JPH04304892A - Biological production of glycine - Google Patents
Biological production of glycineInfo
- Publication number
- JPH04304892A JPH04304892A JP8918891A JP8918891A JPH04304892A JP H04304892 A JPH04304892 A JP H04304892A JP 8918891 A JP8918891 A JP 8918891A JP 8918891 A JP8918891 A JP 8918891A JP H04304892 A JPH04304892 A JP H04304892A
- Authority
- JP
- Japan
- Prior art keywords
- glycine
- glycinamide
- genus
- microorganism
- pseudomonas
- 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
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 239000004471 Glycine Substances 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title claims description 10
- BEBCJVAWIBVWNZ-UHFFFAOYSA-N glycinamide Chemical compound NCC(N)=O BEBCJVAWIBVWNZ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 244000005700 microbiome Species 0.000 claims abstract description 18
- 241000186216 Corynebacterium Species 0.000 claims abstract description 16
- 241000589516 Pseudomonas Species 0.000 claims abstract description 8
- 241000589291 Acinetobacter Species 0.000 claims abstract description 7
- 241000588914 Enterobacter Species 0.000 claims abstract description 7
- 241000316848 Rhodococcus <scale insect> Species 0.000 claims abstract description 7
- 241000588986 Alcaligenes Species 0.000 claims abstract description 5
- 241000186063 Arthrobacter Species 0.000 claims abstract description 5
- 241000187747 Streptomyces Species 0.000 claims abstract description 5
- 238000000034 method Methods 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 230000003301 hydrolyzing effect Effects 0.000 claims description 3
- 150000001408 amides Chemical class 0.000 claims description 2
- 238000012258 culturing Methods 0.000 abstract description 4
- 239000002994 raw material Substances 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 2
- 239000002778 food additive Substances 0.000 abstract description 2
- 235000013373 food additive Nutrition 0.000 abstract description 2
- 239000003905 agrochemical Substances 0.000 abstract 1
- 230000031018 biological processes and functions Effects 0.000 abstract 1
- 230000001580 bacterial effect Effects 0.000 description 16
- 238000006243 chemical reaction Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 8
- 241000187562 Rhodococcus sp. Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 238000006460 hydrolysis reaction Methods 0.000 description 6
- 230000007062 hydrolysis Effects 0.000 description 5
- 239000008363 phosphate buffer Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 241000589774 Pseudomonas sp. Species 0.000 description 4
- 241000186073 Arthrobacter sp. Species 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 101100398412 Arabidopsis thaliana ASK1 gene Proteins 0.000 description 2
- 101100110018 Arabidopsis thaliana ASK3 gene Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 150000008575 L-amino acids Chemical class 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000187392 Streptomyces griseus Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- LELOWRISYMNNSU-UHFFFAOYSA-N hydrogen cyanide Chemical compound N#C LELOWRISYMNNSU-UHFFFAOYSA-N 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- -1 nitrile compounds Chemical class 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000590020 Achromobacter Species 0.000 description 1
- 241000588625 Acinetobacter sp. Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000588810 Alcaligenes sp. Species 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000186312 Brevibacterium sp. Species 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 241000147019 Enterobacter sp. Species 0.000 description 1
- 229940127463 Enzyme Inducers Drugs 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 241000588843 Ochrobactrum Species 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- DFNYGALUNNFWKJ-UHFFFAOYSA-N aminoacetonitrile Chemical compound NCC#N DFNYGALUNNFWKJ-UHFFFAOYSA-N 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- FDIWRLNJDKKDHB-UHFFFAOYSA-N azanium;2-aminoacetate Chemical compound [NH4+].NCC([O-])=O FDIWRLNJDKKDHB-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000003518 caustics Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- NBZBKCUXIYYUSX-UHFFFAOYSA-N iminodiacetic acid Chemical compound OC(=O)CNCC(O)=O NBZBKCUXIYYUSX-UHFFFAOYSA-N 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000011833 salt mixture Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明はグリシンアミドから微生
物を用いることによりグリシンを製造する方法に関する
ものである。グリシンは食品添加物、医農薬合成原料と
して重要なものである。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing glycine from glycinamide using microorganisms. Glycine is important as a food additive and a raw material for the synthesis of medicines and pesticides.
