JPH0424994B2 - - Google Patents
Info
- Publication number
- JPH0424994B2 JPH0424994B2 JP59014765A JP1476584A JPH0424994B2 JP H0424994 B2 JPH0424994 B2 JP H0424994B2 JP 59014765 A JP59014765 A JP 59014765A JP 1476584 A JP1476584 A JP 1476584A JP H0424994 B2 JPH0424994 B2 JP H0424994B2
- Authority
- JP
- Japan
- Prior art keywords
- glutathione
- cysteine
- glycine
- glutamic acid
- bacterial cells
- 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 - Lifetime
Links
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 claims description 51
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 34
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 32
- 108010024636 Glutathione Proteins 0.000 claims description 25
- 229960003180 glutathione Drugs 0.000 claims description 25
- 239000004471 Glycine Substances 0.000 claims description 17
- 229960002989 glutamic acid Drugs 0.000 claims description 17
- PWKSKIMOESPYIA-UHFFFAOYSA-N 2-acetamido-3-sulfanylpropanoic acid Chemical compound CC(=O)NC(CS)C(O)=O PWKSKIMOESPYIA-UHFFFAOYSA-N 0.000 claims description 14
- 244000005700 microbiome Species 0.000 claims description 12
- 239000012736 aqueous medium Substances 0.000 claims description 8
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 241000588986 Alcaligenes Species 0.000 claims description 2
- 241000186063 Arthrobacter Species 0.000 claims description 2
- 241000186146 Brevibacterium Species 0.000 claims description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 2
- 241000235035 Debaryomyces Species 0.000 claims description 2
- 241000159512 Geotrichum Species 0.000 claims description 2
- 241000235649 Kluyveromyces Species 0.000 claims description 2
- 241000192041 Micrococcus Species 0.000 claims description 2
- 241000235648 Pichia Species 0.000 claims description 2
- 241000223252 Rhodotorula Species 0.000 claims description 2
- 241000223230 Trichosporon Species 0.000 claims description 2
- 241001480014 Trigonopsis Species 0.000 claims description 2
- 241000932047 Achromobacter sp. Species 0.000 claims 1
- 241000589159 Agrobacterium sp. Species 0.000 claims 1
- 241000873310 Citrobacter sp. Species 0.000 claims 1
- 241000186249 Corynebacterium sp. Species 0.000 claims 1
- 241001527609 Cryptococcus Species 0.000 claims 1
- 241000488157 Escherichia sp. Species 0.000 claims 1
- 241000589564 Flavobacterium sp. Species 0.000 claims 1
- 241000187681 Nocardia sp. Species 0.000 claims 1
- 241000607714 Serratia sp. Species 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 24
- 238000000034 method Methods 0.000 description 14
- 239000000243 solution Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000588722 Escherichia Species 0.000 description 2
- 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 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 229910001410 inorganic ion Inorganic materials 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 241000590020 Achromobacter Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000588923 Citrobacter Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- RWSXRVCMGQZWBV-PHDIDXHHSA-N L-Glutathione Natural products OC(=O)[C@H](N)CCC(=O)N[C@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-PHDIDXHHSA-N 0.000 description 1
- 239000004201 L-cysteine Substances 0.000 description 1
- 235000013878 L-cysteine Nutrition 0.000 description 1
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- 241000187654 Nocardia Species 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000588769 Proteus <enterobacteria> Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 240000003114 Salix fragilis Species 0.000 description 1
- 241000159586 Schizoblastosporion Species 0.000 description 1
- 241000607720 Serratia Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 230000035508 accumulation Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 239000000729 antidote Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007154 intracellular accumulation Effects 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- -1 iron ions Chemical class 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 229910001425 magnesium ion Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- HPQYKCJIWQFJMS-UHFFFAOYSA-L tetrathionate(2-) Chemical compound [O-]S(=O)(=O)SSS([O-])(=O)=O HPQYKCJIWQFJMS-UHFFFAOYSA-L 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、グルタチオンの製造法に関する。
