JPS6142553B2 - - Google Patents
Info
- Publication number
- JPS6142553B2 JPS6142553B2 JP56114414A JP11441481A JPS6142553B2 JP S6142553 B2 JPS6142553 B2 JP S6142553B2 JP 56114414 A JP56114414 A JP 56114414A JP 11441481 A JP11441481 A JP 11441481A JP S6142553 B2 JPS6142553 B2 JP S6142553B2
- Authority
- JP
- Japan
- Prior art keywords
- tryptophan
- bacterial cells
- tryptophan synthetase
- enzyme
- synthetase
- 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.)
- Expired
Links
- 108010075344 Tryptophan synthase Proteins 0.000 claims description 15
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 7
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 claims description 7
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 claims description 7
- 229960001327 pyridoxal phosphate Drugs 0.000 claims description 7
- 238000007710 freezing Methods 0.000 claims description 5
- 230000008014 freezing Effects 0.000 claims description 5
- 244000005700 microbiome Species 0.000 claims description 5
- 239000007864 aqueous solution Substances 0.000 claims description 4
- 230000003834 intracellular effect Effects 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 description 20
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 16
- 102000004190 Enzymes Human genes 0.000 description 12
- 108090000790 Enzymes Proteins 0.000 description 12
- 230000000694 effects Effects 0.000 description 9
- 229960004799 tryptophan Drugs 0.000 description 8
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 229960001153 serine Drugs 0.000 description 3
- 238000010257 thawing Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- JXDYKVIHCLTXOP-UHFFFAOYSA-N isatin Chemical compound C1=CC=C2C(=O)C(=O)NC2=C1 JXDYKVIHCLTXOP-UHFFFAOYSA-N 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Description
本発明は、トリプトフアン・シンセターゼ（イ
ンドールとＬ−セリンとからＬ−トリプトフアン
を合成する反応を触媒する酵素）を含有する、エ
ツシエリヒア（Escherichia coli）に属する微生
物菌体を、0.0001％以上のピリドキサールリン酸
水溶液中に懸濁させてのち凍結することによつ
て、菌体内に含まれるトリプトフアン・シンセタ
ーゼの活性を低下させることなく保存するトリプ
トフアン・シンセターゼの保存方法に関するもの
である。
近年、Ｌ−トリプトフアンは医薬用のみならず
飼料用としての効果が世の注目を集めるに至り、
工業的規模による安価な本物質生産の期待が高ま
つてきている。Ｌ−トリプトフアンを製造する方
法の中で非常に有望視されている方法の１つにイ
ンドールとＬ−セリンとからＬ−トリプトフアン
を製造する酵素的生産方法がある。該反応を触媒
する酵素がトリプトフアン・シンセターゼであり
主として遺伝的に改良されたエツシエリヒア属に
属する微生物の菌体内に大量に生産される。本酵
素は菌体内に生産されるので菌体そのものを酵素
源として利用するのが工業的に有利であるが、こ
の酵素を商業的規模で使用しうるためには、酵素
活性（単位時間、単位菌体量当りのＬ−トリプト
フアン合成能）を低下させることなく、本酵素を
含む菌体を長時間保存する保存方法の確立がなけ
ればならない。
従来、一般的に酵素を含む菌体を凍結保存する
ことは知られているが、トリプトフアン・シンセ
ターゼを含む菌体の場合は、その保存方法が殆ん
ど知られておらず、本発明者らの試験によれば単
に湿菌体を凍結して保存するだけでは解凍後の菌
体の酵素活性の低下が著しく実用的な保存方法と
は言い難かつた。
本発明者らは、トリプトフアン・シンセターゼ
を含有する菌体を、その酵素活性の低下がなく、
長時間保存する方法を種々検討した結果、該菌体
を0.0001％以上のピリドキサールリン酸水溶液中
に懸濁させて後凍結することにより、湿菌体をそ
のまま凍結した場合に比して、酵素活性の低下が
著しく少なく長時間保存できることを見出し、本
発明を完成した。
本発明に使用するトリプトフアン・シンセター
ゼを菌体内に多量に生産する微生物としては、エ
ツシエリヒア・コリに属する微生物、例えばエツ
シエリヒア・コリMT−10232（FERM BP−
19）、エツシエリヒア・コリMT−10242（FERM
BP−20）などが用いられる。
ピリドキサールリン酸の濃度は0.0001％以上、
好ましくは0.001〜0.1％で使用され、懸濁する際
の菌体濃度としては、乾燥菌体濃度として５−
200ｇ/、好ましくは30〜100ｇ/の範囲であ
り、更に菌体をピリドキサールリン酸水溶液に懸
濁させた後、この懸濁液のPHを７〜９の範囲に調
整するのが望ましい。
菌体懸濁液を凍結する温度としては、−５〜−
50℃、好ましくは−10〜−30℃の範囲であり、凍
結した菌体懸濁液を融解するときの温度として
は、５〜50℃、好ましくは15〜40℃の範囲が使用
される。
本発明の保存方法によれば、菌体内に含まれる
トリプトフアン・シンセターゼを、その酵素活性
の低下も少なくしかも長時間保存することができ
るので、本発明はトリプトフアン・シンセターゼ
の工業的使用に大いに貢献するものと思われる。
以下、実施例により本発明を更に詳細に説明す
る。
実施例 １
トリプトフアン・シンセターゼ生産菌であるエ
ツシエリヒア・コリMT−10232（FERM BP−
19）を500mlの坂口フラスコ中の第１表に示す組
成の培地100mlに接種し、30℃で24時間培養し
た。この培養液200ml（フラスコ２本）を30の
ジヤーフアーメンター中の第２表に示す組成の培
地15に接種し、30℃、PH6.8（28％アンモニア
水でコントロール）で培養した。培養終了後、遠
心して集菌し、170ｇの湿菌体（乾燥菌体で36
ｇ）を得た。この湿菌体３ｇを各試験区の濃度の
ピリドキサールリン酸水溶液で10mlになるように
懸濁し、PHを8.5に調整後、−15℃で各試験区の期
間だけ凍結保存した。この凍結保存物を20℃で解
