公知手法により、部位特異的突然変異誘発法を利用した変異導入用キット、例えばQuikChangeTM Site-Directed Mutagenesis Kit（ストラタジーン社製）、GeneTailorTM Site-Directed Mutagenesis System（インビトロジェン社製）、TaKaRa Site-Directed Mutagenesis System（Mutan-K、Mutan-Super Express Km等：タカラバイオ社製）等を用いて行うことができる。 In order to introduce mutations into DNA, mutation introduction kits using site-directed mutagenesis methods such as QuikChange TM Site-Directed Mutagenesis Kit (Stratagene) are known by known techniques such as Kunkel method and Gapped duplex method. Manufactured), GeneTailor ™ Site-Directed Mutagenesis System (manufactured by Invitrogen), TaKaRa Site-Directed Mutagenesis System (Mutan-K, Mutan-Super Express Km, etc .: manufactured by Takara Bio Inc.) and the like.
本発明のアミダーゼ遺伝子の塩基配列の確認は、慣用の方法により配列決定することにより行うことができる。例えば、ジデオキシヌクレオチドチェーンターミネーション法（Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463）等により行うことができる。また、適当なDNAシークエンサーを利用して配列を解析することもできる。 The base sequence of the amidase gene of the present invention can be confirmed by sequencing by a conventional method. For example, the dideoxynucleotide chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463) can be used. The sequence can also be analyzed using an appropriate DNA sequencer.
上述のアミダーゼを発現させるための宿主−ベクター系の選択は下記のようにして行われる。 Selection of a host-vector system for expressing the amidase described above is performed as follows.
宿主に導入するベクターとしては、上記の酵素遺伝子を保持し、且つ複製可能であれば、特段の制約を受けるものではなく、それぞれの宿主に適したベクターを使用することができる。例えば、プラスミドDNA、バクテリオファージ等が挙げられる。
プラスミドDNAとしては、例えば、大腸菌由来のプラスミド（pBR322、pUC18、pUC19、pUC118、pUC119、pBluescriptなどのColE系プラスミド等）、放線菌由来のプラスミド（pIJ486等）、酵母由来のプラスミド（YEp13、YEp 24、Ycp50等）が挙げられる。ファージDNAとしては、λファージ（Charon4A、Charon21A、EMBL3、EMBL4、λgt10、λgt11等）、レトロトランスポゾンDNA、人工染色体DNA等が挙げられる。
The vector to be introduced into the host is not particularly limited as long as it retains the above enzyme gene and can be replicated, and a vector suitable for each host can be used. For example, plasmid DNA, bacteriophage, etc. are mentioned.
Examples of plasmid DNA include Escherichia coli-derived plasmids (ColE-based plasmids such as pBR322, pUC18, pUC19, pUC118, pUC119, and pBluescript), actinomycete-derived plasmids (pIJ486, etc.), and yeast-derived plasmids (YEp13, YEp 24 , Ycp50, etc.). Examples of the phage DNA include λ phage (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, etc.), retrotransposon DNA, artificial chromosome DNA, and the like.
宿主としては、上記の酵素遺伝子が発現するものであれば良い。例えば、哺乳動物細胞、昆虫細胞、大腸菌、枯草菌、酵母、カビ、ロドコッカス属細菌、植物等を挙げることができる。好ましくは、大腸菌及びロドコッカス属細菌である。
大腸菌宿主としては、例えば大腸菌Ｋ１２株やＢ株、あるいはそれら野生株由来の派生株であるJM109株、XL1-Blue株、C600株、W3110株等を挙げることができる。その他、これら菌株の変異体、組換え体及び遺伝子工学的手法による誘導体等も用いられ得る。
酵母宿主としては、例えばサッカロミセス・セレビシエ（Saccharomyces cerevisiae）、シゾサッカロミセス・ポンベ（Schizosaccharomyces pombe）、ピヒア・パストリス（Pichia pastoris）等を挙げることができる。
ロドコッカス属細菌としては、例えばロドコッカス・ロドクロウス（Rhodococcus rhodochrous）ATCC 12674株やロドコッカス・ロドクロウス（Rhodococcus rhodochrous）J-1株（FERM BP-1478）等を挙げることができる。
Any host that expresses the above-described enzyme gene may be used. For example, mammalian cells, insect cells, Escherichia coli, Bacillus subtilis, yeasts, molds, Rhodococcus bacteria, plants and the like can be mentioned. Preferred are Escherichia coli and Rhodococcus bacteria.
Examples of the E. coli host include E. coli K12 strain and B strain, or JM109 strain, XL1-Blue strain, C600 strain, W3110 strain and the like derived from these wild strains. In addition, mutants of these strains, recombinants, derivatives by genetic engineering techniques, and the like can also be used.
Examples of yeast hosts include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
Examples of the genus Rhodococcus include Rhodococcus rhodochrous ATCC 12674 strain, Rhodococcus rhodochrous J-1 strain (FERM BP-1478), and the like.
以下、より詳細にアミダーゼ遺伝子が導入された形質転換体（あるいは形質導入体）（以下、単に「形質転換体」と称する）の作製方法について説明する。 Hereinafter, a method for producing a transformant (or a transductant) into which an amidase gene has been introduced (hereinafter simply referred to as “transformant”) will be described in more detail.
プラスミドDNAは、宿主細胞中にてプラスミドが増殖するために必要なDNA配列、プロモーター、リボソーム結合配列、転写ターミネーター（転写終結配列）、さらに好ましくは形質転換体の選択マーカーとなる遺伝子を含む。
プロモーター配列としては、大腸菌由来のトリプトファンオペロンのtrpプロモーター、ラクトースオペロンのlacプロモーター、ラムダファージ由来のPLプロモーター及びPRプロモーター、枯草菌由来のグルコン酸合成酵素プロモーター（gnt）、アルカリプロテアーゼプロモーター（apr）、中性プロテアーゼプロモーター（npr）、α−アミラーゼプロモーター（amy）等を挙げることができる。また、tacプロモーター、trcプロモーターのように独自に改変、設計された配列も利用できる。
リボソーム結合配列としては、SD配列やKozak配列が知られており、これらの配列を変異遺伝子の上流に挿入することができる。原核生物を宿主に用いるときにはSD配列を、真核細胞を宿主に用いるときにはKozak配列をPCR法等により付加してもよい。SD配列としては、大腸菌由来、ロドコッカス属細菌又は枯草菌由来の配列等を挙げることができるが、所望の宿主内で機能する配列であれば特に限定されるものではない。例えば、16SリボゾームRNAの３’末端領域に相補的な配列が４塩基以上連続したコンセンサス配列をDNA合成により作製してこれを利用してもよい。
転写終結配列は必ずしも必要ではないが、ρ因子非依存性のもの、例えばリポプロテインターミネーター、trpオペロンターミネーター等が利用できる。
選択マーカーとしては、例えばジヒドロ葉酸還元酵素遺伝子、アンピシリン耐性遺伝子、ネオマイシン耐性遺伝子等を挙げることができる。
The plasmid DNA includes a DNA sequence necessary for the plasmid to grow in the host cell, a promoter, a ribosome binding sequence, a transcription terminator (transcription termination sequence), and more preferably a gene that serves as a selection marker for the transformant.
