JPH01132380A - Heat treatment of cell - Google Patents
Heat treatment of cellInfo
- Publication number
- JPH01132380A JPH01132380A JP28830587A JP28830587A JPH01132380A JP H01132380 A JPH01132380 A JP H01132380A JP 28830587 A JP28830587 A JP 28830587A JP 28830587 A JP28830587 A JP 28830587A JP H01132380 A JPH01132380 A JP H01132380A
- Authority
- JP
- Japan
- Prior art keywords
- enzyme
- bacterial cells
- cell
- tryptophan
- heat treatment
- 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
Links
- 238000010438 heat treatment Methods 0.000 title claims abstract description 24
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 41
- 229960004799 tryptophan Drugs 0.000 claims abstract description 25
- 108010075344 Tryptophan synthase Proteins 0.000 claims abstract description 6
- 230000001580 bacterial effect Effects 0.000 claims description 49
- 238000000034 method Methods 0.000 claims description 29
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 claims description 26
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 15
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 claims description 13
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 claims description 13
- 229960001153 serine Drugs 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 abstract description 40
- 102000004190 Enzymes Human genes 0.000 abstract description 40
- 239000000243 solution Substances 0.000 abstract description 21
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 12
- 230000000977 initiatory effect Effects 0.000 abstract description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 abstract description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 abstract description 4
- 239000000725 suspension Substances 0.000 abstract description 3
- 229910021529 ammonia Inorganic materials 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000002255 enzymatic effect Effects 0.000 abstract 2
- 238000010979 pH adjustment Methods 0.000 abstract 1
- 229940088598 enzyme Drugs 0.000 description 38
- 230000000694 effects Effects 0.000 description 26
- 238000006243 chemical reaction Methods 0.000 description 19
- 239000000203 mixture Substances 0.000 description 10
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 9
- 238000006911 enzymatic reaction Methods 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 238000000108 ultra-filtration Methods 0.000 description 7
- 239000007788 liquid Substances 0.000 description 6
- 238000003786 synthesis reaction Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 230000000813 microbial effect Effects 0.000 description 5
- 238000005119 centrifugation Methods 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 239000012466 permeate Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000000178 monomer Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000000284 resting effect Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 235000010410 calcium alginate Nutrition 0.000 description 2
- 239000000648 calcium alginate Substances 0.000 description 2
- 229960002681 calcium alginate Drugs 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 2
- 239000003518 caustics Substances 0.000 description 2
- 239000000306 component Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 2
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 2
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 2
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 2
- 238000001223 reverse osmosis Methods 0.000 description 2
- 235000010413 sodium alginate Nutrition 0.000 description 2
- 239000000661 sodium alginate Substances 0.000 description 2
- 229940005550 sodium alginate Drugs 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KISBHDLCXWCSEM-UHFFFAOYSA-L calcium dichloride trihydrate Chemical compound O.O.O.[Cl-].[Cl-].[Ca+2] KISBHDLCXWCSEM-UHFFFAOYSA-L 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012527 feed solution Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000003456 ion exchange resin Substances 0.000 description 1
- 229920003303 ion-exchange polymer Polymers 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 235000011118 potassium hydroxide Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- IFGCUJZIWBUILZ-UHFFFAOYSA-N sodium 2-[[2-[[hydroxy-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyphosphoryl]amino]-4-methylpentanoyl]amino]-3-(1H-indol-3-yl)propanoic acid Chemical compound [Na+].C=1NC2=CC=CC=C2C=1CC(C(O)=O)NC(=O)C(CC(C)C)NP(O)(=O)OC1OC(C)C(O)C(O)C1O IFGCUJZIWBUILZ-UHFFFAOYSA-N 0.000 description 1
- 235000011121 sodium hydroxide Nutrition 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000057 synthetic resin Substances 0.000 description 1
- 229920003002 synthetic resin Polymers 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
童栗上■肌■分黙
本発明は、トリプトファン・シンターゼを含む菌体の熱
処理方法に関する。更に詳しくは、インドール及びＬ−
セリンからＬ−トリプトファンを製造するに用いる菌体
（以下、菌体と称することがある）を、保存または酵素
反応に用いるに際し、培養した菌体を熱処理して、酵素
の初期活性を向上せしめると共に、保存または酵素反応
における酵素の活性を低下させること無く長期間保たし
める為の、菌体の熱処理方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for heat treating bacterial cells containing tryptophan synthase. More specifically, indole and L-
When preserving the bacterial cells used to produce L-tryptophan from serine (hereinafter sometimes referred to as bacterial cells) or using them in enzyme reactions, the cultured bacterial cells are heat-treated to improve the initial activity of the enzyme and The present invention relates to a heat treatment method for bacterial cells to preserve them for a long period of time without reducing the activity of enzymes in storage or enzyme reactions.