凍した後、第３表の組成の反応液を使用して、ト
リプトフアン・シンセターゼ活性を測定した。
尚、トリプトフアン・シンセターゼ活性の測定
は、活性測定用反応液を35℃で１時間反応させ、
生成されたＬ−トリプトフアンの量を液体クロマ
トグラフイーで測定し、酵素活性は単位時間、単
位菌体量当りのＬ−トリプトフアン生成量で表示
した。得られた結果を第４表に示した。
第 １ 表
エールリツヒ肉エキス 10ｇ
ポリペプトン 10ｇ
NaCl ５ｇ
蒸留水で１に希釈して使用（PH6.8）
第 ２ 表
グルコース 10 ｇ
（NH4）2SO4 １ ｇ
KH2PO4 0.5ｇ
K2HPO4 0.5ｇ
MgSO4・7H2O １ ｇ
ポリペプトン 0.5ｇ
酵母エキス 0.5ｇ
Ｌ−トリプトフアン 0.1ｇ
アデカノールLG805 ３ ｇ
蒸留水で１に希釈して使用（PH6.8）
第 ３ 表
インドール ２ ％
Ｌ−セリン 1.8 ％
トリトンＸ−100 ５ ％
ピリドキサールリン酸 0.001％
（NH4）2SO4 2.5 ％
菌体解凍液 １ ％
PH8.5
The present invention uses a microorganism belonging to Escherichia coli containing tryptophan synthetase (an enzyme that catalyzes the reaction of synthesizing L-tryptophan from indole and L-serine) to produce a pyridoxal phosphate containing 0.0001% or more of pyridoxal phosphate. The present invention relates to a method for preserving tryptophan synthetase by suspending it in an aqueous solution and then freezing it to preserve it without reducing the activity of tryptophan synthetase contained within bacterial cells. In recent years, L-tryptophan has attracted attention from the world for its effectiveness not only in medicine but also in feed.
Expectations are increasing for the production of this substance at low cost on an industrial scale. One of the most promising methods for producing L-tryptophan is an enzymatic production method for producing L-tryptophan from indole and L-serine. The enzyme that catalyzes this reaction is tryptophan synthetase, which is mainly produced in large quantities within the cells of genetically improved microorganisms belonging to the genus Escherichia. Since this enzyme is produced within the bacterial body, it is industrially advantageous to use the bacterial body itself as an enzyme source. However, in order to use this enzyme on a commercial scale, the enzyme activity (unit time, unit There is a need to establish a preservation method for preserving bacterial cells containing this enzyme for a long period of time without reducing the L-tryptophan synthesis ability per bacterial cell mass. Conventionally, it has been generally known to cryopreserve bacterial cells containing enzymes, but in the case of bacterial cells containing tryptophan synthetase, little is known about how to preserve them, and the present inventors According to the test, simply freezing and preserving wet bacterial cells resulted in a significant decrease in the enzyme activity of the bacterial cells after thawing, making it difficult to call this a practical preservation method. The present inventors have developed bacterial cells containing tryptophan synthetase without a decrease in their enzymatic activity.
As a result of examining various methods for long-term storage, we found that by suspending the bacterial cells in an aqueous solution of pyridoxal phosphate of 0.0001% or more and then freezing them, the enzyme activity was improved compared to when the wet bacterial cells were frozen directly. The present invention was completed based on the discovery that the decrease in the amount of water was significantly reduced and the product could be stored for a long period of time. Microorganisms that produce a large amount of tryptophan synthetase used in the present invention include microorganisms belonging to E. coli, such as E. coli MT-10232 (FERM BP-
19), Etzierihia coli MT-10242 (FERM
BP-20) etc. are used. The concentration of pyridoxal phosphate is 0.0001% or more,
It is preferably used at a concentration of 0.001 to 0.1%, and the bacterial cell concentration when suspended is 5-5% as a dry bacterial cell concentration.