As the promoter sequence, trp promoter of tryptophan operon derived from E. coli, lac promoter of lactose operon, PL promoter and PR promoter derived from lambda phage, gluconate synthase promoter (gnt) derived from Bacillus subtilis, alkaline protease promoter (apr), Examples include neutral protease promoter (npr) and α-amylase promoter (amy). In addition, sequences uniquely modified and designed such as tac promoter and trc promoter can also be used.
SD and Kozak sequences are known as ribosome binding sequences, and these sequences can be inserted upstream of the mutated gene. The SD sequence may be added by PCR or the like when a prokaryotic organism is used as the host, and the Kozak sequence may be added when eukaryotic cells are used as the host. Examples of the SD sequence include sequences derived from Escherichia coli, Rhodococcus spp. Or Bacillus subtilis, but are not particularly limited as long as the sequence functions in a desired host. For example, a consensus sequence in which a sequence complementary to the 3 ′ terminal region of 16S ribosomal RNA is continuous by 4 bases or more may be prepared by DNA synthesis and used.
A transcription termination sequence is not always necessary, but a ρ-factor-independent one such as a lipoprotein terminator, a trp operon terminator, etc. can be used.
Examples of selectable markers include dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like.
大腸菌を宿主に用いた場合には、特に有用なベクターとしては、pTrc99A、pKK233-2、pFY529、pET-12、pET-26b等が例示される。
これらのベクターにアミダーゼをコードする遺伝子断片を組み込むには、アミダーゼ遺伝子を含むDNAを適当な制限酵素で切断し、必要であれば適当なリンカーを付加した後、適当な制限酵素で切断したベクターと結合させることにより行うことができる。
When Escherichia coli is used as a host, examples of particularly useful vectors include pTrc99A, pKK233-2, pFY529, pET-12, and pET-26b.
In order to incorporate a gene fragment encoding amidase into these vectors, the DNA containing the amidase gene is cleaved with an appropriate restriction enzyme, and if necessary, an appropriate linker is added, and then a vector cleaved with an appropriate restriction enzyme is used. This can be done by bonding.
このように作製した発現ベクターを宿主細胞に導入すれば、アミダーゼを高度に発現する形質転換体が得られる。当該形質転換体を培養することにより、これらの酵素を発現させることができる。 When the expression vector thus prepared is introduced into a host cell, a transformant that highly expresses amidase can be obtained. These enzymes can be expressed by culturing the transformant.
発現ベクターの宿主への導入方法としては、DNAを導入する方法であれば特に限定されるものではない。例えばカルシウムイオンを用いる方法、エレクトロポレーション法等を挙げることができる。
酵母への発現プラスミドの導入方法としては、酵母にDNAを導入する方法であれば特に限定されず、例えばエレクトロポレーション法、スフェロプラスト法、酢酸リチウム法等を挙げることができる。
The method for introducing the expression vector into the host is not particularly limited as long as it is a method for introducing DNA. For example, a method using calcium ions, an electroporation method, and the like can be given.
The method for introducing an expression plasmid into yeast is not particularly limited as long as it is a method for introducing DNA into yeast. Examples thereof include an electroporation method, a spheroplast method, and a lithium acetate method.
次に、上記のように作製した形質転換体を培養する方法を説明する。培養に際し使用する培地には、特に制限は無く、宿主菌が資化し得る炭素源、窒素源、無機塩類等を含有し、形質転換体の培養を効率的に行うことができる培地であれば、天然培地、合成培地のいずれを用いてもよい。また培養条件に関しても、形質転換体が生育、増殖可能で且つ酵素産生が良好に行える条件を選択し、培養すればよい。
培養培地に用いる炭素源としては、グルコース、ガラクトース、フラクトース、スクロース、ラフィノース、デンプン等の炭水化物、酢酸、プロピオン酸等の有機酸、エタノール、プロパノール、グリセリン等のアルコール類を挙げることができる。窒素源としては、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム、リン酸アンモニウム等の無機酸若しくは有機酸のアンモニウム塩又はその他の含窒素化合物を挙げることができる。その他、ペプトン、肉エキス、コーンスティープリカー、酵母エキス、各種アミノ酸等を用いてもよい。無機物としては、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅、炭酸カルシウム、硫酸亜鉛等を挙げることができる。その他、ビタミン等を必要に応じて適宜添加してもよい。培養中は必要に応じてアンピシリンやテトラサイクリン等の抗生物質を培地に添加してもよい。
Next, a method for culturing the transformant prepared as described above will be described. The medium used for the culture is not particularly limited, and contains a carbon source, a nitrogen source, inorganic salts, and the like that can be assimilated by the host fungus, so long as the medium can efficiently culture the transformant. Either a natural medium or a synthetic medium may be used. In addition, regarding the culture conditions, it is sufficient to select and culture conditions under which the transformant can grow and proliferate and can produce the enzyme satisfactorily.
Examples of the carbon source used in the culture medium include carbohydrates such as glucose, galactose, fructose, sucrose, raffinose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol, propanol and glycerin. Examples of the nitrogen source include ammonia, ammonium salts of inorganic acids or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, and other nitrogen-containing compounds. In addition, peptone, meat extract, corn steep liquor, yeast extract, various amino acids, and the like may be used. Examples of the inorganic substance include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and zinc sulfate. In addition, vitamins and the like may be added as necessary. During culture, an antibiotic such as ampicillin or tetracycline may be added to the medium as necessary.
プロモーターとして誘導性のプロモーターを用いた発現プラスミドを導入した形質転換体を培養する場合は、必要に応じてインデューサーを培地に添加してもよい。例えば、イソプロピル−β−D−チオガラクトシド（IPTG）で誘導可能なプロモーターを有する発現プラスミドを導入した形質転換体を培養するときは、IPTG等を培地に添加することができる。また、インドール酢酸（IAA）で誘導可能なtrpプロモーターを用いた発現プラスミドを導入した形質転換体を培養するときは、IAA等を培地に添加することができる。 When cultivating a transformant introduced with an expression plasmid using an inducible promoter as a promoter, an inducer may be added to the medium as necessary. For example, when culturing a transformant into which an expression plasmid having a promoter inducible with isopropyl-β-D-thiogalactoside (IPTG) is cultured, IPTG or the like can be added to the medium. In addition, when cultivating a transformant into which an expression plasmid using a trp promoter inducible with indoleacetic acid (IAA) is introduced, IAA or the like can be added to the medium.
大腸菌の培養に際し、通常の固体培養法で培養してもよいが、液体培養法を採用して培養することが好ましい。培地の初発pHは7〜9に調整することが適当である。また、培養は、5℃〜40℃、好ましくは10℃〜37℃で5〜100時間行う。通気攪拌深部培養、振盪培養、静置培養、流加培養等により実施することが好ましい。
ロドコッカス属細菌の培養は、4〜36℃、好ましくは20〜30℃で18〜96時間行う。
When culturing Escherichia coli, the cells may be cultured by a normal solid culture method, but it is preferable to employ a liquid culture method. It is appropriate to adjust the initial pH of the medium to 7-9. The culture is performed at 5 ° C to 40 ° C, preferably 10 ° C to 37 ° C for 5 to 100 hours. It is preferable to carry out by aeration stirring deep culture, shaking culture, static culture, fed-batch culture, or the like.
Rhodococcus bacteria are cultured at 4-36 ° C., preferably 20-30 ° C., for 18-96 hours.