本発明は、所謂バイオリアクターの運転に用いられる酵
素を含む菌体を保存または酵素反応に利用するに際し、
該酵素の初期活性を向上せしめると共に、酵素の活性を
低下させること無く長期間保たしめようとする場合に利
用される。The present invention provides for the preservation or use of enzyme-containing microbial cells used in the operation of so-called bioreactors for enzyme reactions.
It is used when it is desired to improve the initial activity of the enzyme and maintain the enzyme activity for a long period of time without decreasing the activity.
従来■肢術
トリプトファンは輸液成分などの医薬品、睡眠誘発剤な
どの健康食品として従来から利用されて来たが、近年飼
料添加物としての有効性が認められて需要が伸びつつあ
り、安価な製造法の開発が待たれているところである。Traditionally Tryptophan has traditionally been used as an infusion component and other pharmaceuticals, and as a sleep inducer and other health foods, but in recent years its effectiveness as a feed additive has been recognized and demand has been increasing, making it cheaper to manufacture. The development of a law is awaited.
トリプトファンの製造方法としては、化学合成法、発酵
法、および酵素法などが従来から知られている。化学合
成法は合成経路が長く複雑であり、合成されたトリプト
ファンは、Ｌ一体とロ一体の等景況合物であるラセミ体
であり光学分割を行って有効なし一体だけを分離する必
要がある。一方発酵法または酵素法では有効なし一体の
みが生成されるので光学分割の必要も無く有利である。Chemical synthesis methods, fermentation methods, enzymatic methods, and the like are conventionally known as methods for producing tryptophan. The chemical synthesis method has a long and complicated synthesis route, and the synthesized tryptophan is a racemic compound that is an isochemical compound of L and L monomers, and it is necessary to perform optical resolution to separate only the active and non-active monomers. On the other hand, the fermentation method or the enzyme method is advantageous because only effective monomers are produced and there is no need for optical resolution.
しかしながら、発酵法においても、Ｉ！’Ｍなどの培地
成分または微生物菌体と生成物であるトリプトファンが
発酵液中に混合して得られるので、目的のトリプトファ
ンを単離、精製することが非常に困難で効率が悪く、手
間がかかるなどの難点がある。However, even in the fermentation method, I! Since the tryptophan product is obtained by mixing culture medium components such as M or microbial cells and the product tryptophan in the fermentation liquid, it is extremely difficult, inefficient, and time-consuming to isolate and purify the target tryptophan. There are other difficulties.
酵素法で使用される酵素源としては、目的の酵素を生産
する菌体の培養液から遠心分離等で得られる休止菌体、
或いは菌体から抽出法などにより分離された酵素などが
用いられる。また近年は菌体または酵素をアルギン酸カ
ルシウムなどの天然高分子物質などで固定化して用いる
方法であるとか、溶液に懸濁した菌体または酵素を逆浸
透膜または限外ろ過膜などで系内に閉じ込めて使用する
方法なども報告されている。Enzyme sources used in the enzyme method include resting bacterial cells obtained by centrifugation from the culture solution of bacterial cells that produce the desired enzyme;
Alternatively, an enzyme isolated from bacterial cells by an extraction method or the like may be used. In addition, in recent years, methods have been developed in which bacteria or enzymes are immobilized with natural polymeric substances such as calcium alginate, or bacteria or enzymes suspended in a solution are injected into the system using reverse osmosis membranes or ultrafiltration membranes. There have also been reports of methods of using it in a confined manner.
