JPH01179689A - Stabilizer for organism catalyst - Google Patents

Stabilizer for organism catalyst

Info

Publication number
JPH01179689A
JPH01179689A JP204888A JP204888A JPH01179689A JP H01179689 A JPH01179689 A JP H01179689A JP 204888 A JP204888 A JP 204888A JP 204888 A JP204888 A JP 204888A JP H01179689 A JPH01179689 A JP H01179689A
Authority
JP
Japan
Prior art keywords
stabilizer
heavy water
biocatalyst
organism
catalyst
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
Application number
JP204888A
Other languages
Japanese (ja)
Inventor
Toru Hamaya
浜谷 徹
Koki Horikoshi
弘毅 掘越
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Japan Science and Technology Agency
Original Assignee
Research Development Corp of Japan
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Research Development Corp of Japan filed Critical Research Development Corp of Japan
Priority to JP204888A priority Critical patent/JPH01179689A/en
Publication of JPH01179689A publication Critical patent/JPH01179689A/en
Pending legal-status Critical Current

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  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

PURPOSE:To enable prevention of deterioration of organism catalyst during reaction and preservation thereof and to suppress growth of microorganism during preservation of organism catalyst, by adding a given amount of heavy water to an enzyme, cell, etc. to produce a stabilizer for organism catalyst. CONSTITUTION:30-100%, more preferably about 50-100% is added to an enzyme, cell, etc. to give a stabilizer for organism catalyst. Heavy water is directly used as it is or blended with a solvent and an additive and can be used. The stabilizer for organism catalyst can be used as it is in a stable condition of the organism catalyst or can be used as a reaction solution for the organism catalyst by blending the stabilizer with a solution containing a substrate of the organism catalyst so as to make >=30% heavy water concentration or by mixing the stabilizer with a substrate and a pH buffering solution, etc.

Description

【発明の詳細な説明】 〔産業上の利用分野〕 本発明は、生体触媒の安定化剤に関し、更に詳しくは、
保存時及び使用時に生体触媒の活性を長期間安定的に維
持できる生体触媒の安定化剤に関する。
[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a stabilizer for biocatalysts, and more specifically,
The present invention relates to a stabilizer for biocatalysts that can stably maintain the activity of biocatalysts for long periods of time during storage and use.

〔従来の技術〕[Conventional technology]

生体触媒は、常温常圧で使用できることから、食品工業
や化学工業界で広く用いられている。特に再使用が可能
なためコスト面で有利な固定化酵素や、やはりコストの
低減を図る目的から酵素抽出の手間が省ける固定化菌体
が実用化されている。
Biocatalysts are widely used in the food and chemical industries because they can be used at room temperature and pressure. In particular, immobilized enzymes, which are advantageous in terms of cost because they can be reused, and immobilized bacterial cells, which can save the labor of extracting enzymes, have been put into practical use in order to reduce costs.

バイオリアクターとして用いられている酵素または菌体
としては、例えば、異性化糖製造用グルコースイソメラ
ーゼ、低乳糖牛乳製造用ラクターゼ及び酢酸製造用アセ
トバクターアセチ(八cetobacter acet
i)等がある。又、バイオセンサーとしては、ぶどう糖
測定用グルコースオキシダーゼ・パーオキシダーゼ等が
ある。さらにBODの測定にも微生物固定化膜を用いた
センサーが使用されている。
Examples of enzymes or bacterial cells used in bioreactors include glucose isomerase for producing high-fructose sugar, lactase for producing low-lactose milk, and acetobacter acetase for producing acetic acid.
i) etc. Furthermore, examples of biosensors include glucose oxidase and peroxidase for measuring glucose. Furthermore, sensors using microorganism-immobilized membranes are also used to measure BOD.

上記のように生体触媒はますますその応用範囲の拡大が
期待される。しかるに、以下に示す大別して2つの問題
点がある。
As mentioned above, biocatalysts are expected to further expand their range of applications. However, there are two main problems as shown below.

