JP2004024059A

JP2004024059A - Method for sterilizing microbial cell

Info

Publication number: JP2004024059A
Application number: JP2002182355A
Authority: JP
Inventors: Sakaki Sasaki; 佐々木　　賢樹; Takeya Abe; 阿部　　剛也; Takahiro Yamauchi; 山内　　孝弘; Kiyoshi Ito; 伊藤　　潔; Tadashi Araki; 安楽城　　正
Original assignee: Mitsui Chemicals Inc
Current assignee: Mitsui Chemicals Inc
Priority date: 2002-06-24
Filing date: 2002-06-24
Publication date: 2004-01-29
Anticipated expiration: 2022-06-24
Also published as: JP3944716B2

Abstract

<P>PROBLEM TO BE SOLVED: To provide such a method for efficiently sterilizing genetic recombinant microbial cells, as capable of being industrially used without deteriorating the activity of an enzyme, in order to utilize the genetic recombinant microbial cells outside a closed system. <P>SOLUTION: The method for efficiently sterilizing the genetic recombinant microbial cells existing in a high concentration has zealously been examined. Consequently, it has been found that the genetic recombinant microbial cells have efficiently been sterilized approximately without deactivating the catalytic activity of the enzyme, when a nitrile, an amide or its salt is added to the microbial cells in some concentration and then maintained in a specific temperature range. <P>COPYRIGHT: (C)2004,JPO