グルタチオンは、医薬用として肝疾患治療剤あ
るいは解毒剤などに使用されている重用なペプチ
ドである。
グルタチオンの製造法としては、有機合成法、
発酵法による菌体内蓄積方法及び微生物もしくは
酵素変換法が知られている。
有機合成法としては、L−グルタミン酸とL−
システインとグリシンを縮合する際に保護基を導
入、脱離する工程が必要で繁雑な方法となる。発
酵法による菌体内蓄積方法としては、酵母を用い
てL−グルタミン酸あるいはL−システインある
いはグリシンを添加しながら行う方法(特開昭48
−92579,53−94089)が知られているが、このグ
ルタチオン生成系に関与する酵素系は、ATPを
必要とする合成酵素であるので、ATPを生成す
るためにグルタチオン生成を培養と同時に行うこ
とが必須であり、又生成グルタチオンは、少量か
つ菌体内に蓄積するので、グルタチオンの採取が
困難である。
微生物もしくは酵素変換法としては、シユード
モナス属、プロテウス属、バチルス属、エンテロ
バクター属、エルビニア属に属する微生物を水性
媒体中にて、L−グルタミン酸とL−システイン
とグリシンに作用せしめる方法(特開昭57−
2698)あるいはエシエリヒア属に属する微生物を
上記と同様に、反応せしめる方法(特開昭58−
20188)が知られているが、グルタチオン生成系
に関与する酵素系はATPを必要とするので、反
応には高価なATPの添加が必須であつた。
このような背景のもとに、本発明者らは、
ATPの添加を必要とせず、また、菌株を培養し
ながらグルタチオンを生成せしめる方法ではない
安価なグルタチオンの製造法を見出すべく鋭意研
究した結果、微生物をATPを含まない水性媒体
中にてL−グルタミン酸とL−システインとグリ
シンに作用せしめるだけでグルタチオンが効率よ
く生成することを見出し、本発明を完成させるに
至つた。
即ち、本発明は、アクロモバクター属、セラチ
ア属、フラボバクテリウム属、アグロバクテリウ
ム属、エシエリヒア属、シトロバクター属、コリ
ネバクテリウム属、ザルチナ属、ノカルデイア
属、シテロマイセス属、パキソレン属、トリゴノ
プシス属、シゾブラストスポリオン属、デバリオ
マイセス属、トリコスポロン属、キヤンデイダ
属、ロドトルラ属、サツカロマイセス属、ピヒア
属、クリプロコツカス属、ジオトリカム属、トル
ロプシス属、ハンセヌラ属、アルカリゲネス属、
ブレビバクテリウム属、アースロバクター属、ミ
クロコツカス属、クルイベロマイセス属に属しL
−グルタミン酸とL−システインとグリシンを縮
合してグルタチオンを生成する能力を有する微生
物を水性媒体中にてL−グルタミン酸とL−シス
テインとグリシンに作用せしめてグルタチオンを
生成することを特徴とするグルタチオンと製造方
法である。
グルタチオンを生成する能力を有する微生物を
L−グルタミン酸とL−システインとグリシンに
作用せしめてグルタチオンに変換せしめる方法は
水溶性媒体中にてL−グルタミン酸とL−システ
インとグリシンと上記微生物の菌体、培養液ある
いは菌体処理物とを接触せしめればよい。
本発明において用いるL−グルタミン酸とL−
システインとグリシンを縮合してグルタチオンに
変換せしめる能力を有する微生物としては、例え
ば、
The present invention relates to a method for producing glutathione. Glutathione is an important peptide that is used medicinally as a treatment for liver diseases or as an antidote. Methods for producing glutathione include organic synthesis,
A fermentation method for intracellular accumulation and a microbial or enzymatic conversion method are known. As an organic synthesis method, L-glutamic acid and L-
When condensing cysteine and glycine, a step of introducing and removing a protecting group is necessary, making it a complicated method. As a method for accumulation in bacteria by fermentation, a method using yeast and adding L-glutamic acid, L-cysteine, or glycine (Japanese Unexamined Patent Publication No. 48
-92579, 53-94089), but since the enzyme system involved in this glutathione production system is a synthetic enzyme that requires ATP, it is necessary to perform glutathione production at the same time as culturing to produce ATP. is essential, and since the produced glutathione is accumulated in small amounts within the bacterial body, it is difficult to collect glutathione. As a microbial or enzymatic conversion method, a method in which microorganisms belonging to the genus Pseudomonas, Proteus, Bacillus, Enterobacter, and Erwinia are allowed to act on L-glutamic acid, L-cysteine, and glycine in an aqueous medium (Japanese Patent Application Laid-open No. 57−
2698) or a method in which microorganisms belonging to the genus Escherichia are reacted in the same manner as above (JP-A-58-
(20188), but since the enzyme system involved in the glutathione production system requires ATP, the addition of expensive ATP was essential for the reaction. Against this background, the present inventors
As a result of intensive research to find an inexpensive method for producing glutathione that does not require the addition of ATP and does not require the production of glutathione while culturing bacterial strains, we have found that microorganisms are incubated with L-glutamic acid in an aqueous medium that does not contain ATP. The present inventors have discovered that glutathione can be efficiently produced simply by acting on L-cysteine and glycine, leading to the completion of the present invention. That is, the present invention is directed to Achromobacter, Serratia, Flavobacterium, Agrobacterium, Escherichia, Citrobacter, Corynebacterium, Zarcina, Nocardia, Cytelomyces, Paxoren, and Trigonopsis. , Schizoblastosporion spp., Debaryomyces spp., Trichosporon spp., Candeida spp., Rhodotorula spp., Satucharomyces spp., Pychia spp., Cryprococcus spp., Geotrichum spp., Torulopsis spp., Hansenula spp., Alcaligenes spp.