特に後の二つの方法は目的生産物と菌体または酵素との
分離が容易であるとか、或いは菌体または酵素を繰り返
し使用できるので、その使用量が少なくてすむなどの利
点があり、近年特に注目を集めている方法である。In particular, the latter two methods have the advantage that it is easy to separate the target product from the bacterial cells or enzymes, or that the amount used is small because the bacterial cells or enzymes can be used repeatedly. This is a method that is attracting attention.
日がゞ しようとする問題占
しかしながら、酵素法においても、粗または精製酵素を
用いる場合には問題とならないが、休止菌体などの菌体
を反応に使用しようとする場合には以下のような問題点
がある。即ち、菌体を長期間保存する必要が有る場合、
或いは菌体を固定化（以下、固定化酵素と称することが
ある）して反応に長期間使用したい場合に、固定化酵素
の活性を実質的に低下させること無く長期間維持させる
ことは極めて困難である。特に固定化酵素を使用する有
価物製造プロセスにおいては、固定化酵素の活性の寿命
の長短がプロセス全体の成否を決定すると言っても過言
ではなく、反応の場において酵素活性を長期間低下させ
ることなく維持することは極めて重要である。菌体中の
酵素活性の低下の原因については種々考えられるが、本
発明に用いる酵素の場合には、菌体中に含まれる蛋白分
解酵素により目的反応に有効な酵素が分解されてしまう
事によるものと推定される。However, even in the enzymatic method, there is no problem when using crude or purified enzymes, but when using bacterial cells such as resting bacterial cells in the reaction, the following problems occur. There is a problem. In other words, if it is necessary to preserve the bacterial cells for a long period of time,
Alternatively, if you want to immobilize bacterial cells (hereinafter sometimes referred to as immobilized enzymes) and use them for a long period of time in a reaction, it is extremely difficult to maintain the activity of the immobilized enzymes for a long period of time without substantially reducing their activity. It is. Particularly in the process of producing valuables using immobilized enzymes, it is no exaggeration to say that the longevity of the activity of the immobilized enzyme determines the success or failure of the entire process. It is extremely important to maintain this without any problems. There are various possible causes for the decrease in enzyme activity in the bacterial cells, but in the case of the enzyme used in the present invention, it is due to the enzyme effective for the target reaction being degraded by the proteolytic enzymes contained in the bacterial cells. It is estimated that
さて、本発明者らは、休止菌体を用いて酵素法によりイ
ンドール及びＬ−セリンからＬ−トリプトファンを製造
する方法を研究するに際し、培養した菌体を遠心分離し
た後、冷蔵庫などの冷暗所に保存しておいたり、或いは
懸濁または固定化して目的の反応に使用すると短時間の
内に酵素の活性が低下してしまい使用に耐えなくなって
しまうという基本的で重大な問題に直面した。In researching a method for producing L-tryptophan from indole and L-serine by an enzymatic method using resting bacterial cells, the present inventors centrifuged the cultured bacterial cells and then stored them in a cool, dark place such as a refrigerator. We encountered a fundamental and serious problem in that if the enzyme was stored, suspended, or immobilized and used for the desired reaction, the enzyme activity would decrease within a short period of time, making it unusable.
口 占を”するための
そこで本発明者らは、この問題点を解決すべく鋭意研究
した結果、培養した菌体を熱処理することによって、長
期間酵素活性を低下させること無く維持させ、また酵素
の初期活性をも向上せしめる方法を見出し、本発明を完
成するに至った。As a result of intensive research to solve this problem, the present inventors succeeded in maintaining the enzyme activity for a long period of time without reducing the enzyme activity by heat-treating the cultured bacterial cells. The present inventors have discovered a method of improving the initial activity of .
すなわち、本発明は、インドール及びＬ−セリンからＬ
−トリプトファンを生成させるトリプトファン・シンタ
ーゼを含む菌体を、培養液から集菌後保存する場合、或
いはこの菌体を懸濁または固定化して利用しようとする
場合に、培養した菌体をｐＨ６〜９の条件下で４５℃以
上で熱処理することを特徴とした菌体の熱処理方法であ
る。That is, the present invention provides L-serine from indole and L-serine.