〔発明が解決しようとする課題〕[Problem to be solved by the invention]

(1)第1の問題点は、長時間の使用または再使用のた
めの保存中にその活性が劣化することである。
(1) The first problem is that the activity deteriorates during long-term use or storage for reuse.

それに対して従来から劣化防止対策として種々の方法が
用いられている。例えば、硫酸アンモニウム等の塩によ
る塩析状態での保存、有機溶媒によって沈殿させてトル
エン等に!g濁させる方法などがある。しかしこれらの
方法は、固定化生体触媒には利用できないという欠点が
あった。そこで固定化生体触媒の保存方法としては、基
質を添加する方法や、−20℃程度の低温下で保存する
方法等が用いられている。しかし前者は基質の分解が生
じ、後者は、凍結融解時に生体触媒が失活することがあ
り、多量の生体触媒を用いる実プラントにおいては大規
模な設備が必要であり、かつ電気使用料の点から有利な
方法とはいえない。
In response to this, various methods have been used to prevent deterioration. For example, storage in a salted-out state with a salt such as ammonium sulfate, precipitation with an organic solvent, and toluene, etc. There are ways to make it cloudy. However, these methods have the disadvantage that they cannot be used for immobilized biocatalysts. Therefore, as methods for preserving the immobilized biocatalyst, methods such as adding a substrate or preserving it at a low temperature of about -20° C. are used. However, in the former case, the substrate decomposes, and in the latter case, the biocatalyst may be deactivated during freezing and thawing.Actual plants that use a large amount of biocatalysts require large-scale equipment, and there are also concerns about electricity usage costs. Therefore, it cannot be said to be an advantageous method.

(2)第2の問題点は、保存中に、保存液中に微生物が
混入し、生体触媒の劣化がおこることである。
(2) The second problem is that during storage, microorganisms get mixed into the storage solution, causing deterioration of the biocatalyst.

それに対する防止対策としては、保存液中にアジ化ナト
リウム等を添加することが多い。ところがアジ化ナトリ
ウムは、酸と反応して有毒なアジ化水素を発生し、又、
重金属とは爆発性の塩を形成し易い等、取扱いが容易で
ないという問題点があった。
As a preventive measure against this, sodium azide or the like is often added to the storage solution. However, sodium azide reacts with acids to generate toxic hydrogen azide, and
Heavy metals have the problem of not being easy to handle, such as the tendency to form explosive salts.

本発明者らは、上記問題点を解決すべく検討した結果、
重水を用いることにより生体触媒の反応中及び保存中に
その劣化を防止することができ、かつ微生物の生育を抑
制することができることを見出し本発明を完成するに至
った。さらに、重水の使用は、他の安定化法、保存法と
併用でき、かつ再使用が可能であるとの利点をも有する
As a result of studies to solve the above problems, the present inventors found that
The present invention was completed based on the discovery that by using heavy water, deterioration of biocatalysts can be prevented during reaction and storage, and growth of microorganisms can be suppressed. Furthermore, the use of heavy water has the advantage that it can be used in conjunction with other stabilization and preservation methods, and that it can be reused.

〔課題を解決するための手段〕[Means to solve the problem]

本発明は、重水を含む生体触媒安定化剤に関する。 The present invention relates to a biocatalyst stabilizer containing heavy water.

以下本発明について説明する。The present invention will be explained below.

本発明の安定化剤は重水を有効成分として含有し、その
含有率は約30〜100%、好ましくは約50〜100
%であることが適当である。又重水以外の成分としては
用途に応じて例えば水等の溶媒及びその他の添加剤(例
えば基質)を含有させることができる。
The stabilizer of the present invention contains heavy water as an active ingredient, and its content is about 30 to 100%, preferably about 50 to 100%.
% is appropriate. In addition, as components other than heavy water, solvents such as water and other additives (eg, substrates) can be included depending on the purpose.