【０００２】0002
【従来の技術とその問題点】グリシンの工業的製造法と
しては、従来、青酸、ホルムアルデヒドおよびアンモニ
アを主原料にしたストレッカー法によりグリシノニトリ
ルを合成し、続いて苛性アルカリを用い加水分解する方
法が知られている。この方法においては、反応液の著し
い着色、副反応によるイミノジ酢酸等の副生および大量
に生成する塩類の処理等が大きな問題となり、脱色や副
反応物の除去のための精製工程に高度な技術を必要とす
る。[Prior art and its problems] Conventionally, as an industrial method for producing glycine, glycinonitrile is synthesized by the Strecker method using hydrocyanic acid, formaldehyde, and ammonia as main raw materials, and then hydrolyzed using caustic alkali. method is known. In this method, there are major problems such as significant coloration of the reaction solution, by-products such as iminodiacetic acid due to side reactions, and treatment of salts produced in large quantities. Requires.
【０００３】一方、アミドの微生物的加水分解によるア
ミノ酸の製造法については、ＤまたはＬ体のアミノ酸ア
ミドより対応するＤまたはＬ体のアミノ酸を得る方法は
知られている〔特開昭６０−３６４４６号および特開昭
６３−８７９９８号各公報参照〕が、グリシンアミドか
らグリシンを得るという報告はない。わずかに、種々の
基質を用いその基質特異性を調べた実験の中に、ブレビ
バクテリウム（Ｂｒｅｖｉｂａｃｔｅｒｉｕｍ） ｓｐ
．Ｒ−３１２ 、オクロバクトラム・アンスロピ−（Ｏ
ｃｈｒｏｂａｃｔｒｕｍ ａｎｔｈｒｏｐｉ） ＳＣＲ
Ｃ Ｃ１−３８およびアクロモバクタ−（Ａｃｈｒｏｍ
ｏｂａｃｔｅｒ） 属の細菌がグリシンアミドを加水分
解する活性があるとの記載〔Ａ．シイレイら、ザ・ジャ
−ナル・オブ・ベ−シック．マイクロバイオロジ− ２
６ 巻２９９−３１１（１９８６） ；浅野ら、ザ・ジ
ャ−ナル・オブ・バイオロジカル・ケミストリ− ２６
４巻 １４２３３−１４２３９（１９８９）；特開平２
−２３４６７８号公報等参照〕があるのみである。しか
し、その活性は光学活性アミノ酸アミドに比べると極め
て低いものである。On the other hand, regarding a method for producing amino acids by microbial hydrolysis of amides, a method for obtaining the corresponding D- or L-amino acid from a D- or L-amino acid amide is known [JP-A-60-36446]. No. and JP-A No. 63-87998], there is no report that glycine is obtained from glycinamide. Among the few experiments that investigated the substrate specificity using various substrates, Brevibacterium sp.
．． R-312, Ochrobactrum anthropii (O
chromobactrum anthropi) SCR
C C1-38 and Achromobacter (Achrom
It is stated that bacteria of the genus A. obacter have the activity of hydrolyzing glycinamide [A. Shiilei et al., The Journal of Basics. microbiology 2
6 Vol. 299-311 (1986); Asano et al., The Journal of Biological Chemistry 26
Volume 4 14233-14239 (1989);
-234678, etc.]. However, its activity is extremely low compared to optically active amino acid amides.
【０００４】0004
【問題を解決するための手段】本発明者らは、このよう
な従来の製造方法に対し、副生物の生成がなく、エネル
ギー的にも、また収率的にも工業的に有利なグリシンの
製造方法の開発を目的として、グリシンアミドの加水分
解によりグリシンを生成する活性が高く、且つ生成した
グリシンを分解、資化する活性を持たない微生物の探索
と培養および反応条件の研究を鋭意行った。その結果、
ロドコッカス（Ｒｈｏｄｏｃｏｃｃｕｓ） 属、アルス
ロバクター（Ａｒｔｈｒｏｂａｃｔｅｒ）属、カセオバ
クター（Ｃａｓｅｏｂａｃｔｅｒ） 属、シュードモナ
ス（Ｐｓｅｕｄｏｍｏｎａｓ） 属、エンテロバクター
（Ｅｎｔｅｒｏｂａｃｔｅｒ）属、アシネトバクター（
Ａｃｉｎｅｔｏｂａｃｔｅｒ） 属、アルカリゲネス（
Ａｌｃａｌｉｇｅｎｅｓ） 属、コリネバクテリウム（
Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ） 属およびストレプ
トマイセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属に属する微
生物にグリシンアミドからグリシンを生産する高い能力
があることを見出し、本発明を完成した。[Means for Solving the Problem] The present inventors have developed a method for producing glycine that does not produce by-products and is industrially advantageous in terms of energy and yield. For the purpose of developing a production method, we searched for microorganisms that have high activity of producing glycine by hydrolyzing glycinamide, but do not have the activity of decomposing and assimilating the produced glycine, and we conducted intensive research on culture and reaction conditions. . the result,
Rhodococcus spp., Arthrobacter spp., Caseobacter spp., Pseudomonas spp., Enterobacter spp., Acinetobacter spp.