- When storing bacterial cells containing tryptophan synthase, which produces tryptophan, after collecting them from a culture solution, or when intending to suspend or immobilize the bacterial cells and use them, the cultured bacterial cells should be kept at a pH of 6 to 9. This is a heat treatment method for bacterial cells characterized by heat treatment at 45°C or higher under the following conditions.
以下、本発明について詳しく説明する。The present invention will be explained in detail below.
本発明の方法に使用するトリプトファン・シンターゼの
生産菌としては、エシェリヒア・コリＭＴ−１０２３２
（ＦＥＲＭ ＢＰ−１９）、エシェリヒア・コリＭＴ−
１０２４２（ＦＥＲＭ ＢＰ−２０）、ノイスボ・クラ
ッサＡＴＣＣ１４６９２などを挙げることが出来る。As the tryptophan synthase producing bacteria used in the method of the present invention, Escherichia coli MT-10232
(FERM BP-19), Escherichia coli MT-
10242 (FERM BP-20), Neusbo Klassa ATCC 14692, etc.
熱処理に供する菌体の形態としては、菌体を培養した培
養液の状態でも良いし、集菌した菌体を緩衝液に懸濁さ
せた状態でも良い。熱処理後遠心分離などで菌体を集菌
し、保存またはＬ−トリプトファンの合成反応に使用す
る。The form of the bacterial cells to be subjected to the heat treatment may be in the form of a culture solution in which the bacterial cells have been cultured, or in the form of collected bacterial cells suspended in a buffer solution. After heat treatment, the bacterial cells are collected by centrifugation or the like, and stored or used for the synthesis reaction of L-tryptophan.
熱処理時の温度は、４５℃以上、更に好ましくは４５〜
７５℃の範囲である。酵素活性の劣化は温度に敏感に左
右されるので、熱処理温度は十分に管理する必要がある
。本発明に係る酵素の場合は、３０〜４０″Ｃに於いて
最も高い活性を示すが、熱処理温度が４５℃未満である
と目的酵素以外の例えば蛋白分解酵素などを失活させる
ことが出来ず、本発明の目的を達し得ない。また７５℃
を越える温度では本来の酵素の活性が低下してしまうの
で好ましくない。The temperature during heat treatment is 45°C or higher, more preferably 45°C or higher.
The temperature range is 75°C. Since the deterioration of enzyme activity is sensitive to temperature, the heat treatment temperature must be carefully controlled. In the case of the enzyme according to the present invention, the highest activity is shown at 30 to 40"C, but if the heat treatment temperature is lower than 45"C, other enzymes other than the target enzyme, such as proteolytic enzymes, cannot be inactivated. , the object of the present invention cannot be achieved.
Temperatures exceeding this temperature are not preferred because the original enzyme activity decreases.
ない。do not have.
熱処理時のｐＨは、６〜９の範囲が好ましく、更に好ま
しくは６．５〜８．０の範囲である。本発明で使用する
酵素は、ｐＨ７，５〜８．５に於いて最も高い活性を示
すが、熱処理時のｐＨも温度と同様に十分に上記範囲に
管理する必要がある。The pH during heat treatment is preferably in the range of 6 to 9, more preferably in the range of 6.5 to 8.0. The enzyme used in the present invention exhibits the highest activity at a pH of 7.5 to 8.5, but the pH during heat treatment must be sufficiently controlled within the above range as well as the temperature.
ｐＨを調整する為の薬剤としては一般的に多用されてい
る、アンモニア、苛性ソーダ、苛性カリ、塩酸、硫酸な
どが使用できる。Commonly used agents for adjusting pH include ammonia, caustic soda, caustic potash, hydrochloric acid, and sulfuric acid.
また熱処理時間は、５５℃程度で熱処理するときは、１
０〜２０分の範囲が適当であるが、目的酵素の活性を低
下させることなく熱処理の目的を達成させる為には、処
理温度が高い場合には短時間で、処理温度が低い場合に
は、より長い時間で処理することが好ましい。In addition, the heat treatment time is 1
A range of 0 to 20 minutes is appropriate, but in order to achieve the purpose of heat treatment without reducing the activity of the target enzyme, a short time is required when the treatment temperature is high, and a short time is required when the treatment temperature is low. It is preferable to process for a longer time.