（破砕処理方法）
こうして得られた培養物から菌体や細胞を回収するには、遠心分離法や膜ろ過法を用いることができる。回収した形質転換体を、必要に応じて、リン酸−ナトリウム緩衝液、リン酸緩衝液等で洗浄し、懸濁することができる。
菌体又は細胞の破砕方法としては、超音波処理、フレンチプレスやホモジナイザーによる高圧処理、ガラスビーズなどによる磨砕処理、リゾチーム、セルラーゼ、ペクチナーゼ等を用いる酵素処理、凍結融解処理、低張液処理、ファージによる溶菌誘導処理等を利用することができる。破砕処理は、必要に応じて氷冷下で行う。破砕処理を行ったサンプルを「形質転換体破砕物」、「破砕物」又は「細胞抽出液全画分」とも称する。
破砕後、形質転換体破砕物から、必要に応じて菌体又は細胞の破砕残渣（細胞抽出液不溶性画分を含む）を除くことができる。残渣を除去する方法としては、例えば、遠心分離やろ過などが上げられ、必要に応じて、凝集剤やろ過助剤等を使用して残渣除去効率を上げることもできる。残渣を除去した後に得られた上清は、「細胞抽出液可溶性画分」であり、「粗精製したアミダーゼ溶液」とすることができる。
(Crushing method)
Centrifugation and membrane filtration can be used to recover the cells and cells from the culture thus obtained. The recovered transformant can be washed and suspended with a phosphate-sodium buffer solution, a phosphate buffer solution, or the like, if necessary.
As a method for disrupting cells or cells, ultrasonic treatment, high-pressure treatment using a French press or homogenizer, grinding treatment using glass beads, enzyme treatment using lysozyme, cellulase, pectinase, etc., freeze-thawing treatment, hypotonic solution treatment, It is possible to use a lysis inducing treatment with a phage or the like. The crushing process is performed under ice cooling as necessary. The sample subjected to the crushing treatment is also referred to as “transformed product disrupted product”, “crushed product”, or “cell extract whole fraction”.
After crushing, cells or cell crushing residues (including cell extract insoluble fractions) can be removed from the crushed transformant as necessary. As a method for removing the residue, for example, centrifugation or filtration can be used, and if necessary, the residue removal efficiency can be increased by using a flocculant or a filter aid. The supernatant obtained after removing the residue is a “cell extract soluble fraction” and can be a “crudely purified amidase solution”.
２．ニッケル、コバルト若しくはマンガン又はそれらの塩によるアミダーゼ液の処理
（１）ニッケル、コバルト若しくはマンガン又はそれらの塩とアミダーゼとの接触処理
本発明において、「接触」とは、ニッケル、コバルト若しくはマンガン又はそれらの塩とアミダーゼとを共存させること、すなわちニッケル、コバルト若しくはマンガン又はそれらの塩とアミダーゼとを同一の反応系に存在させることを意味し、例えば、アミダーゼ酵素液を含む容器にニッケル、コバルト若しくはマンガン又はそれらの塩を添加すること、ニッケル、コバルト若しくはマンガン又はそれらの塩を含む培養液中でアミダーゼ遺伝子を含む菌体（形質転換体）を培養すること、アミダーゼ遺伝子を含む菌体（形質転換体）又はその破砕物を含有する溶液中にニッケル、コバルト若しくはマンガン又はそれらの塩を添加することなどが含まれる。ニッケル塩、コバルト塩及びマンガン塩の濃度は、それぞれ、好ましくは0.01 mM〜30 mM、好ましくは0.1 mM〜15 mMであり、より好ましくは2 mM〜15 mMであり、さらに好ましくは5 mM〜12 mMである。
ニッケル塩、コバルト塩及びマンガン塩としては、特に限定はされないが、例えば、ニッケル、コバルト及びマンガン各々の、塩酸塩、硫酸塩、硝酸塩、水酸化物塩、炭酸塩及びリン酸塩等が好ましく挙げられる。
このように、アミダーゼと、ニッケル、コバルト若しくはマンガン又はそれらの塩とを接触させることにより、アミダーゼの活性を増加（賦活化）させることができる。このことは、アミダーゼの活性低下（失活）を防止することを意味するものであり、これによりアミダーゼを有効に保存することができる。
なお、本発明において用いられる、ニッケル、コバルト若しくはマンガン又はそれらの塩は、１種のみには限定されず、２種以上を併用してもよく、限定はされない。すなわち、本発明において、アミダーゼと接触させるものとしては、ニッケル、コバルト及びマンガン並びにそれらの塩からなる群より選ばれる少なくとも１種が用いられる。
2. Treatment of amidase solution with nickel, cobalt or manganese or salts thereof (1) Contact treatment of nickel, cobalt or manganese or salts thereof with amidase In the present invention, “contact” means nickel, cobalt or manganese or their It means that a salt and amidase coexist, that is, nickel, cobalt or manganese or their salt and amidase are present in the same reaction system, for example, nickel, cobalt or manganese or a container containing an amidase enzyme solution Adding those salts, culturing cells (transformants) containing the amidase gene in a culture solution containing nickel, cobalt or manganese or salts thereof, cells containing the amidase gene (transformants) Or in a solution containing the crushed material For example, adding Kell, cobalt or manganese or a salt thereof. The concentration of nickel salt, cobalt salt and manganese salt is preferably 0.01 mM to 30 mM, preferably 0.1 mM to 15 mM, more preferably 2 mM to 15 mM, and further preferably 5 mM to 12 mM. mM.
Although it does not specifically limit as nickel salt, cobalt salt, and manganese salt, For example, the hydrochloride, sulfate, nitrate, hydroxide salt, carbonate, phosphate, etc. of nickel, cobalt, and manganese are mentioned preferably, respectively. It is done.
Thus, the amidase activity can be increased (activated) by bringing nickel, cobalt, manganese, or a salt thereof into contact with each other. This means that amidase activity reduction (deactivation) is prevented, whereby the amidase can be stored effectively.
In addition, nickel, cobalt, manganese, or those salts used in this invention are not limited to 1 type, You may use 2 or more types together, There is no limitation. That is, in the present invention, at least one selected from the group consisting of nickel, cobalt, manganese, and salts thereof is used as the amidase.
（２）活性の測定
アミダーゼ活性は、DL−α−アミノ酸アミドやDL−α−ヒドロキシ酸アミドを基質として一定時間反応させた後、生成するα−アミノ酸やα−ヒドロキシ酸を高速液体クロマトグラフィー等により定量し、その定量値を活性の指標として測定することができる。
(2) Activity measurement The amidase activity is determined by reacting the α-amino acid or α-hydroxy acid generated after reacting for a certain period of time using DL-α-amino acid amide or DL-α-hydroxy acid amide as a substrate. The quantitative value can be measured as an index of activity.