Description

【０００１】
【発明の属する技術分野】
本発明は微生物菌体の殺菌方法に関するものであり、例えばニトリル化合物を水和する能力を持つニトリルヒドラターゼ等の、工業的に有用な酵素を生産する遺伝子組換え微生物菌体の殺菌方法に関するものである。
【０００２】
【従来の技術】
近年、微生物菌体を利用した酵素の製造が一般的に行われている。たとえば、ある特定の酵素を触媒として用い、例えばニトリル化合物を原料としてアミド化合物を製造することが実用化されている。
【０００３】
しかし、微生物菌体を用いた酵素の製造においては、製造の各工程における菌体の漏洩の恐れがあり、微生物菌体による汚染をひきおこす可能性がある。さらに、微生物菌体に遺伝子組換え体を利用する場合には、死滅化されていない菌体の使用にあたっては、各種ガイドラインに従い、全ての遺伝子組換え微生物菌体を閉鎖設備内で取り扱う必要がある。　また、閉鎖系外で利用する場合、遺伝子組換え微生物菌体は完全に死滅していなければならない。
【０００４】
さらにこの微生物菌体を死滅させる時には、その微生物菌体のもつ酵素（例えばニトリルヒドラターゼ）の触媒としての活性を失活させることなく、殺菌することが望ましい。この際に、殺菌されたものの中の生菌数は検出されないことが好ましく、また殺菌工程の前後での酵素活性の低下度は２０％以下が好ましいとされ、さらに好ましくは限りなく０％に近いことが良いとされる（ここで言う活性とは、単位微生物菌体重量・単位時間あたりのアミド化合物の生成速度を指す。）。
【０００５】
しかしながら現在までのところ、殺菌方法として、装置的な工夫を施した方法としては、磁気的な殺菌方法（特開平３−２６７１９４）、マイクロ波による殺菌方法（特開平５−２２９５３０）等が知られているが、磁気的、マイクロ波による殺菌方法では大量の液を処理する場合、大掛かりな装置が必要になり、装置自体も高価になる。
【０００６】
また、殺菌時に殺菌剤を加える方法として、塩化ベンザルコニウムを加える殺菌方法（特開昭６１−１５２２７６）、クロルヘキシジンを加える殺菌方法（特開平５−７５３９２）、イソチアン酸アリルを加える殺菌方法（特開平１１−３２２５２１）等が知られているが、これらの殺菌液を加える方法では、殺菌液が製品中に分散されこれを取り除くのは困難であり、不純物として残ってしまい酵素または殺菌済み微生物菌体を用いる反応生成物中まで残ってしまう可能性が強い。特に酵素反応生成物の重合を伴う反応（ポリ乳酸やポリアクリルアミド、ポリメタクリルアミド等）に用いようとすると、これらの物質が還元剤や酸化剤として作用し重合品品質の悪化や重合条件の大幅な変更が必要になる可能性がある。
【０００７】
【発明が解決しようとする課題】
従って、本発明の課題は、微生物菌体を閉鎖系外で利用する為、酵素活性を損なうことなく工業的に使用できるような、効率的な微生物菌体の殺菌方法を提供することを課題とするものである。
【０００８】
【課題を解決するための手段】
本発明者らは、高濃度に存在する微生物菌体の効率的な殺菌方法に関して鋭意検討を行なってきたところ、該微生物菌体にニトリル類、アミド類またはその塩類を加えること、特にある濃度で加え、ある特定の温度範囲で保持することにより、その触媒能はほとんど失活することなく、効率的に殺菌し得ることを見出し、本発明を完成した。
【０００９】
また、本発明において、アミド類を生産する場合には、生産物と同じアミド類もしくは原料ニトリル類を使用することにより不純物がまったく発生しなくなる。
【００１０】
すなわち、本発明は、微生物菌体を含む懸濁液を加熱殺菌する際に、ニトリル類、アミド類、その塩類から選ばれた少なくとも１種の化合物を添加する事を特徴とする微生物菌体の殺菌方法を提供することである。
【００１１】
【発明の実施の形態】
本発明で述べられている酵素とは、化学品等の製造に有用な物質のことである。具体的に本発明に述べる酵素としては、特に制限されるものではないが、例えばニトリルヒドラターゼ、プリンヌクレオシドホスホリラーゼ、トリプトファンシンターゼ等を挙げることができる。
【００１２】
本発明の微生物菌体としては、酵素を生産する微生物菌体であれば特に制限はないが、例えばニトリルヒドラターゼを産生する大腸菌を挙げることができる。
【００１３】
大腸菌の宿主の具体例としては、Ｅｓｃｈｅｒｉｃｈｉａ　ｃｏｌｉ　Ｋ−１２由来Ｗ３１１０株（ＡＴＣＣ２７３２５）、同ＨＢ１０１株（ＡＴＣＣ３３６９４）、同ＪＭ１０９株（ＡＴＣＣ５３２２３）または同ＷＡ８０２株（ＡＴＣＣ３３５２６）株などがあげられる。
【００１４】
ニトリルヒドラターゼを産生する遺伝子組換え大腸菌の具体例としては、ニトリルヒドラターゼ遺伝子が大腸菌に導入されているＭＴ−１０８２２株（ＦＥＲＭ　ＢＰ−５７８５、特開平１１−２５３１６８参照）等があげられる。