Belongs to the genus Brevibacterium, Arthrobacter, Micrococcus, and Kluyveromyces L
- Glutathione, which is characterized in that a microorganism capable of condensing glutamic acid, L-cysteine, and glycine to produce glutathione is made to act on L-glutamic acid, L-cysteine, and glycine in an aqueous medium to produce glutathione. This is the manufacturing method. A method for converting L-glutamic acid, L-cysteine, and glycine into glutathione by allowing a microorganism capable of producing glutathione to act on L-glutamic acid, L-cysteine, and glycine is to prepare L-glutamic acid, L-cysteine, glycine, and the bacterial cells of the above microorganism in an aqueous medium. It may be brought into contact with a culture solution or a treated bacterial cell material. L-glutamic acid and L- used in the present invention
Examples of microorganisms that have the ability to condense cysteine and glycine into glutathione include:
【表】
これらの微生物の菌体を作用させてグルタチオ
ン生成せしめるにほ、通常の培地を用いて、培養
後の菌体を用いてL−グルタミン酸とL−システ
インとグリシンとを反応せしめる方法でよい。
培養後の菌体を用いる方法に用いられる培地は
L−グルタミン酸とL−システインとグリシンを
含むほかは通常の炭素源、窒素源、無機イオンを
含有する通常の培地である。
更にビタミン、アミノ酸等の有機微量栄養素を
添加すると望ましい結果が得られる場合が多い。
炭素源としては、グルコース、シユクロース等
の炭水化物、酢酸等の有機酸、アルコール類、そ
の他が適宜使用される。窒素源としては、アンモ
ニアガス、アンモニア水、アンモニウム塩、その
他が用いられる。無機イオンとしては、マグネシ
ウムイオン、燐酸イオン、カリイオン、鉄イオ
ン、その他が必要に応じ適宜使用される。
培養は好気的条件下に、PH4ないし9、温度25
ないし40℃の適当な範囲に制御しつつ1ないし10
日培養を行えば望ましい結果が得られる。
菌体としては、培養終了後の培養液そのまま、
培養液より分離された菌体、洗浄された菌体など
いずれも使用可能である。菌体処理物としては凍
結乾燥菌体、アセトン乾燥菌体、トルエン、界面
活性剤等と接触せしめた菌体、リゾチームで処理
した菌体、超音波にさらした菌体、機械的に摩砕
した菌体等のほか、これら菌体処理物から得られ
たL−グルタミン酸とL−システインとグリシン
をグルタチオンに変換せしめる酵素活性を有する
酵素蛋白区分、更には、これらの菌体の固定化
物、菌体処理物の不溶化物、その他いずれも使用
できる。
水溶性媒体としては、水、バツフアー及びエタ
ノール等の有機溶媒を含むものが使用できる。更
に必要に応じて、微生物の生育に必要な栄養素、
抗酸化剤、界面活性剤、補酵素、ヒドロキシルア
ミン及び金属イオン等を水性媒体に添加すること
もできる。
上記微生物の培養液、培養菌体あるいは菌体処
理物をL−グルタミン酸とL−システインとグリ
シンと接触せしめて作用せしめる方法には、L−
グルタミン酸とL−システインとグリシンと培養
液、培養菌体あるいは菌体処理物を溶解または懸
濁した水性媒体を10℃ないし70℃の適当な温度に
調節し、PHを4ないし9に保ちつつ、暫時静置ま
たは撹拌すればよい。かくして5ないし100時間
も経過すれば水性媒体中に多量のグルタチオンが
生成蓄積される。
生成したグルタチオンは、公知の分離方法によ
り分離精製することができる。生成したグルタチ
オンはアミノ酸アナライザーを用いて測定した。
実施例 1
グルコース 2.0g/dl、(NH4)2SO4 0.5g/
dl、KH2PO4 0.1g/dl、K2HPO4 0.1g/dl、
MgSO4・7H2O 0.05g/dl、FeSO4・7H2O 1
mg/dl、MnSO4・4H2O 1mg/dl、酵母エキス
1.0g/dll、ペプトン1.0g/dll、炭酸カルシ
ウム4.0g/dll、(別殺菌)を含む培地(PH7.0)
を500ml容フラスコに50ml入れ120℃で15分間殺菌
した。
これにブイヨン寒天培地で30℃にて、24時間培
養した表2の微生物を1白金耳接種し、30℃で20
時間培養した。この培養液より菌体を遠心分離に
より採取し、培養液と同量の生理食塩水で1回洗
浄し、菌体を集めた。
これらの菌体を表1に示す反応液Aに5g/dl
になるように添加し(終末PH6.0、5ml)、37℃に
16時間保持反応した。
この時に生成したグルタチオンをテトラチオネ
ート法(Fahey,R.C.;J.Bacteriol.121:144−
151,1975)で定量し、表2に示した。[Table] To produce glutathione by allowing the cells of these microorganisms to react, it is sufficient to use a normal medium and react L-glutamic acid, L-cysteine, and glycine using the cells after culturing. . The medium used in the method using the cultured bacterial cells is a normal medium containing L-glutamic acid, L-cysteine, and glycine, as well as normal carbon sources, nitrogen sources, and inorganic ions. Additionally, desirable results can often be obtained by adding organic micronutrients such as vitamins and amino acids. As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols, and others are used as appropriate. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, and others are used. As the inorganic ions, magnesium ions, phosphate ions, potassium ions, iron ions, and others are used as appropriate. Cultivation is carried out under aerobic conditions, pH 4 to 9, temperature 25