（３）加熱処理
本発明においては、アミダーゼを、ニッケル、コバルト若しくはマンガン又はそれらの塩で処理する際は、常温下（20±15℃、例えば25℃程度）でも保温下（30〜40℃、例えば37℃程度）でもよいし、加熱処理を行ってもよい。適度な保温又は加熱処理を行うことにより、高活性アミダーゼを得ることができる。ここで、加熱処理とは、アミダーゼと、ニッケル、コバルト若しくはマンガン又はそれらの塩との混合物を、60℃〜80℃、好ましくは65℃〜75℃の温度範囲内の環境下に曝すことを意味し、例えば、アミダーゼ溶液に上記塩を添加した後、上記温度条件で5〜360分、好ましくは30〜60分処理する態様が挙げられる。
アミダーゼを上記塩による処理、又は上記塩による処理と加熱処理とを施すことにより、アミダーゼを24時間〜300日、好ましくは10〜100日保存することができる。また、アミダーゼ活性は、上記処理を施さないアミダーゼと比較して、好ましくは110〜5,000％、の活性に上昇させることが可能となる。その結果、アミダーゼの失活予防効果をより高くすることができる。
(3) Heat treatment In the present invention, when amidase is treated with nickel, cobalt, manganese, or a salt thereof, it is kept warm (30-40 ° C, about 20 ° C, for example, about 25 ° C) even at room temperature. For example, about 37 ° C.) or heat treatment may be performed. A highly active amidase can be obtained by performing an appropriate heat retention or heat treatment. Here, the heat treatment means that a mixture of amidase and nickel, cobalt, manganese or a salt thereof is exposed to an environment within a temperature range of 60 ° C to 80 ° C, preferably 65 ° C to 75 ° C. For example, after adding the said salt to an amidase solution, the aspect processed for 5 to 360 minutes by the said temperature conditions, Preferably it is 30 to 60 minutes is mentioned.
The amidase can be stored for 24 hours to 300 days, preferably 10 to 100 days by treating the amidase with the salt, or the salt treatment and the heat treatment. In addition, the amidase activity can be increased to an activity of preferably 110 to 5,000% as compared with the amidase not subjected to the above treatment. As a result, the inactivation preventing effect of amidase can be further increased.
３．キット
本発明において、ニッケル、コバルト若しくはマンガン又はそれらの塩は、アミダーゼの保存用キット、アミダーゼの活性賦活用キットあるいはアミダーゼの失活予防用キットの構成成分として使用される。
本発明のキットは、上記ニッケル、コバルト若しくはマンガン又はそれらの塩に加えて、一般の測定において慣用的な成分、例えば緩衝液、生理食塩水、pH調整剤等を含んでいてもよい。また、本発明のキットは、アミダーゼを保存する方法及び／又はアミダーゼの活性の測定方法等を記載した取扱説明書などを含んでいてもよい。
3. Kit In the present invention, nickel, cobalt, manganese, or a salt thereof is used as a component of an amidase storage kit, an amidase activity utilization kit, or an amidase inactivation prevention kit.
The kit of the present invention may contain components commonly used in general measurement, such as a buffer solution, physiological saline, a pH adjuster, and the like, in addition to the above nickel, cobalt, manganese, or salts thereof. In addition, the kit of the present invention may include an instruction manual describing a method for preserving amidase and / or a method for measuring amidase activity.
以下に、実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されるものではない。
Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.
＜プラスミドpTA002の作製＞
（１）Thermus sp.属（Thermus sp.0-3-1株）由来耐熱性アミダーゼ遺伝子断片の調製
Thermus sp.0-3-1株由来のアミダーゼ遺伝子を含むプラスミドpM501KN（WO 03/020929参照）を鋳型として、下記の配列番号３に示される塩基配列からなる合成DNA（TAM-01）及び配列番号４に示される塩基配列からなる合成DNA（TAM-02）をプライマーとして用い、ポリメラーゼ伸長鎖反応（PCR）により、アミダーゼ遺伝子を含むDNA断片を増幅した。
<Preparation of plasmid pTA002>
(1) Preparation of thermostable amidase gene fragment from Thermus sp. Genus (Thermus sp. 0-3-1 strain)
Synthetic DNA (TAM-01) consisting of the base sequence shown in SEQ ID NO: 3 and SEQ ID NO: 3 using the plasmid pM501KN (see WO 03/020929) containing the amidase gene derived from Thermus sp. 0-3-1 strain as a template A DNA fragment containing the amidase gene was amplified by polymerase chain reaction (PCR) using the synthetic DNA (TAM-02) consisting of the base sequence shown in 4 as a primer.
プライマー：
TAM-01： GCGGATCCATGGAGGGCTACAGGACCATTC （配列番号３）
TAM-02： GGACCTGCAGGTCCATCAGGCGAAAATATC （配列番号４）
Primer:
TAM-01: GCGGATCCATGGAGGGCTACAGGACCATTC (SEQ ID NO: 3)
TAM-02: GGACCTGCAGGTCCATCAGGCGAAAATATC (SEQ ID NO: 4)
PCR用の反応混合物は、5μLの10×Pfu Turboバッファー、4μLのdNTP mix（2.5 mM each）、1μLのPfu Turbo DNAポリメラーゼ、37μLの蒸留水、1μLの各プライマー、および鋳型としてpLA205を1μL添加したものを用いた。PCRは、95℃で1分の変性を行った後、94℃で1分、60℃で1分、72℃で5分の反応を、計30サイクル行った。
反応終了後、1μLの反応終了液を1.5 %アガロースゲル電気泳動により分析し、増幅産物を確認した。増幅産物をGFX PCR DNA and Gel Band Purification Kit（GEヘルスケア バイオサイエンス社）を用いて精製し、約50μLのDNA溶液を得た。
次に、43μLのDNA溶液に、10×Kバッファー（タカラバイオ社）を5μL、制限酵素NcoIおよびSse8783Iを各1μL加え、37℃で2時間反応後、エタノール沈殿によりDNA断片を回収した。反応終了後1.5 %アガロースゲル電気泳動を行い、約0.93kbの目的バンド（アミダーゼ遺伝子断片）を切り出し、GFX PCR DNA and Gel Band Purification Kitを用いて精製した。
The reaction mixture for PCR added 5 μL of 10 × Pfu Turbo buffer, 4 μL of dNTP mix (2.5 mM each), 1 μL of Pfu Turbo DNA polymerase, 37 μL of distilled water, 1 μL of each primer, and 1 μL of pLA205 as a template. A thing was used. PCR was performed by denaturing at 95 ° C for 1 minute, followed by a total of 30 cycles of 94 ° C for 1 minute, 60 ° C for 1 minute, and 72 ° C for 5 minutes.
After completion of the reaction, 1 μL of the reaction completion solution was analyzed by 1.5% agarose gel electrophoresis to confirm the amplified product. The amplification product was purified using GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bioscience) to obtain about 50 μL of DNA solution.
Next, 5 μL of 10 × K buffer (Takara Bio Inc.), 1 μL each of restriction enzymes NcoI and Sse8783I were added to 43 μL of DNA solution, reacted at 37 ° C. for 2 hours, and then DNA fragments were collected by ethanol precipitation. After completion of the reaction, 1.5% agarose gel electrophoresis was performed, and an about 0.93 kb target band (amidase gene fragment) was excised and purified using GFX PCR DNA and Gel Band Purification Kit.