【００１５】
本発明の微生物菌体は通常、分子生物学、生物工学、遺伝子工学の分野において、公知の一般的な方法を利用して調製される。例えば、ＬＢ培地やＭ９培地等の通常液体培地に該微生物菌体を植菌した後、適当な培養温度（一般的には３０〜５０℃であるが、好熱菌の場合は５０℃以上でもよい。）で生育させることにより得られる。
【００１６】
殺菌処理を実施する場合、ここでの懸濁液とは培養液、培養液から遠心分離等利用し回収した集菌液、さらに適当な緩衝液で洗浄した洗浄菌体、その処理物が挙げられる。処理物とは例えば、細胞膜の透過性向上や遺伝子組換え菌が生産する有用物質の安定性向上等の為に培養液、菌体、洗浄菌体を熱処理、薬剤処理をおこなったもの、破砕菌体処理したもの等が上げられる。さらに、薬剤処理、凍結処理等で部分的に死滅化した懸濁液と本殺菌法を併用することも可能である。
【００１７】
本殺菌時の菌体濃度は、特に制限はない。加えるニトリル類としてはアクリロニトリル、メタクリロニトリル等が、加えるアミド類としてはアクリルアミド、メタクリルアミド等が挙げられる。加えるニトリル類、アミド類、またはその塩類の濃度は、０．１重量％〜１０．０重量％、より好ましくは、０．１重量％〜５．０重量％が望ましい。この範囲であると、十分な殺菌が行われ、且つ酵素活性も余り低下しない。
【００１８】
また、殺菌時の液温度は殺菌を実施する懸濁液で４５℃〜６０℃が望ましい。この範囲であると、十分な殺菌が行われ、且つ酵素活性も余り低下しない。その際の培養液、菌体等の懸濁液のｐＨは、遺伝子組換え微生物菌体が生産する酵素の変性、失活等がおこりにくいｐＨに設定することが望ましい。具体的には、ｐＨは、６．０から８．０が望ましい。この範囲であると、十分な殺菌が行われ、且つ酵素活性も余り低下しない。
【００１９】
さらに、殺菌時のホールド時間は、１時間〜４時間が望ましい。この範囲であると、十分な殺菌が行われ、且つ酵素活性も余り低下しない。
【００２０】
また、微生物菌体が生産する酵素の変性、失活等が起りにくいよう安定剤、例えば酵素であればその基質、生成物等を添加することも有効である。まれに、一回の殺菌操作で遺伝子組換え菌の完全な死滅化が実施されないことがあるが、その際には本殺菌法を複数回実施すれば完全な死滅化が達成される。
【００２１】
本発明で用いられる微生物菌体とは、特定の酵素を生産するように遺伝子組換えした微生物菌体であり、具体例としてニトリルヒドラターゼ活性を有する菌体が挙げられるが、これに限定するものではない。
【００２２】
ここでニトリルヒドラターゼ活性を有する微生物菌体とは、シュードノカルディア・サーモフィラＪＣＭ３０９５由来のニトリルヒドラターゼを酵素として有する菌体が代表例として挙げられ、菌体としては大腸菌の他、枯草菌、酵母や放線菌等が挙げられる。
【００２３】
上記した菌体は通常、分子生物学、生物工学、遺伝子工学の分野において、公知の一般的な方法を利用して調製される。例えば、ＬＢ培地やＭ９培地等の通常液体培地に該微生物菌体を植菌した後、適当な培養温度（一般的には２０〜５０℃であるが、好熱菌の場合は５０℃以上でもよい。）で生育させ、続いて該微生物菌体を遠心分離等によって培養液より分離することにより得られる。
【００２４】
本殺菌法を実施する場合の懸濁液として培養液、培養液から遠心分離等利用し回収した集菌液、さらに適当な緩衝液で洗浄した洗浄菌体、その処理物が挙げられる。処理物とは、例えば、細胞膜の透過性向上、遺伝子組換え菌が生産する酵素の安定性向上等の為、培養液、菌体、洗浄菌体に薬剤処理をおこなったもの、破砕菌体処理したもの等が上げられる。さらに、薬剤処理、凍結処理等で部分的に死滅化した処理物と本殺菌方法を併用することも可能である。
【００２５】
以下、本発明における殺菌方法を簡単にまとめると、まず、培養槽や反応釜など加熱できる容器の中に入った該懸濁液にニトリル類、アミド類、その塩類などを加える。次に懸濁液を加温して、先述の温度範囲（４５〜６０℃）に保持する。
【００２６】
さらに、本発明では上記の範囲の温度に、先述のように一定時間保持（１時間〜４時間）する。その後、保持時間を過ぎたら速やかに冷却することによって、酵素活性の低下を最小限に留めることが出来る。
【００２７】
【実施例】
以下、実施例により本発明の大腸菌の殺菌方法を更に詳細に説明するが、本発明はこれら実施例に限定されるものではない。
【００２８】
（実施例１）
本実施例に使用する酵素触媒はニトリルヒドラターゼ遺伝子が大腸菌ＨＢ１０１株に導入され、三井化学株式会社が寄託しているＭＴ−１０８２２株（ＦＥＲＭ　ＢＰ−５７８５、特開平１１−２５３１６８参照）を用いた。
【００２９】
本菌株の培養は２ｌのバッフル付三角フラスコに下記の組成の培地５００ｍｌを調製し、１２１℃・２０分間のオートクレーブにより滅菌した。
【００３０】