1 to 10 while controlling the temperature within an appropriate range of 40℃ to 40℃.
The desired results can be obtained by culturing for 1 day. As bacterial cells, the culture solution as it is after cultivation is completed,
Both bacterial cells isolated from the culture solution and washed bacterial cells can be used. The bacterial cell treatments include freeze-dried bacterial cells, acetone-dried bacterial cells, bacterial cells that have been brought into contact with toluene, surfactants, etc., bacterial cells that have been treated with lysozyme, bacterial cells that have been exposed to ultrasound, and mechanically ground bacterial cells. In addition to bacterial cells, enzyme protein fractions having enzyme activity that converts L-glutamic acid, L-cysteine, and glycine obtained from these processed bacterial cells into glutathione, as well as immobilized products of these bacterial cells, bacterial cells, etc. Insolubilized products of treated products and others can also be used. As the water-soluble medium, those containing water, buffers, and organic solvents such as ethanol can be used. Furthermore, as necessary, nutrients necessary for the growth of microorganisms,
Antioxidants, surfactants, coenzymes, hydroxylamine, metal ions, etc. can also be added to the aqueous medium. The method of bringing the culture solution, cultured cells, or treated bacterial cells of the above microorganisms into contact with L-glutamic acid, L-cysteine, and glycine to cause them to act includes the following:
Adjust an aqueous medium in which glutamic acid, L-cysteine, glycine, culture solution, cultured bacterial cells, or treated bacterial cells are dissolved or suspended to an appropriate temperature of 10°C to 70°C, and maintain the pH between 4 and 9. It may be left standing for a while or stirred. Thus, after 5 to 100 hours, a large amount of glutathione is produced and accumulated in the aqueous medium. The generated glutathione can be separated and purified by a known separation method. The generated glutathione was measured using an amino acid analyzer. Example 1 Glucose 2.0g/dl, (NH 4 ) 2 SO 4 0.5g/
dl, KH 2 PO 4 0.1g/dl, K 2 HPO 4 0.1g/dl,
MgSO 4・7H 2 O 0.05g/dl, FeSO 4・7H 2 O 1
mg/dl, MnSO 4・4H 2 O 1mg/dl, yeast extract
Medium containing 1.0g/dll, peptone 1.0g/dll, calcium carbonate 4.0g/dll, (separately sterilized) (PH7.0)
50 ml of the mixture was placed in a 500 ml flask and sterilized at 120°C for 15 minutes. One platinum loopful of the microorganisms listed in Table 2 that had been cultured on a bouillon agar medium at 30°C for 24 hours was inoculated into this, and the mixture was incubated at 30°C for 20 hours.