（２）ベクター断片の調製とライゲーション
一方、5μLのベクターpTrc99A（GEバイオサイエンス社）に10×Kバッファー（タカラバイオ社）を3μL、滅菌水を23μL、制限酵素NcoIおよびPstIを各1μL加え、37℃で2時間反応後、アルカリフォスファターゼ処理（Shrimp Alkaline Phosphatase、プロメガ株式会社）を行い、エタノール沈殿によりDNA断片を回収した。
NcoIとPstIとにより切断されたこれらのDNA断片、すなわち上記（１）で得られたアミダーゼ遺伝子断片および本項（２）で得られたベクターDNA断片を各2μLと、solution I（DNA Ligation Kit ver.2（タカラバイオ社））（10μL）とを混合して、ライゲーション混合物を調製した。該混合物を、16℃で2時間インキュベートすることでアミダーゼ遺伝子断片とベクターpTrc99Aを結合させた。
(2) Preparation and Ligation of Vector Fragment On the other hand, 3 μL of 10 × K buffer (Takara Bio), 23 μL of sterilized water, 1 μL each of restriction enzymes NcoI and PstI were added to 5 μL of vector pTrc99A (GE Bioscience), 37 After reacting at 0 ° C. for 2 hours, alkaline phosphatase treatment (Shrimp Alkaline Phosphatase, Promega Corporation) was performed, and DNA fragments were recovered by ethanol precipitation.
2 μL each of these DNA fragments cleaved by NcoI and PstI, that is, the amidase gene fragment obtained in (1) above and the vector DNA fragment obtained in (2) above, and solution I (DNA Ligation Kit ver. .2 (Takara Bio Inc.)) (10 μL) was mixed to prepare a ligation mixture. The mixture was incubated at 16 ° C. for 2 hours to bind the amidase gene fragment and the vector pTrc99A.
（３）大腸菌 JM109株のコンピテントセルの作製
大腸菌JM109株をLB培地(1%バクトトリプトン、0.5%バクトイーストエキス、0.5% NaCl) 1 mLに接種し37℃、5時間好気的に前培養した。得られた前培養液 0.4 mLをSOB培地 40 mL(2%バクトトリプトン、0.5%バクトイーストエキス、10mM NaCl 、2.5mM KCl 、1mM MgSO4 、1mM MgCl2) に加え、18℃で20時間培養した。当該培養物を遠心分離（3,700 G、10分間、4℃）により集菌した後、冷TF溶液 (20 mM PIPES−KOH (pH 6.0)、200 mM KCl 、10 mM CaCl2 、40mM MnCl2)を13 mL加え、0℃で10分間放置した。その後、再度遠心分離（3,700 G、10分間、4℃）し、上清を除いた。沈殿した大腸菌を冷TF溶液 3.2 mLに懸濁し、0.22 mLのジメチルスルホキシドを加え0℃で10分間放置した。その後、液体窒素下で冷凍した後、-80℃で保存した。
(3) Preparation of competent cells of E. coli JM109 strain Inoculate E. coli JM109 strain into 1 mL of LB medium (1% bactotryptone, 0.5% bacto yeast extract, 0.5% NaCl) and aerobically incubate at 37 ° C for 5 hours. Cultured. Add 0.4 mL of the resulting preculture to 40 mL of SOB medium (2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO 4 , 1 mM MgCl 2 ), and incubate at 18 ° C for 20 hours did. The culture was collected by centrifugation (3,700 G, 10 minutes, 4 ° C.), and then a cold TF solution (20 mM PIPES-KOH (pH 6.0), 200 mM KCl, 10 mM CaCl 2 , 40 mM MnCl 2 ) was added. 13 mL was added and left at 0 ° C. for 10 minutes. Then, it was centrifuged again (3,700 G, 10 minutes, 4 ° C.), and the supernatant was removed. The precipitated E. coli was suspended in 3.2 mL of a cold TF solution, 0.22 mL of dimethyl sulfoxide was added, and the mixture was allowed to stand at 0 ° C. for 10 minutes. Then, after freezing under liquid nitrogen, it preserve | saved at -80 degreeC.
（４）組換えプラスミドの調製
上記（３）で作製したコンピテントセル 200μLを氷上で解凍し、上記（２）で作製したライゲーション産物10μLに加え、0℃で30分放置した。続いて、当該コンピテントセルに42℃で30秒間ヒートショックを与え、0℃で2分間冷却した。その後、SOC 培地 (20 mM グルコース、2%バクトトリプトン、0.5%バクトイーストエキス、10 mM NaCl、2.5 mM KCl 、1 mM MgSO4、1mM MgCl2) 1 mLを添加し、37℃にて1時間振盪培養した。培養後の培養液を各200μLずつ、LB Amp寒天培地（アンピシリン 100 mg/L 、1.5％寒天を含有するLB培地）にまき、37℃で一晩培養した。寒天培地上に生育した形質転換体コロニー複数個を 1.5 mLのLB Amp培地（アンピシリン 100 mg/Lを含有するLB培地）にて37℃で一晩培養した。得られた培養液を各々集菌後、Flexi Prep（GEヘルスケア バイオサイエンス社製）を用いて組換えプラスミドを回収した。得られたプラスミドの塩基配列をCEQ DTCS Quick Start Kitおよび蛍光シーケンサCEQ 2000XL DNA Analysis system（いずれもBECKMAN COULTER、米国）を用いて解析した。目的とするプラスミドをpTA002と命名した（図１参照）。
(4) Preparation of recombinant plasmid 200 μL of the competent cell prepared in (3) above was thawed on ice, added to 10 μL of the ligation product prepared in (2) above, and left at 0 ° C. for 30 minutes. Subsequently, the competent cell was subjected to heat shock at 42 ° C. for 30 seconds and cooled at 0 ° C. for 2 minutes. Then, 1 mL of SOC medium (20 mM glucose, 2% bactotryptone, 0.5% bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 1 mM MgSO 4 , 1 mM MgCl 2 ) was added, and the mixture was incubated at 37 ° C for 1 hour. Cultured with shaking. Each 200 μL of the culture solution after culturing was plated on LB Amp agar medium (LB medium containing ampicillin 100 mg / L, 1.5% agar) and cultured overnight at 37 ° C. A plurality of transformant colonies grown on the agar medium were cultured overnight at 37 ° C. in 1.5 mL of LB Amp medium (LB medium containing ampicillin 100 mg / L). After collecting each of the obtained culture solutions, the recombinant plasmid was recovered using Flexi Prep (manufactured by GE Healthcare Bioscience). The base sequence of the obtained plasmid was analyzed using CEQ DTCS Quick Start Kit and fluorescent sequencer CEQ 2000XL DNA Analysis system (both BECKMAN COULTER, USA). The target plasmid was named pTA002 (see FIG. 1).