Acinetobacter) genus, Alcaligenes (
Alcaligenes) genus, Corynebacterium (
The present invention was completed based on the discovery that microorganisms belonging to the genus Corynebacterium and Streptomyces have a high ability to produce glycine from glycinamide.
【０００５】すなわち、本発明は、微生物由来の加水分
解酵素の作用によりグリシンアミドからグリシンを生成
させる方法において、使用する微生物がロドコッカス（
Ｒｈｏｄ−ｏｃｏｃｃｕｓ）属、アルスロバクター（Ａ
ｒｔｈｒｏｂａｃｔｅｒ）属、カセオバクター（Ｃａｓ
ｅｏｂａｃｔｅｒ） 属、シュードモナス（Ｐｓｅｕｄ
ｏｍｏｎａｓ） 属、エンテロバクター（Ｅｎｔｅｒｏ
ｂａｃｔｅｒ）属、アシネトバクター（Ａｃｉｎｅｔｏ
ｂａｃｔｅｒ） 属、アルカリゲネス（Ａｌｃａｌｉｇ
ｅｎｅｓ） 属、コリネバクテリウム（Ｃｏｒｙｎｅｂ
ａｃｔｅｒｉｕｍ） 属またはストレプトマイセス（Ｓ
ｔｒｅｐｔｏｍｙｃｅｓ）属に属し該ニトリルを加水分
解する能力を有するものであること、を特徴とするグリ
シンの生物学的製造法である。That is, the present invention provides a method for producing glycine from glycinamide by the action of a hydrolase derived from a microorganism, in which the microorganism used is Rhodococcus (
Genus Rhod-ococcus, Arthrobacter (A
rthrobacter), Caseobacter (Cas
eobacter) genus, Pseudomonas (Pseud
omonas), Enterobacter (Enterobacter)
bacter), Acinetobacter (Acinetobacter)
bacter), Alcaligenes (Alcalig)
enes), genus Corynebacterium (Coryneb
acterium) or Streptomyces (S
This is a biological production method for glycine, characterized in that the glycine belongs to the genus Treptomyces and has the ability to hydrolyze the nitrile.
【０００６】本発明において使用される微生物は、上記
の各属に属するグリシン生産菌であるが、具体的にはロ
ドコッカス ｓｐ． ＳＫ４９ （微工研菌寄第１１３
０３ 号）、ロドコッカス ｓｐ． ＳＫ７０ （微工
研菌寄第１１３０４ 号）、ロドコッカス ｓｐ． Ｓ
Ｋ９２ （微工研条寄第３３２４号） 、ロドコッカス
ｓｐ． ＨＲ１１ （微工研菌寄第１１３０６ 号）
、ロドコッカス ｓｐ． ＥＡ４ （微工研菌寄第１２
１３６ 号）、アルスロバクター ｓｐ． ＳＫ１０３
（微工研菌寄第１１３００ 号） 、アルスロバクター
ｓｐ． ＨＲ１（微工研条寄第３３２３号）、アルス
ロバクター ｓｐ． ＨＲ４（微工研菌寄第１１３０２
号）、カセオバクター ｓｐ． ＢＣ４（微工研条寄
第３３１６号）、シュードモナス ｓｐ． ＳＫ１０
（微工研菌寄第１１３０７ 号）、シュードモナス ｓ
ｐ． ＳＫ１１ （微工研菌寄第１１３０８ 号）、シ
ュードモナス ｓｐ． ＳＫ１３ （微工研条寄第３３
２５号）、シュードモナス ｓｐ． ＳＫ ３１（微工
研菌寄第１１３１０ 号）、シュードモナス ｓｐ．
ＳＫ８７ （微工研菌寄第１１３１１ 号）、エンテロ
バクタ−ｓｐ． ＳＫ１２（微工研条寄第３３２２号）
、アシネトバクター ｓｐ． ＢＣ９−２（微工研条寄
第３３１７号）、アルカリゲネス ｓｐ． ＢＣ１６−
２ （微工研条寄第３３２１号）、コリネバクテリウム
ニトリロフィラス（ＡＴＣＣ ２１４１９）およびスト
レプトマイセス グリセウス（ＩＦＯ ３３５５）あ
るいはこれらの変異株を挙げることができる。The microorganisms used in the present invention are glycine-producing bacteria belonging to each of the above genera, and specifically Rhodococcus sp. SK49 (Fiber Engineering Laboratory No. 113
No. 03), Rhodococcus sp. SK70 (Feikoken Bacteria No. 11304), Rhodococcus sp. S
K92 (Feikoken Jokyo No. 3324), Rhodococcus sp. HR11 (Microtechnical Research Institute No. 11306)