【００３１】
この培地に終濃度が５０μｇ／ｍｌとなるようにアンピシリンを添加した後、上記菌株を一白金耳植菌し、３７℃・１３０ｒｐｍにて２０時間培養した。また、この際、培養開始約１５時間後にＩＰＴＧ（Ｉｓｏｐｒｏｐｙｌ−β−Ｄ−ｔｈｉｏｇａｌａｃｔｏｐｙｒａｎｏｓｉｄｅ）を終濃度１００μｍｏｌ／ｌになるよう添加し培養する（以下、培養液と略）。遠心分離（１５０００Ｇ×１５分間）により菌体のみを培養液より分離し、分離した液に流動性を持たせるため固形分率が約１５重量％となるように集菌時の上澄み液を加えて湿菌体を得た（以下、集菌液と略）。これらの一部をサンプリングし下記方法にて酵素活性測定した。
【００３２】
その後、集菌液にアクリルアミド（以下、ＡＡＭと略）を０．１重量％になるように加え、加熱温度５０℃、ホールド時間３ｈで殺菌処理を実施し、殺菌処理後のサンプルをサンプリングしその活性を測定した。
【００３３】
次式、活性残存率（％）＝（殺菌後の活性／殺菌前の活性）×１００にて活性残存率を求めた。
【００３４】
生菌数は、市販のマッコンキー寒天培地「ダイゴ」（日本製薬製）に殺菌前と殺菌後の菌体液を適切に希釈し、１００μｌ播き、寒天培地上にコンラージ棒でよく広げた後、３０℃で２日間培養して、生育してきたコロニー数を数えて求めた。結果を表１に示す。
（酵素活性測定方法）
サンプリングした菌体液それぞれの、酵素活性を以下の手順で測定する。
各画分液を５０ｍＭリン酸カリウム緩衝液（ｐＨ７．５）により適当に希釈し、これに１重量％のアクリロニトリルを添加して１０℃で１０分間反応させる。反応液にこれと等量の１Ｍリン酸水溶液を添加して反応を停止させ、生成したアクリルアミド濃度を高速液体クロマトグラフィー（以下、ＨＰＬＣと略）分析により測定する。ＨＰＬＣカラムはＵＬＴＲＯＮ　８０ＨＧ（５０×８φｍｍ）を用い、１０ｍＭリン酸水溶液を展開液として使用する。アクリルアミドは２２０ｎｍの吸光度により検出する。
【００３５】
（実施例２〜５、比較例１）
集菌液に加えるアクリルアミド濃度を表１のようにかえたこと以外は前記実施例１と同様の方法で殺菌処理を行った後、同様の方法で生菌数と残存活性を測定した。その結果を表１に示す。
【００３６】
【表１】