本発明の安定化剤は、重水をそのまま用いても、あるい
は重水と上記溶媒及び添加剤を適宜混合して得ることも
できる。さらに本発明の安定化剤としては、重水を得る
ための濃縮工程で得られる重水(D20)及び水(H2
O)の混合物をそのまま用いることもできる。
The stabilizer of the present invention can be obtained by using heavy water as it is, or by appropriately mixing heavy water with the above solvents and additives. Further, as the stabilizer of the present invention, heavy water (D20) obtained in the concentration step for obtaining heavy water and water (H2
A mixture of O) can also be used as is.

本発明の安定化剤は、そのまま生体触媒の保存液として
該生体触媒の安定条件(pH,温度等)で用いることが
できる。又、本発明の安定化剤は、生体触媒の基質を含
有する溶液に重水濃度30%以上、好ましくは50%以
上となるように添加混合することによって、あるいは該
安定化剤に基質及びpH緩衝剤等を添加することにより
生体触媒の反応液として用いることもできる。
The stabilizer of the present invention can be used as it is as a storage solution for the biocatalyst under stable conditions (pH, temperature, etc.) for the biocatalyst. Furthermore, the stabilizer of the present invention can be added to a solution containing a substrate for a biocatalyst at a heavy water concentration of 30% or more, preferably 50% or more, or mixed with the substrate and a pH buffer. It can also be used as a reaction solution for biocatalysts by adding agents and the like.

本発明の安定化剤を適用し得る生体触媒としては、例え
ば酵素及び菌体を挙げることができ、酵素は固定化酵素
も含み、菌体には固定化菌体及び休止菌体も含む。
Biocatalysts to which the stabilizer of the present invention can be applied include, for example, enzymes and bacterial cells; enzymes include immobilized enzymes, and bacterial cells also include immobilized bacterial cells and resting bacterial cells.

前記生体触媒の保存液または反応液として用いた安定化
剤は、逆浸透等の処理をすることによって再生使用する
ことができる。
The stabilizer used as the storage solution or reaction solution for the biocatalyst can be reused by performing treatment such as reverse osmosis.

重水は水の同位体の一種であり、天然水中0.01〜0
.015%の割合で含まれている。通常重水は、重水を
含む水を水酸化すI−IJウム等のアルカリ溶液として
電気分解することにより、濃縮、純粋化して得られる。
Heavy water is a type of water isotope, and is present in natural water from 0.01 to 0.
.. It is included at a rate of 0.015%. Usually, heavy water is obtained by concentrating and purifying water containing heavy water by electrolyzing it as an alkaline solution such as I-IJium hydroxide.

現在重水は低価格とはいえない。Currently, heavy water is not cheap.

この点からは、本発明の安定化剤は、特に小型かつ高価
格のバイオセンサーの保存液として優れている。しかし
、前記のように本発明の安定化剤は再使用が可能である
。従って、重水が比較的高価格であっても、小型のバイ
オセンサーの保存液以外の目的で工業的に使用すること
も可能である。
From this point of view, the stabilizer of the present invention is particularly excellent as a storage solution for small and expensive biosensors. However, as mentioned above, the stabilizer of the present invention can be reused. Therefore, even though heavy water is relatively expensive, it can be used industrially for purposes other than as a storage solution for small biosensors.

さらに、本発明には、低い濃縮率の重水そのまま用いる
こともできコストの低減が可能である。また、微生物を
用いた重水濃縮法も発明され(特開昭50−13240
0号)、重水の価格の低減化が期待される。
Furthermore, in the present invention, heavy water with a low concentration ratio can be used as it is, and costs can be reduced. In addition, a heavy water concentration method using microorganisms was invented (Japanese Patent Application Laid-Open No. 13240-1980).
0), it is expected that the price of heavy water will be reduced.

以下本発明を実施例によりさらに説明する。The present invention will be further explained below with reference to Examples.