, Rhodococcus sp. EA4 (Fiber Engineering Laboratory No. 12
No. 136), Arthrobacter sp. SK103
(Feikoken Bacterial Serial No. 11300), Arthrobacter sp. HR1 (Feikoken Jokyo No. 3323), Arthrobacter sp. HR4 (Microtechnical Research Institute No. 11302)
), Caseobacter sp. BC4 (Feikoken Article No. 3316), Pseudomonas sp. SK10
(Feikoken Bibori No. 11307), Pseudomonas s
p. SK11 (Feikoken Bibori No. 11308), Pseudomonas sp. SK13 (Feikokenjoyori No. 33
No. 25), Pseudomonas sp. SK 31 (Feikoken Bibori No. 11310), Pseudomonas sp.
SK87 (Feikoken Bacterial Serial No. 11311), Enterobacter sp. SK12 (Feikokenjoyori No. 3322)
, Acinetobacter sp. BC9-2 (Feikoken Article No. 3317), Alcaligenes sp. BC16-
2 (Feikoken Jokyo No. 3321), Corynebacterium nitrilophilus (ATCC 21419), and Streptomyces griseus (IFO 3355), or mutant strains thereof.
【０００７】これらの微生物のうち、コリネバクテリウ
ム ニトリロフィラス（ＡＴＣＣ ２１４１９）およ
びストレプトマイセス グリセウス（ＩＦＯ ３３５
５）以外は、本発明者らにより自然界から新たに分離さ
れたものであり、それぞれ上記寄託番号にて工業技術院
微生物工業技術研究所に寄託されており、その菌学的性
質は以下のとおりである。Among these microorganisms, Corynebacterium nitrilophilus (ATCC 21419) and Streptomyces griseus (IFO 335
All substances other than 5) were newly isolated from nature by the present inventors, and have been deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology under the above deposit numbers, and their mycological properties are as follows. It is.
【表１】[Table 1]
【表２】[Table 2]
【表３】[Table 3]
【表４】[Table 4]
【表５】[Table 5]
【０００８】以上の菌学的性質をバージーズ・マニュア
ル オブ システマティック バクテリオロジー
（Ｂｅｒｇｅｙ’ｓ Ｍａｎｕａｌ ｏｆ Ｓｙｓｔｅ
ｍａｔｉｃ Ｂａｃｔｅｒｉｏｌｏｇｙ， １９８６）
に従って分類し、ＳＫ４９、ＳＫ７０、ＳＫ９２、ＨＲ
１１およびＥＡ４ 株はロドコッカス（Ｒｈｏｄｏｃｏ
ｃｃｕｓ） 属、ＳＫ１０３ 、ＨＲ１ およびＨＲ４
株はアルスロバクター（Ａｒｔｈｒｏｂａｃｔｅｒ）
属、ＢＣ４ 株はカセオバクター（Ｃａｓｅｏｂａｃｔ
ｅｒ） 属、ＳＫ１０、ＳＫ１１、ＳＫ１３、ＳＫ３１
およびＳＫ８７株はシュードモナス（Ｐｓｅｕｄｏｍｏ
ｎａｓ） 属、ＳＫ１２株はエンテロバクター（Ｅｎｔ
ｅｒｏｂａｃｔｅｒ）属、ＢＣ９−２株はアシネトバク
ター（Ａｃｉｎｅｔｏｂａｃｔｅｒ） 属およびＢＣ１
６−２株はアルカリゲネス（Ａｌ−ｃａｌｉｇｅｎｅｓ
）属に属する細菌とそれぞれ同定した。The above mycological properties are summarized in Bergey's Manual of Systematic Bacteriology.
matic Bacteriology, 1986)
Classified according to SK49, SK70, SK92, HR
11 and EA4 strains are Rhodococcus
ccus) genus, SK103, HR1 and HR4
The strain is Arthrobacter
Genus, BC4 strain is Caseobacter (Caseobacter).
er) Genus, SK10, SK11, SK13, SK31
and SK87 strain is Pseudomonas (Pseudomonas).
nas) genus, strain SK12 is Enterobacter (Ent
erobacter genus, BC9-2 strain is Acinetobacter genus and BC1
Strain 6-2 is Al-caligenes (Al-caligenes).