【００３７】
なお、アクリルアミドをメタクリルアミド、アクリロニトリル、メタクリロニトリルまたは、その塩類に変えても同様の結果が得られた。
さらに、集菌液を培養液に変えても同様の結果が得られた。
【００３８】
（実施例６〜９）
殺菌時の処理温度を変えた事以外は前記実施例３と同様の方法で殺菌処理を行った後、同様の方法で生菌数と残存活性を測定した。その結果を表２に示す。
【００３９】
【表２】

【００４０】
なお、アクリルアミドをメタクリルアミド、アクリロニトリル、メタクリロニトリルまたは、その塩類に変えても同様の結果が得られた。
さらに、集菌液を培養液に変えても同様の結果が得られた。
【００４１】
（実施例１０〜１２）
集菌液のｐＨを変えたこと以外は前記実施例３と同様の方法で殺菌処理を行った後、同様の方法で生菌数と残存活性を測定した。
【００４２】
なお、集菌液のｐＨは水酸化ナトリウム溶液もしくは、硫酸を使って調整した。
その結果、表３のような結果が得られた。
【００４３】
【表３】

【００４４】
なお、アクリルアミドをメタクリルアミド、アクリロニトリル、メタクリロニトリルまたは、その塩類に変えても同様の結果が得られた。
さらに、集菌液を培養液に変えても同様の結果が得られた。
【００４５】
（実施例１３〜１５）
殺菌時のホールド時間を変えた事以外は前記実施例３と同様の方法で殺菌処理を行った後、同様の方法で生菌数と残存活性を測定した。その結果を表４に示す。
【００４６】
【表４】