上記のように熱処理を実施した後、遠心分離などで得た
菌体を保存またはＬ−トリプトファンを製造する反応に
使用すると、酵素の活性が低下すること無く長期間維持
される。When the microbial cells obtained by centrifugation or the like after heat treatment as described above are stored or used in a reaction to produce L-tryptophan, the enzyme activity is maintained for a long period of time without decreasing.
菌体を保存する方法としては、合成樹脂またはステンレ
スなどの金属製密閉容器に該菌体を格納して、冷凍庫ま
たは冷蔵庫内に保管すれば良い。As a method for preserving the bacterial cells, the bacterial cells may be stored in a sealed container made of synthetic resin or metal such as stainless steel, and stored in a freezer or refrigerator.
また酵素反応によりインドール及びＬ−セリンからＬ−
トリプトファンを製造するのに用いる方法としては、特
に制限は無いが、例えば、該菌体を反応仕込液中に懸濁
して用いる方法、アルギン酸カルシウムゲル、アクリル
アミド重合物などで固定化して用いる方法、更にまた逆
浸透膜または限外ろ過膜などで反応系内に菌体を実質的
に閉じ込めて使用するか、または使用した菌体を回収再
利用する方法などを挙げることが出来る。本発明の方法
は菌体を固定化して長時間、反応に使用しようとする場
合に特に有効である。Also, by enzymatic reaction, indole and L-serine are converted to L-
There are no particular restrictions on the method used to produce tryptophan, but examples include a method in which the bacterial cells are suspended in a reaction preparation solution, a method in which they are immobilized with calcium alginate gel, an acrylamide polymer, etc. Other methods include using a reverse osmosis membrane or an ultrafiltration membrane to substantially confine the bacterial cells within the reaction system, or collecting and reusing the used bacterial cells. The method of the present invention is particularly effective when immobilizing bacterial cells and using them for long-term reactions.
詐■
本発明の方法により熱処理した菌体は、酵素の初期活性
が向上する上に、冷蔵庫などで酵素の活性を長期間低下
させること無く保存することが出来、また菌体を固定化
して使用する場合にも長期間活性を維持させることが可
能となり、固定化酵素を用いたＬ−トリプトファンの工
業的な製造が可能となったのである。更に、菌体を懸濁
して利用する場合にも、例えば限外ろ過膜で菌体を回収
して再利用することが可能となった。In addition to improving the initial activity of the enzyme, the microbial cells heat-treated by the method of the present invention can be stored in a refrigerator for a long period of time without reducing the enzyme activity, and the microbial cells can be immobilized for use. It has become possible to maintain the activity for a long period of time even when the enzyme is immobilized, and it has become possible to industrially produce L-tryptophan using an immobilized enzyme. Furthermore, even when the bacterial cells are used in suspension, it has become possible to collect and reuse the bacterial cells using, for example, an ultrafiltration membrane.
実隻開 以下、本発明を実施例により更に具体的に説明する。Actual ship opening EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例１
トリプトファン・シンターゼ生産菌であるエシェリヒア
・コリＭＴ−１０２３２（ＦＥＲＭ ＢＰ−１９）を５
００ｍ （ｌの坂ロフラスコ中の第１表に示す組成の培
地１００ｍ ｌに接種し、３５℃で２４時間培養した。Example 1 Escherichia coli MT-10232 (FERM BP-19), which is a tryptophan synthase-producing bacterium, was
The mixture was inoculated into 100 ml of a medium having the composition shown in Table 1 in a Sakaro flask, and cultured at 35°C for 24 hours.
この培養液２００ｍ１（フラスコ２本）を３０１！、の
ジャーファーメンタ−中の第２表に示す組成の培地１５
Ｉ！、に接種し、３５℃，ｐＨ６，８（２８χアンモニ
ア水で調整）で３０時間培養した。200ml of this culture solution (2 flasks) costs 301! Medium 15 with the composition shown in Table 2 in Jar Fermentor of
I! , and cultured at 35°C and pH 6.8 (adjusted with 28x ammonia water) for 30 hours.