) were identified as bacteria belonging to the genus.
【０００９】本発明に使用される微生物の培養には、ニ
トリル化合物、またはアミド化合物等の酵素誘導物質、
通常資化しうる炭素源、窒素源、および微生物の生育に
必要な有機および無機栄養素を含有する培地が用いられ
る。培養は好気的条件下でｐＨ４〜１０、温度２０〜５
０℃で、それぞれの微生物に適した範囲に制御しつつ行
えばよい。[0009] For culturing the microorganisms used in the present invention, enzyme inducers such as nitrile compounds or amide compounds,
A medium containing normally assimilable carbon sources, nitrogen sources, and organic and inorganic nutrients necessary for the growth of microorganisms is used. Cultivation is carried out under aerobic conditions at pH 4-10 and temperature 20-5.
It may be carried out at 0° C. while controlling the temperature within a range suitable for each microorganism.
【００１０】加水分解反応は、上記により微生物を培養
し、その培養液、培養液から分離した菌体または菌体
処理物（菌体破砕物、抽出酵素）または常法により固
定化した菌体あるいは酵素をを水、緩衝液または生理食
塩水に懸濁し、これにグリシンアミドを共存させればよ
い。グリシンアミドに前記菌体等を作用させる条件とし
ては、菌体等を乾燥菌体換算０．０１〜１０重量％、グ
リシンアミド０．１ 〜２０重量％を含む水性懸濁液を
温度氷点〜６０℃、好ましくは１０〜４０℃、ｐＨ５〜
１１、好ましくは６〜１０で、０．５ 〜５０時間反応
させればよい。[0010] The hydrolysis reaction is carried out by culturing microorganisms as described above, and using the culture solution, bacterial cells isolated from the culture solution, or bacterial cells.
A treated product (crushed bacterial cells, extracted enzyme) or bacterial cells or enzymes immobilized by a conventional method may be suspended in water, a buffer solution, or physiological saline, and glycinamide may be allowed to coexist therein. The conditions for allowing the bacterial cells, etc. to act on glycinamide are as follows: 0.01 to 10% by weight of the bacterial cells, etc. in terms of dry bacterial cells, and an aqueous suspension containing 0.1 to 20% by weight of glycinamide, at a temperature of freezing point to 60%. ℃, preferably 10-40℃, pH 5-
11, preferably 6 to 10, and the reaction may be carried out for 0.5 to 50 hours.
【００１１】かくして、グリシンアミドは、ほぼ １０
０％のモル収率でグリシンとアンモニアに転換され、グ
リシンアンモニウム塩の高濃度水溶液として生成蓄積さ
せることができる。グリシンアンモニウムを含有した反
応液からのグリシンの単離は濃縮、イオン交換、抽出、
晶析などの公知の方法を利用することにより目的物であ
るグリシンを取得できる。[0011] Thus, glycinamide has approximately 10
It is converted into glycine and ammonia with a molar yield of 0% and can be produced and stored as a highly concentrated aqueous solution of glycine ammonium salt. Isolation of glycine from a reaction solution containing ammonium glycine involves concentration, ion exchange, extraction,
The target product, glycine, can be obtained by using known methods such as crystallization.
【００１２】0012
【発明の作用及び効果】本発明は、グリシンアミドの加
水分解活性を有する微生物を用いることを特徴とし、グ
リシンアミドからグリシンを生成せしめるものであり、
反応活性およびグリシンへの選択性が高い上に、グリシ
ンの蓄積濃度が極めて高く、工業的に十分満足し得るグ
リシンの製造方法を提供するものである。[Operations and Effects of the Invention] The present invention is characterized by using a microorganism having glycinamide hydrolysis activity, and is capable of producing glycine from glycinamide.
The present invention provides a method for producing glycine that has high reaction activity and high selectivity to glycine, and has an extremely high accumulated concentration of glycine, and is industrially satisfactory.