実施例1 市販のインベルターゼを用い、その熱安定性を調べるた
め、加速試験を行った。すなわち、インベルターゼをp
H5,5の酢酸緩衝液にて溶解した後、水または重水で
希釈した。この時点での重水濃度は、0.30.50及
び80%とした。各溶液を、65℃中に10分間保持し
、残存活性を比較した。
Example 1 An accelerated test was conducted using commercially available invertase to examine its thermal stability. That is, invertase is p
After dissolving in H5.5 acetate buffer, it was diluted with water or heavy water. The heavy water concentration at this point was 0.30.50 and 80%. Each solution was kept at 65° C. for 10 minutes and the remaining activity was compared.

残存活性の相対値を表1に示す。Table 1 shows the relative values of residual activity.

表   1 実施例2 酵母をYPD培地で48時間培養後、その菌体を遠心分
離法によって集め、常法により、アルギン酸ナトリウム
に固定化した。この固定化菌体を、重水濃度O及び80
%の酢酸緩衝液(pH5,5)中にいれ、70℃中に1
0〜30分間保持した後、固定化菌体中のインベルター
ゼの残存活性を測定した。残存活性の相対値を表2に示
す。
Table 1 Example 2 After culturing yeast in YPD medium for 48 hours, the cells were collected by centrifugation and immobilized on sodium alginate by a conventional method. The immobilized bacterial cells were mixed with heavy water at a concentration of O and 80
% acetate buffer (pH 5,5) and at 70°C.
After holding for 0 to 30 minutes, the residual activity of invertase in the immobilized bacterial cells was measured. The relative values of residual activity are shown in Table 2.

表    2 実施例3 重水濃度0及び80%の培地中での大腸菌及び酵母の生
育速度を調べた。大腸菌にはLB培地を、酵母にはYP
D培地を用い共に30℃で培養した。
Table 2 Example 3 The growth rates of Escherichia coli and yeast in medium with heavy water concentrations of 0 and 80% were investigated. LB medium for E. coli, YP for yeast
Both were cultured at 30°C using D medium.

培養経過を第1図(大腸菌)及び第2図(酵母)に示す
。重水の存在により微生物(大腸菌及び酵母)の生育が
抑制されることがわかる。
The progress of the culture is shown in Figure 1 (E. coli) and Figure 2 (yeast). It can be seen that the presence of heavy water suppresses the growth of microorganisms (E. coli and yeast).

〔発明の効果〕〔Effect of the invention〕

本発明の生体触媒安定化剤は、生体触媒の反応中及び保
存中にその劣化を防止でき、かつ生体触媒保存中の微生
物の生育を抑制することもできる。
The biocatalyst stabilizer of the present invention can prevent deterioration of the biocatalyst during reaction and storage, and can also suppress the growth of microorganisms during the storage of the biocatalyst.

【図面の簡単な説明】[Brief explanation of the drawing]

第1図は大腸菌の培養結果を示し、第2図は酵素の培養
結果を示す。
FIG. 1 shows the results of culturing E. coli, and FIG. 2 shows the results of culturing enzymes.

Claims (9)

【特許請求の範囲】[Claims] (1)重水を含む生体触媒安定化剤。(1) A biocatalyst stabilizer containing heavy water. (2)重水を30〜100重量%含む請求項1に記載の
安定化剤。
(2) The stabilizer according to claim 1, containing 30 to 100% by weight of heavy water.
(3)生体触媒が酵素又は菌体である請求項1に記載の
安定化剤。
(3) The stabilizer according to claim 1, wherein the biocatalyst is an enzyme or a bacterial cell.
(4)酵素が固定化酵素である請求項3に記載の安定化
剤。
(4) The stabilizer according to claim 3, wherein the enzyme is an immobilized enzyme.
(5)菌体が休止菌体である請求項3に記載の安定化剤
(5) The stabilizer according to claim 3, wherein the bacterial cells are dormant bacterial cells.
(6)菌体が固定化菌体である請求項3に記載の安定化
剤。
(6) The stabilizer according to claim 3, wherein the bacterial cells are immobilized bacterial cells.
(7)生体触媒を重水と共存させることを特徴とする生
体触媒の安定化法。
(7) A method for stabilizing a biocatalyst, characterized by allowing the biocatalyst to coexist with heavy water.
(8)生体触媒を重水を含む保存液中で保存する請求項
7に記載の安定化法。
(8) The stabilization method according to claim 7, wherein the biocatalyst is stored in a storage solution containing heavy water.
(9)生体触媒を重水を含む反応液中で使用する請求項
7に記載の安定化法。
(9) The stabilization method according to claim 7, wherein the biocatalyst is used in a reaction solution containing heavy water.
JP204888A 1988-01-08 1988-01-08 Stabilizer for organism catalyst Pending JPH01179689A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP204888A JPH01179689A (en) 1988-01-08 1988-01-08 Stabilizer for organism catalyst