【００４７】
なお、アクリルアミドをメタクリルアミド、アクリロニトリル、メタクリロニトリルまたは、その塩類に変えても同様の結果が得られた。
さらに、集菌液を培養液に変えても同様の結果が得られた。
【００４８】
【発明の効果】
本発明によれば、遺伝子組換え微生物菌体にアクリルアミド類もしくはニトリル類、その塩類などを加え、一定温、一定時間で殺菌することにより酵素や有用な物質を変性、失活させることなく完全に死滅化することが可能である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for sterilizing microbial cells, for example, a method for sterilizing genetically modified microbial cells that produce industrially useful enzymes, such as nitrile hydratase having the ability to hydrate nitrile compounds. It is.
[0002]
[Prior art]
BACKGROUND ART In recent years, production of enzymes using microbial cells has been generally performed. For example, production of an amide compound using a specific enzyme as a catalyst and using, for example, a nitrile compound as a raw material has been put to practical use.
[0003]
However, in the production of enzymes using microbial cells, there is a risk of microbial cells leaking in each step of the production, which may cause contamination by microbial cells. Furthermore, when using genetically modified microorganisms for microbial cells, it is necessary to handle all genetically modified microbial cells in closed facilities in accordance with various guidelines when using non-killed cells. . When used outside a closed system, the genetically modified microbial cells must be completely killed.
[0004]
Further, when the microbial cells are killed, it is desirable that the microbial cells be sterilized without losing the catalytic activity of an enzyme (eg, nitrile hydratase) possessed by the microbial cells. At this time, it is preferable that the number of viable bacteria in the sterilized one is not detected, and the degree of decrease in enzyme activity before and after the sterilization step is preferably 20% or less, more preferably as close as possible to 0%. (The term “activity” refers to the rate of formation of amide compounds per unit microbial cell weight per unit time.)
[0005]
However, up to now, as a sterilization method, a magnetic sterilization method (JP-A-3-267194), a microwave sterilization method (JP-A-5-229530), and the like have been known as methods that have been devised as devices. However, the magnetic and microwave sterilization methods require a large-scale apparatus when processing a large amount of liquid, and the apparatus itself becomes expensive.
[0006]
As a method of adding a disinfectant at the time of disinfection, a disinfection method of adding benzalkonium chloride (JP-A-61-152276), a disinfection method of adding chlorhexidine (JP-A-5-75392), and a disinfection method of adding allyl isothiocyanate (JP-A-5-75392) However, according to the method of adding these sterilizing solutions, the sterilizing solution is dispersed in the product and it is difficult to remove the sterilizing solution. There is a strong possibility that it will remain in the reaction product using the body. In particular, when used in reactions involving the polymerization of enzymatic reaction products (polylactic acid, polyacrylamide, polymethacrylamide, etc.), these substances act as reducing agents and oxidizing agents, deteriorating the quality of the polymerized products and significantly increasing polymerization conditions. Major changes may be required.
[0007]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide an efficient method for disinfecting microbial cells, which utilizes the microbial cells outside of a closed system and can be used industrially without impairing the enzyme activity. Is what you do.
[0008]
[Means for Solving the Problems]
The present inventors have made intensive studies on an efficient method for sterilizing microbial cells present at a high concentration, and added nitriles, amides or salts thereof to the microbial cells, particularly at a certain concentration. In addition, it has been found that by maintaining the temperature in a certain specific temperature range, the catalytic activity can be efficiently sterilized with almost no deactivation, and the present invention has been completed.
[0009]
In the present invention, when amides are produced, no impurities are generated at all by using the same amides or starting nitriles as the product.
[0010]
That is, the present invention relates to a method for producing a suspension of microbial cells, which comprises adding at least one compound selected from nitriles, amides, and salts thereof when heating and suspending the suspension containing the microbial cells. It is to provide a sterilization method.
[0011]
BEST MODE FOR CARRYING OUT THE INVENTION
The enzyme described in the present invention is a substance useful for producing chemicals and the like. Although the enzyme specifically described in the present invention is not particularly limited, examples thereof include nitrile hydratase, purine nucleoside phosphorylase, tryptophan synthase and the like.
[0012]
The microbial cell of the present invention is not particularly limited as long as it is a microbial cell that produces an enzyme, and examples thereof include E. coli that produces nitrile hydratase.
[0013]
Specific examples of the Escherichia coli host include Escherichia coli K-12 derived strain W3110 (ATCC 27325), strain HB101 (ATCC 33694), strain JM109 (ATCC 53223), and strain WA802 (ATCC 33526).
[0014]
Specific examples of the recombinant Escherichia coli producing nitrile hydratase include MT-10822 strain in which the nitrile hydratase gene has been introduced into E. coli (FERM BP-5785, see JP-A-11-253168).
[0015]
The microbial cell of the present invention is usually prepared using a known general method in the fields of molecular biology, biotechnology, and genetic engineering. For example, after inoculating the microbial cells in a normal liquid medium such as LB medium or M9 medium, a suitable culture temperature (generally 30 to 50 ° C., but in the case of thermophilic bacteria, even at 50 ° C. or higher) Good).
[0016]
When the sterilization treatment is performed, the suspension herein includes a culture solution, a bacterial collection solution collected from the culture solution by centrifugation or the like, a washed cell body further washed with an appropriate buffer, and a processed product thereof. . Processed products include, for example, those obtained by subjecting culture solutions, cells, and washed cells to heat treatment and chemical treatment to improve the permeability of cell membranes and the stability of useful substances produced by genetically modified bacteria, A body-treated one can be raised. Furthermore, the present sterilization method can be used in combination with a suspension partially killed by drug treatment, freezing treatment, or the like.
[0017]
There is no particular limitation on the cell concentration during the main sterilization. The nitriles to be added include acrylonitrile and methacrylonitrile, and the amides to be added include acrylamide and methacrylamide. The concentration of the nitriles, amides, or salts thereof to be added is preferably 0.1% by weight to 10.0% by weight, more preferably 0.1% by weight to 5.0% by weight. In this range, sufficient sterilization is performed, and the enzyme activity does not decrease much.
[0018]
Further, the liquid temperature during sterilization is desirably 45 ° C to 60 ° C for the suspension to be sterilized. In this range, sufficient sterilization is performed, and the enzyme activity does not decrease much. At this time, the pH of the suspension of the culture solution, the cells and the like is desirably set to a pH that does not easily cause denaturation, inactivation, and the like of the enzyme produced by the recombinant microorganism cells. Specifically, the pH is desirably 6.0 to 8.0. In this range, sufficient sterilization is performed, and the enzyme activity does not decrease much.
[0019]
Further, the hold time during sterilization is desirably 1 hour to 4 hours. In this range, sufficient sterilization is performed, and the enzyme activity does not decrease much.
[0020]
It is also effective to add a stabilizer such as a substrate or a product of an enzyme, for example, so that the enzyme produced by the microbial cells hardly undergoes denaturation or inactivation. In rare cases, complete sterilization of a genetically modified bacterium may not be achieved by a single sterilization operation. In such a case, complete sterilization can be achieved by performing the sterilization method several times.
[0021]
The microbial cell used in the present invention is a microbial cell genetically modified to produce a specific enzyme, and specific examples include a cell having nitrile hydratase activity, but are not limited thereto. is not.
[0022]
Here, the microbial cells having nitrile hydratase activity include, as representative examples, cells having nitrile hydratase derived from Pseudonocardia thermophila JCM3095 as an enzyme, and other than Escherichia coli, Bacillus subtilis, Examples include yeast and actinomycetes.
[0023]
The above-mentioned cells are usually prepared using a known general method in the fields of molecular biology, biotechnology and genetic engineering. For example, after inoculating the microbial cells in a normal liquid medium such as an LB medium or an M9 medium, an appropriate culture temperature (generally 20 to 50 ° C., but in the case of thermophilic bacteria, even at 50 ° C. or higher) Good), followed by separating the microbial cells from the culture solution by centrifugation or the like.
[0024]
Suspensions for carrying out the present disinfection method include a culture solution, a harvested solution collected from the culture solution by centrifugation or the like, a washed cell body washed with an appropriate buffer, and a processed product thereof. Treated products include, for example, those obtained by subjecting a culture solution, bacterial cells, and washed bacterial cells to a chemical treatment, for improving cell membrane permeability, improving the stability of enzymes produced by genetically modified bacteria, and treating disrupted bacterial cells. And so on. Furthermore, it is also possible to use the treated product partially killed by a chemical treatment, a freezing treatment or the like together with the present sterilization method.
[0025]
Hereinafter, the sterilization method of the present invention will be briefly summarized. First, nitriles, amides, salts thereof, and the like are added to the suspension contained in a heatable container such as a culture tank or a reaction vessel. Next, the suspension is heated and maintained in the aforementioned temperature range (45 to 60 ° C.).
[0026]
Further, in the present invention, the temperature is kept in the above-mentioned range for a fixed time (1 hour to 4 hours) as described above. Thereafter, by cooling immediately after the holding time, a decrease in enzyme activity can be minimized.
[0027]
【Example】
Hereinafter, the method for killing Escherichia coli of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
[0028]
(Example 1)
The enzyme catalyst used in this example was the MT-10822 strain (FERM BP-5785, JP-A-11-253168) in which the nitrile hydratase gene was introduced into Escherichia coli HB101 and deposited by Mitsui Chemicals, Inc. .
[0029]
For cultivation of this strain, 500 ml of a medium having the following composition was prepared in a 2 l Erlenmeyer flask with baffles, and sterilized by an autoclave at 121 ° C. for 20 minutes.
[0030]