【００１３】[0013]
【実験例】以下、実施例により本発明を具体的に説明す
るが、本発明はこれらの例のみに限定されるものではな
い。EXPERIMENTAL EXAMPLES The present invention will be specifically explained below with reference to Examples, but the present invention is not limited to these Examples.
【００１４】実施例１
（１） 培養
表−１に示す各微生物を下記のプレート培地に接種し、
培養した。
培地
グリセロール
５ ｇ／ｌアセトアミド
０．５ ｇ／ｌ酵母エキス
０．２ ｇ／ｌＫ２ＨＰＯ４
３ ｇ／ｌＮａ２ＳＯ４
０．３ ｇ／ｌＭｇＣｌ２
０．２
ｇ／ｌＣａＣｌ２
４０ｍｇ／ｌＭｎＳＯ４ ・４Ｈ２
Ｏ ４ ｍｇ／ｌ
ＦｅＣｌ３ ・７Ｈ２Ｏ
０．７ ｍｇ／ｌＺｎＳＯ４
０．１ｍｇ／ｌ寒
天
１８ ｇ／ｌｐＨ
７．２培養
３０℃／３日間Example 1 (1) Culture Each microorganism shown in Table 1 was inoculated into the following plate medium,
Cultured. medium glycerol
5 g/l acetamide
0.5 g/l yeast extract
0.2 g/lK2HPO4
3 g/lNa2SO4
0.3 g/lMgCl2
0.2
g/lCaCl2
40mg/lMnSO4 ・4H2
O4mg/l
FeCl3 ・7H2O
0.7 mg/lZnSO4
0.1mg/l agar
18 g/lpH
7.2 Culture at 30℃/3 days
【００１５】（１） グリシンアミドの加水分解各プレ
ートより菌体を一定量採取し、各々０．０５Ｍリン酸緩
衝液（ｐＨ７．７）で洗浄した後、遠心分離して菌体を
集め、１ｍｌの上記リン酸緩衝液に懸濁した。次に、２
００ｍＭグリシンアミドを含む０．０５Ｍリン酸緩衝液
（ｐＨ ７．７） ０．５ｍｌに上記菌体懸濁液を ０
．５ｍｌ添加して３０℃で４８時間反応させた。反応終
了後、それぞれの反応液を遠心分離して菌体を除去した
後、上清液を液体クロマトグラフィーにて分析し反応液
中のグリシンを測定した。結果を表−１に示した。(1) Hydrolysis of glycinamide A certain amount of bacterial cells was collected from each plate, washed with 0.05M phosphate buffer (pH 7.7), centrifuged to collect the bacterial cells, and 1 ml was collected. of the above phosphate buffer. Next, 2
Add the above cell suspension to 0.5 ml of 0.05 M phosphate buffer (pH 7.7) containing 00 mM glycinamide.
．． 5ml was added and reacted at 30°C for 48 hours. After the reaction was completed, each reaction solution was centrifuged to remove bacterial cells, and the supernatant was analyzed by liquid chromatography to measure glycine in the reaction solution. The results are shown in Table-1.
【表６】[Table 6]
【００１６】実施例２
（１） 培養
ロドコッカス ｓｐ． ＳＫ９２ 株を下記の条件で培
養した。
培地
アセトアミド
７ ｇ／ｌ酵母エキス
３ ｇ／ｌＫＨ２ＰＯ４
０．５ ｇ／ｌ
Ｋ２ＨＰＯ４
０．５ ｇ／ｌ無機塩混合液＊
５ ｍｌ／ｌｐＨ
７
．０＊ ＭｇＣｌ２・６Ｈ２Ｏ ８．１０ｇ， Ｃａ
Ｃｌ２ １．５５ｇ， ＭｎＳＯ４・４Ｈ２Ｏ ０．
１５ｇ
ＦｅＳＯ４・７Ｈ２Ｏ ０．２７ｇ， ＺｎＳＯ４・
７Ｈ２Ｏ ０．００６ｇ， ＣｏＣｌ２・６Ｈ２Ｏ
０．２４ｇ
を水 ２００ｍｌに溶解し調製したもの。
培養
３０℃／２日間Example 2 (1) Cultured Rhodococcus sp. The SK92 strain was cultured under the following conditions. medium acetamide
7 g/l yeast extract
3 g/lKH2PO4
0.5 g/l
K2HPO4
0.5 g/l inorganic salt mixture*
5ml/lpH
7
．． 0* MgCl2・6H2O 8.10g, Ca
Cl2 1.55g, MnSO4.4H2O 0.