培養液を分取して、第３表に示した条件で熱処理後、遠
心集菌して酵素の活性を測定し、熱処理の効果及び熱処
理条件の比較を行った。The culture solution was separated, heat-treated under the conditions shown in Table 3, and then centrifuged to collect the bacteria to measure enzyme activity, and the effects of heat treatment and heat treatment conditions were compared.
酵素の活性の測定は以下の通り行った。Enzyme activity was measured as follows.
インドール、Ｌ−セリン、ピリドキサール−５゛−リン
酸（ＰＬＰ）及び菌体を乾燥菌体として０．２ｇ＃！含
む反応液を１０ｍ１調合し、振とう培養機で、３５℃１
１時間振とうして反応を行い、生成したし一トリプトフ
ァンを高速液体クロマトグラフィーで分析した。Indole, L-serine, pyridoxal-5'-phosphate (PLP) and bacterial cells as dried bacterial cells 0.2g #! Prepare 10ml of the reaction solution containing the
The reaction was carried out by shaking for 1 hour, and the produced tryptophan was analyzed by high performance liquid chromatography.
第１表 培養培地の組成
蒸留水１１に希釈して使用（ｐＨ６，８）第２表 増殖
培地の組成
蒸留水１１に希釈して使用（ｐＨ６，８）（以下余白）
第３表 熱処理の効果
実施例２
実施例１と同様に培養して、熱処理をしない菌体と熱処
理を行った菌体を得た。熱処理条件は、ｐｕ ７．０、
温度５５℃ｌ２Ｏ分である。これらの菌体を密封容器に
入れ３〜５℃の冷蔵庫に保管して所定時間経過後の活性
を測定し、その結果を第４表に示した。Table 1 Composition of culture medium used diluted in distilled water 11 (pH 6, 8) Table 2 Composition of growth medium used diluted in distilled water 11 (pH 6, 8) (blank below) Table 3 Effect of heat treatment Example 2 Cells were cultured in the same manner as in Example 1 to obtain cells that were not heat-treated and cells that were heat-treated. The heat treatment conditions were pu 7.0,
The temperature was 55°C and 120 minutes. These bacterial cells were placed in a sealed container and stored in a refrigerator at 3 to 5°C, and the activity was measured after a predetermined period of time, and the results are shown in Table 4.
（以下余白） 第４表 菌体の保存結果 保存後の活性は、初期の活性に対する相対値で示した。(Margin below) Table 4: Storage results of bacterial cells The activity after storage was expressed as a relative value to the initial activity.
実施例３
実施例１と同様にして得た、熱処理をしない菌体と、ｐ
Ｈを変えて熱処理を実施した菌体を用いて、固定化酵素
を作成し、インドールとし一セリンからＬ−トリプトフ
ァンを合成する反応を実施して、酵素活性の寿命を見た
。インドールを基準としたＬ−トリプトファンの収率が
５０χに落ちるまでの時間を酵素の寿命としその結果を
第５表に示した。Example 3 Bacterial cells obtained in the same manner as in Example 1 without heat treatment and p.
An immobilized enzyme was prepared using bacterial cells that had been heat-treated with different amounts of H, and a reaction was carried out to synthesize L-tryptophan from indole and monoserine to examine the lifetime of the enzyme activity. The time required for the yield of L-tryptophan to fall to 50x based on indole was defined as the life of the enzyme, and the results are shown in Table 5.
固定化酵素の作成は以下の要領で行った。The immobilized enzyme was prepared as follows.
遠心集菌した湿菌体１部と生理食塩液１部とを撹はん混
合した。一方蒸留水７．７６部と、アルギン酸ナトリウ
ム（記文フードケミファ社製Ｎ５ＰＬＬ ）０．２４部
とを撹はん混合しｐＨを８．５に苛性カリで調整した。One part of wet bacterial cells collected by centrifugation and one part of physiological saline were mixed by stirring. On the other hand, 7.76 parts of distilled water and 0.24 parts of sodium alginate (N5PLL manufactured by Kibun Food Chemifa) were mixed with stirring, and the pH was adjusted to 8.5 with caustic potassium.