＜アミダーゼ酵素溶液の調製＞
（１）形質転換体の作製及び培養
実施例１と同様にして、大腸菌JM109株のコンピテントセルに対しプラスミドpTA002による形質転換を行った。得られた形質転換体JM109/pTA002のコロニーを、5 mL 2×LB培地(20 g/Lポリペプトン、10 g/L乾燥酵母エキス、20 g/L NaCl、50μg/mLアンピシリン)に植菌し、37℃ 、250rpmで12時間振盪培養を行い前培養とした。前培養液を集菌(8,000rpm 10分間)し菌体を沈殿させ、約100μLとなるよう上清を除去した。菌体ペレットをピペットマンで懸濁し、2 L modified TB培地(12 g/Lポリペプトン、24 g/L乾燥酵母エキス、40 g/Lグリセロール、12.5 g/L K2HPO4、2.3 g/L KH2PO4、0.5 g/L ポリプロピレングリコール12000、50μg/mLアンピシリン)に植菌し、37℃、180 rpmで培養し、これを本培養とした。培養開始より4時間後にIPTGを1 mMとなるよう添加し14時間の誘導を行った。
菌体懸濁液の調製は、サンプリングして得られた培養液から遠心分離（3,700 G、10分間、4℃）により菌体を回収し、100 mM トリス塩酸緩衝液（pH 8.0）で洗浄した後、同緩衝液に懸濁することにより行った。
<Preparation of amidase enzyme solution>
(1) Production and culture of transformant In the same manner as in Example 1, a competent cell of Escherichia coli JM109 was transformed with plasmid pTA002. The resulting colony of transformant JM109 / pTA002 was inoculated into 5 mL 2 × LB medium (20 g / L polypeptone, 10 g / L dry yeast extract, 20 g / L NaCl, 50 μg / mL ampicillin), Pre-culture was performed by shaking culture at 37 ° C. and 250 rpm for 12 hours. The preculture was collected (8,000 rpm for 10 minutes) to precipitate the cells, and the supernatant was removed to about 100 μL. Suspend the cell pellet with Pipetman and add 2 L modified TB medium (12 g / L polypeptone, 24 g / L dry yeast extract, 40 g / L glycerol, 12.5 g / LK 2 HPO 4 , 2.3 g / L KH 2 PO 4 , 0.5 g / L polypropylene glycol 12000, 50 μg / mL ampicillin) and cultured at 37 ° C. and 180 rpm, and this was used as main culture. After 4 hours from the start of culture, IPTG was added to 1 mM to induce 14 hours.
To prepare the cell suspension, the cells were collected from the culture solution obtained by sampling by centrifugation (3,700 G, 10 minutes, 4 ° C) and washed with 100 mM Tris-HCl buffer (pH 8.0). Then, it was performed by suspending in the same buffer.
（２）アミダーゼ酵素溶液の調製
(i) 無細胞抽出液の調製
本培養を行った培養液に対して6,000 rpmで20分間、遠心分離を行い、菌体の洗浄として50 mMトリス塩酸(pH 8.0)溶液200 mLで懸濁した後、再度6,000rpmで20分間、遠心分離した。集菌した菌体ペレットを50 mMトリス塩酸(pH 8.0)で湿菌体重の2倍量に懸濁し、150Wで20分間、超音波破砕を行った。この超音波破砕液を無細胞抽出液とした。
(2) Preparation of amidase enzyme solution
(i) Preparation of cell-free extract Centrifugation was performed at 6,000 rpm for 20 minutes to the culture solution in which main culture was performed, and the cells were suspended in 200 mL of 50 mM Tris-HCl (pH 8.0) solution. Thereafter, it was centrifuged again at 6,000 rpm for 20 minutes. The collected cell pellet was suspended in 50 mM Tris-hydrochloric acid (pH 8.0) twice as much as the weight of wet bacteria, and sonicated at 150 W for 20 minutes. This ultrasonic disruption solution was used as a cell-free extract.
(ii) 粗酵素溶液の調製
無細胞抽出液に対して、破砕液を14,000 rpmで15分間、遠心分離を行い、沈殿物を除去した。70℃で40分間のヒートショックを行った後、14,000 rpmで20分間、遠心分離を行った。沈殿したタンパク質を除去し、上清を採取した。
(ii) Preparation of crude enzyme solution The cell-free extract was centrifuged at 14,000 rpm for 15 minutes to remove precipitates. After heat shock at 70 ° C. for 40 minutes, centrifugation was performed at 14,000 rpm for 20 minutes. The precipitated protein was removed and the supernatant was collected.
(iii) 精製酵素溶液の調製
上記（ii）で得られた無細胞抽出液熱処理物を、硫酸アンモニウム沈殿法により４℃で分別した。硫酸アンモニウムを３０％の飽和濃度になるまで攪拌しながらゆっくりと加え、次いで、この溶液を４℃、12,000 Gで、２０分間で遠心分離し、得られたペレットを200 mLの50 mMトリス塩酸緩衝液(pH 8.0)に懸濁した。
得られた硫安沈殿画分のタンパク質量5 mg当たり1 mLのDEAEトヨパール650H(TOSOH)をカラムに充填し、担体の5倍量の50 mMトリス塩酸緩衝液(pH 8.0)で平衡化を行った。その後、タンパク質溶液をアプライし、平衡化で用いたものと同組成のパッファーを担体の15倍量添加し洗浄を行った。洗浄後、溶出用バッファー(50 mM トリス塩酸緩衝液(pH 8.0)、100 mM KCl)を7.5倍量アプライし、ステップワイズで本酵素の溶出を行った。最後に、押し出しとして溶出用緩衝液(50 mMトリス塩酸緩衝液(pH 8.0)、500 mM KCl)を添加した。溶出液は約7 mL(200ドロップ)ずつ試験管に分取し、タンパク質の溶出は各画分のO.D.280 nmを測定することにより決定した。
アミダーゼ活性は、50 mMトリス塩酸緩衝液(pH 8.0)、100 mM KClにより溶出を開始し、アミダーゼ活性を有する画分を集めてフェニルトヨパールによる精製を行った。
(iii) Preparation of purified enzyme solution The cell-free extract heat-treated product obtained in (ii) above was fractionated at 4 ° C. by ammonium sulfate precipitation. Ammonium sulfate is slowly added with stirring until a saturation concentration of 30%, then the solution is centrifuged at 12,000 G for 20 minutes at 4 ° C. and the resulting pellet is 200 mL of 50 mM Tris-HCl buffer. Suspended in (pH 8.0).
The column was packed with 1 mL of DEAE Toyopearl 650H (TOSOH) per 5 mg of protein in the resulting ammonium sulfate precipitate fraction, and equilibrated with 50 mM Tris-HCl buffer (pH 8.0), 5 times the amount of the carrier. . Thereafter, the protein solution was applied, and a buffer having the same composition as that used for equilibration was added 15 times the amount of the carrier, followed by washing. After washing, 7.5 times volume of elution buffer (50 mM Tris-HCl buffer (pH 8.0), 100 mM KCl) was applied, and the enzyme was eluted stepwise. Finally, elution buffer (50 mM Tris-HCl buffer (pH 8.0), 500 mM KCl) was added as an extrusion. About 7 mL (200 drops) of the eluate was collected in a test tube, and protein elution was determined by measuring the OD280 nm of each fraction.
The amidase activity was eluted with 50 mM Tris-HCl buffer (pH 8.0) and 100 mM KCl, and fractions having amidase activity were collected and purified with phenyltoyopearl.