15g FeSO4・7H2O 0.27g, ZnSO4・
7H2O 0.006g, CoCl2・6H2O
Prepared by dissolving 0.24g in 200ml of water. Culture at 30℃/2 days
【００１７】（２） グリシンアミドの加水分解培養終
了後、得られた培養液から遠心分離によって菌体を集め
、０．０５Ｍリン酸緩衝液（ｐＨ ７．７）で洗浄した
後、乾燥重量として ０．５重量％となるように前記緩
衝液に懸濁した。この菌体懸濁液４０ｍｌにグリシンア
ミド １０．４ｇを添加し、３０℃でｐＨを７．９ 〜
８．１ に制御しながら反応を行ったところ、反応開始
４５時間でグリシンアミドはほぼ完全に加水分解し、モ
ル収率ほぼ １００％（２２５ｇ／ｌ）でグリシンが生
成していた。(2) After completion of hydrolysis culture of glycinamide, bacterial cells were collected from the obtained culture solution by centrifugation, washed with 0.05M phosphate buffer (pH 7.7), and then expressed as dry weight. It was suspended in the buffer solution at a concentration of 0.5% by weight. 10.4 g of glycinamide was added to 40 ml of this bacterial cell suspension, and the pH was adjusted to 7.9 to 7.9 at 30°C.
When the reaction was carried out while controlling the temperature to 8.1, glycinamide was almost completely hydrolyzed 45 hours after the start of the reaction, and glycine was produced at a molar yield of approximately 100% (225 g/l).
【００１８】実施例３
（１） 培養
ロドコッカス ｓｐ． ＥＡ４株を下記の条件で培養し
た。
培地
グリセロ−ル
５ ｇ／ｌ酵母エキス
０．０２ ｇ／ｌアセトアミド
５ ｇ／ｌＭｇＳＯ４
・７Ｈ２Ｏ ０．
５ ｇ／ｌＫＨ２ＰＯ４
０．５ ｇ／ｌＫ２ＨＰＯ４
０．５
ｇ／ｌｐＨ
７．０培養
３０℃／２日間Example 3 (1) Cultured Rhodococcus sp. EA4 strain was cultured under the following conditions. medium glycerol
5 g/l yeast extract
0.02 g/l acetamide
5 g/lMgSO4
・7H2O 0.
5 g/lKH2PO4
0.5 g/lK2HPO4
0.5
g/lpH
7.0 Culture at 30℃/2 days
【００１９】（２） グリシンアミドの加水分解培養終
了後、得られた培養液から遠心分離により菌体を集め、
０．０５Ｍリン酸緩衝液（ｐＨ ７．７）で洗浄した後
、乾燥重量として０．０９重量％となるように前記緩衝
液に懸濁した。この菌体懸濁液４０ｍｌにグリシンアミ
ド ２．９８ｇを添加し、３０℃でｐＨを７．９ 〜８
．１ に制御しながら反応を行ったところ、反応開始
２．７時間でグリシンアミドはほぼ完全に加水分解し、
モル収率ほぼ １００％（７５ｇ／ｌ） でグリシンが
生成していた。(2) After completion of hydrolysis culture of glycinamide, bacterial cells are collected from the obtained culture solution by centrifugation,
After washing with 0.05M phosphate buffer (pH 7.7), it was suspended in the buffer to a dry weight of 0.09%. 2.98 g of glycinamide was added to 40 ml of this bacterial cell suspension, and the pH was adjusted to 7.9 to 8 at 30°C.
．． When the reaction was carried out under control at 1, the reaction started.
Glycinamide was almost completely hydrolyzed in 2.7 hours,
Glycine was produced at a molar yield of approximately 100% (75 g/l).