菌体の懸濁液２部と、上記のアルギン酸ナトリウムの溶
解液８部を撹はん混合し、注射器に充填、内径が０．５
〜０．８ｍｍ程度の注射針の先端より、ゲル化液に滴下
した。ゲル化液は、０．５モル濃度の塩化カルシウム三
水塩水溶液を６規定苛性カリ水溶液でｐＨを８．５に調
整し、１０℃に保った液を５０部使用した。ゲル化液に
滴下されて生成した粒子は液中で約１時間撹はん熟成後
、液中より取り出し固定化酵素源として使用した。Stir and mix 2 parts of the suspension of bacterial cells and 8 parts of the above sodium alginate solution, and fill it into a syringe with an inner diameter of 0.5.
It was dropped into the gelatinized liquid from the tip of an approximately 0.8 mm injection needle. The gelling solution used was 50 parts of a 0.5 molar calcium chloride trihydrate aqueous solution whose pH was adjusted to 8.5 with a 6N caustic potassium aqueous solution and kept at 10°C. The particles produced by dropping into the gelling solution were stirred and aged in the solution for about 1 hour, and then taken out from the solution and used as an immobilized enzyme source.
また上記で作成した固定化酵素を用いてＬ−トリプトフ
ァンを合成して酵素活性の寿命を見る反応試験は以下の
要領で実施した。In addition, a reaction test was conducted in the following manner to synthesize L-tryptophan using the immobilized enzyme prepared above and to determine the lifetime of the enzyme activity.
５００ｍ１の撹はん機付ガラス製反応器に第６表に示し
た組成からインドールだけを除いた溶液１００ｍ１と固
定化酵素５０ｍ２を装入し、この反応器を温水浴中に保
持して温度を常に３０℃に保った。次に第６表で示した
組成の反応供給液を、内容を撹はんしている反応器に毎
時５０ｍ ｌの速度で連続的に供給し、一方反応器から
も同速度で連続的に抜液を行い、一定時間毎のサンプル
をとり生成したＬ−トリプトファン濃度及び残存インド
ール、Ｌ−セリン濃度を液体クロマトグラフ法により求
めた。A 500 ml glass reactor equipped with a stirrer was charged with 100 ml of the solution shown in Table 6 with only indole removed and 50 m2 of the immobilized enzyme, and the reactor was kept in a hot water bath to maintain the temperature. The temperature was always kept at 30°C. Next, the reaction feed liquid having the composition shown in Table 6 was continuously fed at a rate of 50 ml/hour to the reactor whose contents were being stirred, while it was continuously withdrawn from the reactor at the same rate. The solution was sampled at regular intervals, and the concentration of L-tryptophan produced and the concentrations of residual indole and L-serine were determined by liquid chromatography.
反応の結果は、供給したインドール量を基準として生成
したＬ−トリプトファンの収率を求め、この収率が良好
に維持される時間の長さで検討した。The reaction results were determined by determining the yield of L-tryptophan produced based on the amount of indole supplied, and examining the length of time during which this yield was maintained satisfactorily.
この収率が１００χである場合とは、反応器から抜液し
た液中のＬ−トリプトファン濃度が４．３６ｇ／ ｌに
達する場合である。The case where the yield is 100χ is the case where the L-tryptophan concentration in the liquid drained from the reactor reaches 4.36 g/l.