Cultured for hours. Bacterial cells were collected from this culture solution by centrifugation, washed once with physiological saline in the same amount as the culture solution, and collected. These bacterial cells were added to reaction solution A shown in Table 1 at 5 g/dl.
(final pH 6.0, 5 ml) and heated to 37℃.
The reaction was maintained for 16 hours. The glutathione produced at this time was converted to the tetrathionate method (Fahey, RC; J. Bacteriol. 121: 144-
151, 1975) and are shown in Table 2.
【表】【table】
【表】
コンスピキユラ
[Table] Conspicyura
【表】
ス〓フラジリス
[Table] S. fragilis
Claims (1)
クテリウム属、アグロバクテリウム属、エシエリ
ヒア属、シトロバクター属、コリネバクテリウム
属、ザルチナ属、ノカルデイア属、シテロマイセ
ス属、バキソレン属、トリゴノプシス属、シゾブ
ラストスポリオン属、デバリオマイセス属、トリ
コスポロン属、キヤンデイダ属、ロドトルラ属、
サツカロマイセス属、ビヒア属、クリプトコツカ
ス属、ジオトリカム属、トルロプシス属、ハンセ
ヌラ属、アルカリゲネス属、ブレビバクテリウム
属、アースロバクター属、ミクロコツカス属、ク
ルイベロマイセス属に属し、ATPの不存在下で
L−グルタミン酸とL−システインとグリシンか
らグルタチオンを生成する能力を有する微生物を
ATPを含まない水性媒体中にてL−グルタミン
酸とL−システインとグリシンに作用せしめて、
グルタチオンを生成することを特徴とするグルタ
チオンの製造法。1 Achromobacter sp., Serratia sp., Flavobacterium sp., Agrobacterium sp., Escherichia sp., Citrobacter sp., Corynebacterium sp., Zarutina sp., Nocardia sp., Sitelomyces sp., Baxoren sp., Trigonopsis sp., Schizoblastospo Lyon, Debaryomyces, Trichosporon, Candeida, Rhodotorula,
It belongs to the genera Satucharomyces, Vichia, Cryptococcus, Geotrichum, Torulopsis, Hansenula, Alcaligenes, Brevibacterium, Arthrobacter, Micrococcus, and Kluyveromyces, and in the absence of ATP. Microorganisms that have the ability to produce glutathione from L-glutamic acid, L-cysteine, and glycine
By acting on L-glutamic acid, L-cysteine, and glycine in an aqueous medium that does not contain ATP,
A method for producing glutathione, characterized by producing glutathione.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1476584A JPS60160894A (en) | 1984-01-30 | 1984-01-30 | Preparation of gluthathione |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP1476584A JPS60160894A (en) | 1984-01-30 | 1984-01-30 | Preparation of gluthathione |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60160894A JPS60160894A (en) | 1985-08-22 |
JPH0424994B2 true JPH0424994B2 (en) | 1992-04-28 |
Family
ID=11870167
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP1476584A Granted JPS60160894A (en) | 1984-01-30 | 1984-01-30 | Preparation of gluthathione |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60160894A (en) |
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EP1945201B1 (en) * | 2005-11-09 | 2011-09-28 | Ajinomoto Co., Inc. | Calcium receptor activator |
CN101541827A (en) | 2006-10-16 | 2009-09-23 | 协和发酵生化株式会社 | Crystal of glutathione and process for production thereof |
CN105581344A (en) * | 2015-12-16 | 2016-05-18 | 开平牵牛生化制药有限公司 | Probiotic product containing reduced glutathione and preparation method of probiotic product |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5394088A (en) * | 1977-01-27 | 1978-08-17 | Kanebo Ltd | Preparation of glutathione |
JPS5486691A (en) * | 1977-12-19 | 1979-07-10 | Tokuyama Soda Co Ltd | Preparation of glutathione |
JPS5682099A (en) * | 1979-12-07 | 1981-07-04 | Tanabe Seiyaku Co Ltd | Production of glutathion |
-
1984
- 1984-01-30 JP JP1476584A patent/JPS60160894A/en active Granted
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5394088A (en) * | 1977-01-27 | 1978-08-17 | Kanebo Ltd | Preparation of glutathione |
JPS5486691A (en) * | 1977-12-19 | 1979-07-10 | Tokuyama Soda Co Ltd | Preparation of glutathione |
JPS5682099A (en) * | 1979-12-07 | 1981-07-04 | Tanabe Seiyaku Co Ltd | Production of glutathion |
Also Published As
Publication number | Publication date |
---|---|
JPS60160894A (en) | 1985-08-22 |
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