[0031]
After ampicillin was added to this medium to a final concentration of 50 μg / ml, one loopful of the above strain was inoculated and cultured at 37 ° C. and 130 rpm for 20 hours. At this time, about 15 hours after the start of the culture, IPTG (Isopropyl-β-D-thiogalactopyranoside) is added so that the final concentration is 100 μmol / l, and the cells are cultured (hereinafter, abbreviated as a culture solution). Only the cells were separated from the culture solution by centrifugation (15000 G × 15 minutes), and the supernatant at the time of cell collection was added so that the solid content was about 15% by weight so that the separated solution had fluidity. Wet cells were obtained (hereinafter abbreviated as a cell collection solution). Some of these were sampled and the enzyme activity was measured by the following method.
[0032]
Thereafter, acrylamide (hereinafter, abbreviated as AAM) was added to the harvested solution to a concentration of 0.1% by weight, sterilization was performed at a heating temperature of 50 ° C. and a hold time of 3 hours, and a sample after the sterilization was sampled. Activity was measured.
[0033]
The residual activity rate was determined by the following equation, residual activity rate (%) = (activity after sterilization / activity before sterilization) × 100.
[0034]
The viable cell count was determined by appropriately diluting the bacterial cell fluid before and after sterilization on a commercially available MacConkey agar medium “Digo” (manufactured by Nippon Pharmaceutical Co., Ltd.), seeding 100 μl, spreading the mixture on the agar medium with a cona rod, and then 30 ° C. For 2 days, and the number of growing colonies was counted and determined. Table 1 shows the results.
(Method for measuring enzyme activity)
The enzyme activity of each of the sampled cell fluids is measured by the following procedure.
Each fraction is appropriately diluted with a 50 mM potassium phosphate buffer (pH 7.5), and 1% by weight of acrylonitrile is added thereto, followed by reaction at 10 ° C. for 10 minutes. The reaction is stopped by adding an equivalent volume of a 1 M phosphoric acid aqueous solution to the reaction solution, and the concentration of the generated acrylamide is measured by high performance liquid chromatography (hereinafter abbreviated as HPLC) analysis. The HPLC column uses ULRON 80HG (50 × 8 φmm), and a 10 mM phosphoric acid aqueous solution is used as a developing solution. Acrylamide is detected by absorbance at 220 nm.
[0035]
(Examples 2 to 5, Comparative Example 1)
Except for changing the concentration of acrylamide added to the cell collection solution as shown in Table 1, sterilization was carried out in the same manner as in Example 1, and the viable cell count and residual activity were measured in the same manner. Table 1 shows the results.
[0036]
[Table 1]