The amount is 200g/, preferably 30 to 100g/, and after suspending the bacterial cells in an aqueous pyridoxal phosphate solution, it is desirable to adjust the pH of this suspension to a range of 7 to 9. The temperature for freezing the bacterial cell suspension is -5 to -
The temperature is 50°C, preferably -10 to -30°C, and the temperature used for thawing the frozen bacterial cell suspension is 5 to 50°C, preferably 15 to 40°C. According to the preservation method of the present invention, the tryptophan synthetase contained within the bacterial cells can be preserved for a long time with little decrease in its enzyme activity, so the present invention greatly contributes to the industrial use of tryptophan synthetase. It seems to be. Hereinafter, the present invention will be explained in more detail with reference to Examples. Example 1 Tryptophan synthetase producing bacterium E. coli MT-10232 (FERM BP-
19) was inoculated into 100 ml of a medium having the composition shown in Table 1 in a 500 ml Sakaguchi flask, and cultured at 30°C for 24 hours. 200 ml (2 flasks) of this culture solution was inoculated into medium 15 having the composition shown in Table 2 in a 30 jar fermenter, and cultured at 30°C and pH 6.8 (controlled with 28% aqueous ammonia). After culturing, collect the bacteria by centrifugation and collect 170g of wet bacteria (36g of dry bacteria).
g) was obtained. 3 g of this wet bacterial body was suspended in a pyridoxal phosphate aqueous solution having the concentration of each test group to a total volume of 10 ml, and after adjusting the pH to 8.5, it was stored frozen at -15°C for the period of each test group. After thawing this frozen product at 20°C, tryptophan synthetase activity was measured using a reaction solution having the composition shown in Table 3.
To measure tryptophan synthetase activity, react the reaction solution for activity measurement at 35°C for 1 hour.
The amount of L-tryptophan produced was measured by liquid chromatography, and the enzyme activity was expressed as the amount of L-tryptophan produced per unit time and unit amount of bacterial cells. The results obtained are shown in Table 4. Table 1 Ehrlitsug meat extract 10g Polypeptone 10g NaCl 5g Diluted to 1 with distilled water (PH6.8) Table 2 Glucose 10g (NH 4 ) 2 SO 4 1g KH 2 PO 4 0.5g K 2 HPO 4 0.5g MgSO 4・7H 2 O 1g Polypeptone 0.5g Yeast extract 0.5g L-tryptophan 0.1g Adekanol LG805 3g Diluted to 1 with distilled water (PH6.8) Table 3 Indole 2% L-serine 1.8 % Triton _
【表】
実施例 ２
トリプトフアン・シンセターゼ生産菌であるエ
ツシエリヒア・コリMT−10242（FERM BP−
20）を用いて、実施例１と同様の操作を行なつ
た。得られた結果を第５表に示した。[Table] Example 2 Tryptophan synthetase producing bacterium E. coli MT-10242 (FERM BP-
20), the same operation as in Example 1 was performed. The results obtained are shown in Table 5.
【表】【table】
Claims (1)
ツシエリヒア・コリに属する微生物菌体を0.0001
％以上のピリドキサールリン酸水溶液中に懸濁さ
せてのち凍結することを特徴とする菌体内トリプ
トフアン・シンセターゼの保存方法。1. 0.0001 microorganisms belonging to Etscherichia coli containing tryptophan synthetase.
A method for preserving intracellular tryptophan synthetase, which comprises suspending it in an aqueous solution of pyridoxal phosphate with a concentration of at least 10% and then freezing it.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56114414A JPS5816676A (en) | 1981-07-23 | 1981-07-23 | Storage of tryptophan-synthetase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56114414A JPS5816676A (en) | 1981-07-23 | 1981-07-23 | Storage of tryptophan-synthetase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5816676A JPS5816676A (en) | 1983-01-31 |
| JPS6142553B2 true JPS6142553B2 (en) | 1986-09-22 |
Family
ID=14637088
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56114414A Granted JPS5816676A (en) | 1981-07-23 | 1981-07-23 | Storage of tryptophan-synthetase |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5816676A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6410755U (en) * | 1987-07-10 | 1989-01-20 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS59147389A (en) * | 1983-02-10 | 1984-08-23 | シャープ株式会社 | Dot matrix display unit |
-
1981
- 1981-07-23 JP JP56114414A patent/JPS5816676A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6410755U (en) * | 1987-07-10 | 1989-01-20 |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5816676A (en) | 1983-01-31 |
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