タンパク質量5 mg当たり1 mLのフェニルトヨパール650H(TOSOH)をカラムに充填し、担体の5倍量の洗浄緩衝液(50 mMトリス塩酸緩衝液(pH 8.0)、10％硫酸アンモニウム)で平衡化を行った。次に、タンパク質溶液に10%となるように硫酸アンモニウムを添加し、懸濁した。その後、硫酸アンモニウムを添加したタンパク質溶液をカラムにアプライし、平衡化で用いたものと同組成の緩衝液を担体の15倍量添加し洗浄を行った。洗浄後、溶出用緩衝液(50 mMトリス塩酸緩衝液(pH 8.0))を7.5倍量アプライし、ステップワイズで本酵素の溶出を行った。溶出液は約7 mL(200ドロップ)ずつ試験管に分取し、タンパク質の溶出は各画分のO.D.280 mmを測定することにより決定した。
アミダーゼ活性は、50 mMトリス塩酸緩衝液(pH 8.0)、10％硫酸アンモニウムにより溶出を開始し、アミダーゼ活性を有する画分を集めてSephacryI S-200 HRによる精製を行った。
約700 mLのSephacryI S-200 High Resolutiomをカラムに充填し、担体の1〜4%までサンプルを濃縮した。担体の2倍量の50 mMトリス塩酸緩衝液(pH 8.0)で、1 mL/分の流速で平衡化を行った。
その後、マーカー(5 mg Blue Dextran 2000／700μL平衡化緩衝液)、タンパク質溶液を順にアプライし、平衡化で用いたものと同組成の緩衝液を担体の2倍量添加し、流速は一定のまま溶出を行った。溶出液は約7 mL(200ドロップ)ずつ試験管に分取し、タンパク質の溶出は各画分のO.D.280 mmを測定することにより決定した。また、それぞれのタンパク質のピークに合わせて活性測定を行い、活性の高い画分を濃縮して、精製酵素溶液とした。
Pack 1 mL of phenyltoyopearl 650H (TOSOH) per 5 mg of protein and equilibrate with 5 times the washing buffer (50 mM Tris-HCl buffer (pH 8.0), 10% ammonium sulfate). went. Next, ammonium sulfate was added to the protein solution so as to be 10% and suspended. Thereafter, a protein solution to which ammonium sulfate was added was applied to the column, and a buffer solution having the same composition as that used for equilibration was added 15 times the amount of the carrier, followed by washing. After washing, 7.5 times volume of elution buffer (50 mM Tris-HCl buffer (pH 8.0)) was applied, and the enzyme was eluted stepwise. About 7 mL (200 drops) of the eluate was collected in a test tube, and protein elution was determined by measuring OD280 mm of each fraction.
The amidase activity was eluted with 50 mM Tris-HCl buffer (pH 8.0) and 10% ammonium sulfate, and fractions having amidase activity were collected and purified by SephacryI S-200 HR.
About 700 mL of SephacryI S-200 High Resolutiom was packed in the column, and the sample was concentrated to 1-4% of the carrier. Equilibration was performed with 50 mM Tris-HCl buffer (pH 8.0) twice the amount of the carrier at a flow rate of 1 mL / min.
Then, apply marker (5 mg Blue Dextran 2000 / 700μL equilibration buffer) and protein solution in order, add twice the amount of the same buffer as that used for equilibration, and keep the flow rate constant. Elution was performed. About 7 mL (200 drops) of the eluate was collected in a test tube, and protein elution was determined by measuring OD280 mm of each fraction. Moreover, activity was measured according to the peak of each protein, and the fraction with high activity was concentrated to obtain a purified enzyme solution.
（３）酵素活性の測定方法
アミダーゼの活性測定は、0.32 mM ロイシン-p-ニトロアニリン、100 mM トリス塩酸緩衝液（pH 9.0）に適当量のアミダーゼ溶液を加え、80℃にて30分間反応させた。氷中に入れることにより反応を停止させ、反応液の405nmの吸光度を測定して、生成したp-ニトロアニリン量を決定し、アミダーゼ活性とした。
(3) Enzyme activity measurement method The amidase activity was measured by adding an appropriate amount of amidase solution to 0.32 mM leucine-p-nitroaniline and 100 mM Tris-HCl buffer (pH 9.0) and reacting at 80 ° C for 30 minutes. It was. The reaction was stopped by placing it in ice, and the absorbance at 405 nm of the reaction solution was measured to determine the amount of p-nitroaniline produced and used as amidase activity.
（４）タンパク質濃度（酵素濃度）の定量方法
Protein Assayキット（Bio-Rad）を用いた。適当な濃度に希釈した0.8 mLの酵素溶液に0.2 mLのアッセイ試薬を添加し、10〜15分室温に静置して、O.D.595 nmを測定することにより試料のタンパク質濃度を決定した。検量線の作製には、BSA（1.25, 2.5, 5.0, 7.5, 10μg/mL）を用いた。
(4) Method for quantifying protein concentration (enzyme concentration)
Protein Assay kit (Bio-Rad) was used. The protein concentration of the sample was determined by adding 0.2 mL of assay reagent to 0.8 mL of the enzyme solution diluted to an appropriate concentration, allowing to stand at room temperature for 10 to 15 minutes, and measuring OD595 nm. BSA (1.25, 2.5, 5.0, 7.5, 10 μg / mL) was used for preparing the calibration curve.
実施例２で調製したアミダーゼ精製酵素画分を用いて、金属塩の添加効果を検討した。
すなわち、アミダーゼ精製酵素画分150μLに、塩化コバルト、塩化マンガン、塩化ニッケルを、それぞれ、終濃度5 mMとなるように添加し、３７℃及び８０℃で、それぞれ、１時間インキュベートし、次いで、10,000 rpmで5分間遠心処理を行い、沈殿物を除去した。その後、各々150 mLのトリス塩酸緩衝液（pH 8.0）に対して透析を２時間実施し、緩衝液を交換して同様にもう一度透析を行った。その後、各処理酵素液のタンパク質濃度及び活性を測定した。塩化コバルト、塩化マンガン及び塩化ニッケルの添加により、それぞれ、７００％、７４０％及び６４０％（３７℃）、並びに８６０％、１２３０％及び４８００％（８０℃）の活性向上が認められた。
Using the amidase purified enzyme fraction prepared in Example 2, the effect of adding a metal salt was examined.
That is, cobalt chloride, manganese chloride, and nickel chloride were added to 150 μL of the amidase purified enzyme fraction, respectively, to a final concentration of 5 mM, and incubated at 37 ° C. and 80 ° C. for 1 hour, respectively, and then 10,000 Centrifugation was performed at rpm for 5 minutes to remove precipitates. Thereafter, dialysis was performed for 2 hours against 150 mL of Tris-HCl buffer (pH 8.0), and the dialysis was performed again in the same manner after exchanging the buffer solution. Thereafter, the protein concentration and activity of each treatment enzyme solution were measured. With the addition of cobalt chloride, manganese chloride and nickel chloride, 700%, 740% and 640% (37 ° C) and 860%, 1230% and 4800% (80 ° C) activity improvements were observed, respectively.
実施例２で調製したアミダーゼ精製酵素画分を用いて、酵素溶液をEDTAで処理した後、金属塩の添加効果を検討した。
すなわち、1.8 mLのアミダーゼ精製酵素画分に100 mM EDTA (pH 8.0)を終濃度 5 mMとなるように添加した後、80℃で60分間静置し、その後、500 mLのトリス塩酸緩衝液（pH8.0）に対して透析を２時間実施し、緩衝液を交換して同様にもう一度透析を行なうことによりEDTAを除去した。EDTA処理した、アミダーゼ精製酵素画分150μLに塩化コバルト、塩化マンガン、塩化ニッケル、塩化カルシウム、塩化マグネシウム、塩化亜鉛をそれぞれ、終濃度５ｍＭとなるように添加し、３７℃及び８０℃で、それぞれ、１時間インキュベートし、次いで、10000 rpmで5分間遠心処理を行い、沈殿物を除去した。その後、各々150 mLのトリス塩酸緩衝液（pH 8.0）に対して透析を２時間実施し、緩衝液を交換して同様にもう一度透析を行った。その後、各処理酵素液のタンパク質濃度及び活性を測定した。
Using the amidase purified enzyme fraction prepared in Example 2, the enzyme solution was treated with EDTA, and then the effect of adding a metal salt was examined.