[0037]
Similar results were obtained when acrylamide was changed to methacrylamide, acrylonitrile, methacrylonitrile, or salts thereof.
Further, similar results were obtained even when the harvested solution was changed to a culture solution.
[0038]
(Examples 6 to 9)
After the sterilization treatment was performed in the same manner as in Example 3 except that the treatment temperature during sterilization was changed, the viable cell count and residual activity were measured in the same manner. Table 2 shows the results.
[0039]
[Table 2]

[0040]
Similar results were obtained when acrylamide was changed to methacrylamide, acrylonitrile, methacrylonitrile, or salts thereof.
Further, similar results were obtained even when the harvested solution was changed to a culture solution.
[0041]
(Examples 10 to 12)
After the sterilization treatment was performed in the same manner as in Example 3 except that the pH of the cell collection solution was changed, the number of viable cells and the residual activity were measured in the same manner.
[0042]
The pH of the harvested solution was adjusted using a sodium hydroxide solution or sulfuric acid.
As a result, the results shown in Table 3 were obtained.
[0043]
[Table 3]

[0044]
Similar results were obtained when acrylamide was changed to methacrylamide, acrylonitrile, methacrylonitrile, or salts thereof.
Further, similar results were obtained even when the harvested solution was changed to a culture solution.
[0045]
(Examples 13 to 15)
Except that the hold time during sterilization was changed, sterilization was performed in the same manner as in Example 3, and the viable cell count and residual activity were measured in the same manner. Table 4 shows the results.
[0046]
[Table 4]

[0047]
Similar results were obtained when acrylamide was changed to methacrylamide, acrylonitrile, methacrylonitrile, or salts thereof.
Further, similar results were obtained even when the harvested solution was changed to a culture solution.
[0048]
【The invention's effect】
According to the present invention, acrylamides or nitriles, salts thereof, etc. are added to the genetically modified microbial cells, and the enzyme or useful substance is completely denatured and inactivated by sterilizing at a constant temperature and for a predetermined time, without completely inactivating it. It is possible to die.

Claims

A method for sterilizing microbial cells, comprising adding at least one compound selected from nitriles, amides, and salts thereof when heat-sterilizing a suspension containing microbial cells.

2. The method for killing microbial cells according to claim 1, wherein the microbial cells are genetically modified microorganisms.

3. The method according to claim 2, wherein the genetically modified microorganism is a microorganism that produces an enzyme.

The method according to claim 3, wherein the enzyme according to claim 3 is nitrile hydratase, purine nucleoside phosphorylase, or tryptophan synthase.

5. The method according to claim 2, wherein the genetically modified microorganism is a genetically modified E. coli.

The host of the genetically modified Escherichia coli is Escherichia coli K-12 derived strain W3110 (ATCC 27325), strain HB101 (ATCC 33694), strain JM109 (ATCC 53223) or strain WA802 (ATCC 33526). The method for disinfecting microbial cells according to the above.

The method for sterilizing microbial cells according to any one of claims 1 to 5, wherein the nitrile is at least one selected from acrylonitrile and methacrylonitrile, and the amide is at least one selected from acrylamide and methacrylamide.

The method for killing microbial cells according to any one of claims 1 to 7, wherein the concentration of nitriles, amides or salts thereof in the suspension is 0.1 to 10.0% by weight.

The method for disinfecting microbial cells according to any one of claims 1 to 8, wherein the pH of the suspension is 6.0 to 8.0.

The method for sterilizing microbial cells according to any one of claims 1 to 9, wherein the temperature at the time of heat sterilization is 45 to 60 ° C.

The method for sterilizing microbial cells according to any one of claims 1 to 10, wherein the heating and holding time is 1 to 4 hours.