第５表 固定化酵素の寿命
酵素の寿命：インドールに対する収率が５０χに低下す
るまでの時間
熱処理条件：温度５５℃１時間２０分
第６表 反応供給液の組成
ｐＨ８，５
実施例４
熱処理条件をｐｔ＋ ７．５、温度５５℃１時間２０分
として処理して得た菌体を、第６表に示した組成の液か
らインドール及び塩化カルシウムを除いた組成の反応液
５０！に乾燥菌体として１０ｇ／ ｌの濃度で仕込み、
ジャケット及び撹はん機付ステンレス製反応器内で撹は
んしなから３５℃に保った。この反応器に第６表で示し
た組成の液から塩化カルシウムを除いた組成の反応液を
、毎時１８１の速度で供給し、Ｌ−トリプトファンの合
成反応を行なわしめた。同時に反応液の一部を連続的に
抜き出して、限外ろ過装置に供給して液中に生成したＬ
−１−リプトファンを限外ろ過膜の透過液側に透過せし
め、菌体を非透過液側に残した。限外ろ過装置はＤＤＳ
社製ＧＲ８１ＰＰ膜を０．３６ｍ２セツトし、供給液量
８１分、３５℃１５Ｋｇ／ｃｍ２の条件で使用した。透
過液中に溶解しているＬ−トリプトファンはイオン交換
樹脂吸着法などの常法により分離回収した。また菌体を
含んだ非透過液はそのまま反応器に循環してＬ−トリプ
トファンの合成反応に供した。反応器、限外ろ過装置は
連続運転を行い、インドールに対するＬ−トリプトファ
ンの収率が５０χに低下するまで１２０時間の運転を継
続することができた。Table 5 Lifespan of immobilized enzyme Enzyme lifespan: Time until yield decreases to 50χ Heat treatment conditions: Temperature 55°C 1 hour 20 minutes Table 6 Composition of reaction feed solution pH 8.5 Example 4 Heat treatment conditions The bacterial cells obtained by treating the cells at a temperature of 55° C. for 1 hour and 20 minutes at a pt+ of 7.5 and a reaction solution having a composition shown in Table 6 but excluding indole and calcium chloride were added to the reaction solution 50! Prepared as dried bacterial cells at a concentration of 10g/l,
The temperature was maintained at 35° C. without stirring in a stainless steel reactor with jacket and stirrer. A reaction solution having the composition shown in Table 6 except for calcium chloride was supplied to this reactor at a rate of 181/hour to carry out the synthesis reaction of L-tryptophan. At the same time, a part of the reaction solution is continuously extracted and supplied to the ultrafiltration device to generate L produced in the solution.
-1-Liptophan was allowed to permeate into the permeate side of the ultrafiltration membrane, and the bacterial cells were left on the non-permeate side. The ultrafiltration device is DDS
0.36 m2 of GR81PP membrane manufactured by Co., Ltd. was set, and the amount of liquid supplied was 81 minutes, and it was used at 35° C. and 15 kg/cm2. L-tryptophan dissolved in the permeate was separated and recovered by a conventional method such as ion exchange resin adsorption method. Further, the non-permeated liquid containing bacterial cells was directly circulated to the reactor and subjected to the synthesis reaction of L-tryptophan. The reactor and ultrafiltration device were operated continuously, and the operation could be continued for 120 hours until the yield of L-tryptophan based on indole decreased to 50χ.
特許出願人 三井東圧化学株式会社Patent applicant: Mitsui Toatsu Chemical Co., Ltd.
Claims (1)
を製造するに用いるトリプトファン・シンターゼを含む
菌体を、ｐＨ６〜９の条件下で４５℃以上で加熱処理す
ることを特徴とする菌体の熱処理方法。1) A method for heat treatment of bacterial cells, which comprises heating bacterial cells containing tryptophan synthase used for producing L-tryptophan from indole and L-serine at 45° C. or higher under conditions of pH 6 to 9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28830587A JPH01132380A (en) | 1987-11-17 | 1987-11-17 | Heat treatment of cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28830587A JPH01132380A (en) | 1987-11-17 | 1987-11-17 | Heat treatment of cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01132380A true JPH01132380A (en) | 1989-05-24 |
Family
ID=17728449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28830587A Pending JPH01132380A (en) | 1987-11-17 | 1987-11-17 | Heat treatment of cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01132380A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7900804B2 (en) | 2004-07-20 | 2011-03-08 | Max Co., Ltd. | Stapler and staple cartridge |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58129972A (en) * | 1982-01-28 | 1983-08-03 | Mitsui Toatsu Chem Inc | Method for suppressing activity of serine decomposing enzyme in bacterial cell |
-
1987
- 1987-11-17 JP JP28830587A patent/JPH01132380A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS58129972A (en) * | 1982-01-28 | 1983-08-03 | Mitsui Toatsu Chem Inc | Method for suppressing activity of serine decomposing enzyme in bacterial cell |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7900804B2 (en) | 2004-07-20 | 2011-03-08 | Max Co., Ltd. | Stapler and staple cartridge |
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