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP204888A JPH01179689A (en) 1988-01-08 1988-01-08 Stabilizer for organism catalyst

Publications (1)

Publication Number Publication Date
JPH01179689A true JPH01179689A (en) 1989-07-17

Family

ID=11518445

Family Applications (1)

Application Number Title Priority Date Filing Date
JP204888A Pending JPH01179689A (en) 1988-01-08 1988-01-08 Stabilizer for organism catalyst

Country Status (1)

Country Link
JP (1) JPH01179689A (en)

Cited By (8)

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WO2003018614A1 (en) * 2001-08-24 2003-03-06 Wako Pure Chemical Industries, Ltd. Method of stabilizing substance altering in aqueous medium
US8911759B2 (en) 2005-11-01 2014-12-16 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US9011537B2 (en) 2009-02-12 2015-04-21 Warsaw Orthopedic, Inc. Delivery system cartridge
US9034358B2 (en) 2003-12-31 2015-05-19 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US9333082B2 (en) 2007-07-10 2016-05-10 Warsaw Orthopedic, Inc. Delivery system attachment
US9415136B2 (en) 2003-12-31 2016-08-16 Warsaw Orthopedic, Inc. Osteoinductive demineralized cancellous bone
US9554920B2 (en) 2007-06-15 2017-01-31 Warsaw Orthopedic, Inc. Bone matrix compositions having nanoscale textured surfaces
US9717822B2 (en) 2007-06-15 2017-08-01 Warsaw Orthopedic, Inc. Bone matrix compositions and methods

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003018614A1 (en) * 2001-08-24 2003-03-06 Wako Pure Chemical Industries, Ltd. Method of stabilizing substance altering in aqueous medium
US9034358B2 (en) 2003-12-31 2015-05-19 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US9415136B2 (en) 2003-12-31 2016-08-16 Warsaw Orthopedic, Inc. Osteoinductive demineralized cancellous bone
US8911759B2 (en) 2005-11-01 2014-12-16 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US8992965B2 (en) 2005-11-01 2015-03-31 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US10328179B2 (en) 2005-11-01 2019-06-25 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US9717822B2 (en) 2007-06-15 2017-08-01 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US10357511B2 (en) 2007-06-15 2019-07-23 Warsaw Orthopedic, Inc. Bone matrix compositions and methods
US10220115B2 (en) 2007-06-15 2019-03-05 Warsaw Orthopedic, Inc. Bone matrix compositions having nanoscale textured surfaces
US9554920B2 (en) 2007-06-15 2017-01-31 Warsaw Orthopedic, Inc. Bone matrix compositions having nanoscale textured surfaces
US9492278B2 (en) 2007-07-10 2016-11-15 Warsaw Orthopedic, Inc. Delivery system
US10028837B2 (en) 2007-07-10 2018-07-24 Warsaw Orthopedic, Inc. Delivery system attachment
US9333082B2 (en) 2007-07-10 2016-05-10 Warsaw Orthopedic, Inc. Delivery system attachment
US10098681B2 (en) 2009-02-12 2018-10-16 Warsaw Orthopedic, Inc. Segmented delivery system
US9220598B2 (en) 2009-02-12 2015-12-29 Warsaw Orthopedic, Inc. Delivery systems, tools, and methods of use
US9011537B2 (en) 2009-02-12 2015-04-21 Warsaw Orthopedic, Inc. Delivery system cartridge

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