Claims (1)
りグリシンアミドからグリシンを生成させる方法におい
て、使用する微生物がロドコッカス（Ｒｈｏｄｏｃｏｃ
ｃｕｓ） 属、アルスロバクター（Ａｒｔｈｒｏｂａｃ
ｔｅｒ）属、カセオバクター（Ｃａｓｅｏｂａｃｔｅｒ
） 属、シュードモナス（Ｐｓｅｕｄｏｍｏｎａｓ）
属、エンテロバクター（Ｅｎｔｅｒｏｂａｃｔｅｒ）属
、アシネトバクター（Ａｃｉｎｅｔｏｂａｃｔｅｒ）
属、アルカリゲネス（Ａｌｃａｌｉｇｅｎｅｓ） 属、
コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ
） 属またはストレプトマイセス（Ｓｔｒｅｐｔｏｍｙ
ｃｅｓ）属に属し該アミドを加水分解する能力を有する
ものであること、を特徴とするグリシンの生物学的製造
法。Claim 1: A method for producing glycine from glycinamide by the action of a hydrolytic enzyme derived from a microorganism, in which the microorganism used is Rhodococcus.
cus), the genus Arthrobacter
ter) genus, Caseobacter
) Genus, Pseudomonas
Genus, Enterobacter, Acinetobacter
Genus, Alcaligenes,
Corynebacterium
) or Streptomyces (Streptomyces
1. A method for biologically producing glycine, characterized in that the glycine belongs to the genus Ces) and has the ability to hydrolyze said amide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8918891A JPH04304892A (en) | 1991-03-29 | 1991-03-29 | Biological production of glycine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP8918891A JPH04304892A (en) | 1991-03-29 | 1991-03-29 | Biological production of glycine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04304892A true JPH04304892A (en) | 1992-10-28 |
Family
ID=13963753
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP8918891A Pending JPH04304892A (en) | 1991-03-29 | 1991-03-29 | Biological production of glycine |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04304892A (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001211892A (en) * | 2000-02-03 | 2001-08-07 | Asahi Kasei Corp | Method of microbiologically producing glycine |
| JP2001258586A (en) * | 2000-03-24 | 2001-09-25 | Asahi Kasei Corp | Method for microbiologically producing glycine with simultanously separating ammonia formed by reaction |
| JP2001269190A (en) * | 2000-03-29 | 2001-10-02 | Asahi Kasei Corp | Microbiological method for production of glycine prevented from discoloration |
| JP2001275692A (en) * | 2000-03-29 | 2001-10-09 | Asahi Kasei Corp | Method for microbiologically producing glycine with no occurrence of discoloration of glycine |
| JP2001299378A (en) * | 2000-04-24 | 2001-10-30 | Asahi Kasei Corp | Method of microbiological production for preventing glycine from coloring |
| JP2001299377A (en) * | 2000-04-28 | 2001-10-30 | Asahi Kasei Corp | Method of microbiological production for glycine |
| JP2001340096A (en) * | 2000-06-01 | 2001-12-11 | Asahi Kasei Corp | Method for microbiologically producing glycine |
| JP2001340097A (en) * | 2000-06-01 | 2001-12-11 | Asahi Kasei Corp | Method for microbiologically producing glycine |
| WO2018149819A1 (en) | 2017-02-16 | 2018-08-23 | Nestec S.A. | Natural flavor base and process for its preparation |
-
1991
- 1991-03-29 JP JP8918891A patent/JPH04304892A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001211892A (en) * | 2000-02-03 | 2001-08-07 | Asahi Kasei Corp | Method of microbiologically producing glycine |
| JP2001258586A (en) * | 2000-03-24 | 2001-09-25 | Asahi Kasei Corp | Method for microbiologically producing glycine with simultanously separating ammonia formed by reaction |
| JP2001269190A (en) * | 2000-03-29 | 2001-10-02 | Asahi Kasei Corp | Microbiological method for production of glycine prevented from discoloration |
| JP2001275692A (en) * | 2000-03-29 | 2001-10-09 | Asahi Kasei Corp | Method for microbiologically producing glycine with no occurrence of discoloration of glycine |
| JP4544685B2 (en) * | 2000-03-29 | 2010-09-15 | 旭化成ケミカルズ株式会社 | Microbiological production method preventing coloring of glycine |
| JP2001299378A (en) * | 2000-04-24 | 2001-10-30 | Asahi Kasei Corp | Method of microbiological production for preventing glycine from coloring |
| JP2001299377A (en) * | 2000-04-28 | 2001-10-30 | Asahi Kasei Corp | Method of microbiological production for glycine |
| JP2001340096A (en) * | 2000-06-01 | 2001-12-11 | Asahi Kasei Corp | Method for microbiologically producing glycine |
| JP2001340097A (en) * | 2000-06-01 | 2001-12-11 | Asahi Kasei Corp | Method for microbiologically producing glycine |
| WO2018149819A1 (en) | 2017-02-16 | 2018-08-23 | Nestec S.A. | Natural flavor base and process for its preparation |
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