Specifically, 100 mM EDTA (pH 8.0) was added to 1.8 mL of the amidase purified enzyme fraction to a final concentration of 5 mM, and then allowed to stand at 80 ° C. for 60 minutes, and then 500 mL of Tris-HCl buffer ( Dialysis was performed against pH 8.0) for 2 hours, and the EDTA was removed by performing another dialysis in the same manner while exchanging the buffer. Cobalt chloride, manganese chloride, nickel chloride, calcium chloride, magnesium chloride, and zinc chloride were added to 150 μL of the amidase purified enzyme fraction treated with EDTA to a final concentration of 5 mM, respectively, at 37 ° C. and 80 ° C., respectively. Incubated for 1 hour and then centrifuged at 10000 rpm for 5 minutes to remove the precipitate. Thereafter, dialysis was performed for 2 hours against 150 mL of Tris-HCl buffer (pH 8.0), and the dialysis was performed again in the same manner after exchanging the buffer solution. Thereafter, the protein concentration and activity of each treatment enzyme solution were measured.
EDTA処理を行なうことにより、アミダーゼ活性は、処理前の6.6％に減少した。
このEDTA処理したアミダーゼ精製酵素画分に金属塩を添加することにより、塩化コバルト、塩化マンガン、塩化ニッケル、塩化カルシウム、塩化マグネシウム及び塩化亜鉛の添加により、無処理アミダーゼ精製酵素画分の活性に対して、それぞれ、１９６％、７７％、１１５％、１３％、１６％及び３７％（３７℃）、並びに３９７％、２８７％、２３６％、４４％、２８％及び９４％（８０℃）の活性回復が認められた。
By performing EDTA treatment, amidase activity was reduced to 6.6% before treatment.
By adding a metal salt to the EDTA-treated amidase purified enzyme fraction, the addition of cobalt chloride, manganese chloride, nickel chloride, calcium chloride, magnesium chloride, and zinc chloride reduces the activity of the untreated amidase purified enzyme fraction. Activity of 196%, 77%, 115%, 13%, 16% and 37% (37 ° C) and 397%, 287%, 236%, 44%, 28% and 94% (80 ° C), respectively. Recovery was observed.
実施例２で調製したアミダーゼ精製酵素画分を用いて、実施例４に記載した方法と同様の方法にほり酵素溶液をEDTAで処理した後、塩化コバルト、塩化マンガン、塩化ニッケルの添加濃度の影響を検討した。
すなわち、アミダーゼ精製酵素画分150μLに塩化コバルト、塩化マンガン、塩化ニッケルをそれぞれ、終濃度1 mM、5 mMあるいは10 mMとなるように添加し、80℃で、１時間インキュベートし、次いで、10,000 rpmで5分間遠心処理を行い、沈殿物を除去した。その後、各々150 mLのトリス塩酸緩衝液（pH 8.0）に対して透析を２時間実施し、緩衝液を交換して同様にもう一度透析を行った。その後、各処理酵素液のタンパク質濃度及び活性を測定した。塩化コバルトの添加により、それぞれ、100％（1 mM）、４９０％（5 mM）及び９８０％（10 mM）、塩化ニッケルの添加により、それぞれ、５０％（１ｍM）、２８０％（5 mM）及び３０５％（10 mM）、並びに塩化マンガンの添加により、それぞれ、１００％（1 mM）、２２０％（5 mM）及び２０５％（10 mM）の活性向上が認められた。
Using the amidase-purified enzyme fraction prepared in Example 2, after treating the holy enzyme solution with EDTA in the same manner as described in Example 4, the effect of the addition concentration of cobalt chloride, manganese chloride, and nickel chloride It was investigated.
That is, cobalt chloride, manganese chloride, and nickel chloride were added to 150 μL of the amidase purified enzyme fraction to a final concentration of 1 mM, 5 mM, or 10 mM, respectively, incubated at 80 ° C. for 1 hour, and then 10,000 rpm. Centrifuged for 5 minutes to remove the precipitate. Thereafter, dialysis was performed for 2 hours against 150 mL of Tris-HCl buffer (pH 8.0), and the dialysis was performed again in the same manner after exchanging the buffer solution. Thereafter, the protein concentration and activity of each treatment enzyme solution were measured. 100% (1 mM), 490% (5 mM) and 980% (10 mM), respectively, with addition of cobalt chloride, 50% (1 mM), 280% (5 mM) and The addition of 305% (10 mM) and manganese chloride was found to increase activity by 100% (1 mM), 220% (5 mM) and 205% (10 mM), respectively.
配列番号３：合成DNA
配列番号４：合成DNA
Sequence number 3: Synthetic DNA
Sequence number 4: Synthetic DNA
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| JPH05328972A (en) * | 1991-01-11 | 1993-12-14 | Natl Food Res Inst | Novel aminoacylase and its production |
| WO2003020929A1 (en) * | 2001-08-28 | 2003-03-13 | Mitsubishi Rayon Co., Ltd. | Novel amide hydrolase gene |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH05328972A (en) * | 1991-01-11 | 1993-12-14 | Natl Food Res Inst | Novel aminoacylase and its production |
| WO2003020929A1 (en) * | 2001-08-28 | 2003-03-13 | Mitsubishi Rayon Co., Ltd. | Novel amide hydrolase gene |
Non-Patent Citations (5)
| Title |
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| JPN6010000565; ＫＯＲＥＩＳＨＩ， Ｍ． ｅｔ ａｌ．: '"Ａ ｎｏｖｅｌ ｅｐｓｉｌｏｎ-ｌｙｓｉｎｅ ａｃｙｌａｓｅ ｆｒｏｍ Ｓｔｒｅｐｔｏｍｙｃｅｓ ｍｏｂａｒａｅｎｓｉｓ ｆｏｒ ｓｙｎｔｈｅｓｉｓ ｏｆ Ｎｅｐｓｉｌｏｎ-ａｃｙｌ-Ｌ-ｌｙｓｉｎ' Ｊ． ＡＭ． ＯＩＬ ＣＨＥＭ． ＳＯＣ． Ｖｏｌ．８２， Ｎｏ．９, 200509, Ｐ．６３１-６３７ * |
| JPN6014003610; Ａｐｐｌ． Ｅｎｖｉｒｏｎ． Ｍｉｃｒｏｂｉｏｌ． （２００５） ｖｏｌ．７１， ｎｏ．１２， ｐ．７９６１-７９７３ * |
| JPN6014003611; Ａｎｎ． Ｎ． Ｙ． Ａｃａｄ． Ｓｃｉ． （１９９２） ｖｏｌ．６７２， ｐ．１２６-１３６ * |
| JPN6014003613; 社団法人日本生化学会編， 新生化学実験講座１ タンパク質Ｉ-分離・精製・性質-， １９９０年２月， 株式会社東 * |
| JPN6014003614; 掘越弘毅ほか編， 酵素 科学と工学， １９９２年４月， 株式会社講談社， ｐ．１６０-１６１ * |
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