JPS63283591A - Production of l-alpha-amino-epsilon-caprolactam - Google Patents
Production of l-alpha-amino-epsilon-caprolactamInfo
- Publication number
- JPS63283591A JPS63283591A JP7446887A JP7446887A JPS63283591A JP S63283591 A JPS63283591 A JP S63283591A JP 7446887 A JP7446887 A JP 7446887A JP 7446887 A JP7446887 A JP 7446887A JP S63283591 A JPS63283591 A JP S63283591A
- Authority
- JP
- Japan
- Prior art keywords
- acl
- amino
- caprolactam
- epsilon
- alpha
- 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
- BOWUOGIPSRVRSJ-YFKPBYRVSA-N L-2-aminohexano-6-lactam Chemical compound N[C@H]1CCCCNC1=O BOWUOGIPSRVRSJ-YFKPBYRVSA-N 0.000 title claims abstract description 8
- 238000004519 manufacturing process Methods 0.000 title claims description 9
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 6
- BUVWZEQVFJESEQ-UHFFFAOYSA-N 3-amino-2-oxoazepane-1-carbaldehyde Chemical compound C(=O)N1C(C(CCCC1)N)=O BUVWZEQVFJESEQ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 230000001580 bacterial effect Effects 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 10
- 239000000243 solution Substances 0.000 abstract description 19
- 241000193764 Brevibacillus brevis Species 0.000 abstract description 2
- 239000007864 aqueous solution Substances 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 241000894006 Bacteria Species 0.000 abstract 2
- 230000003301 hydrolyzing effect Effects 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 238000006243 chemical reaction Methods 0.000 description 20
- 230000000694 effects Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 9
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 8
- 239000004472 Lysine Substances 0.000 description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 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 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 5
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 5
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 5
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 239000003456 ion exchange resin Substances 0.000 description 5
- 229920003303 ion-exchange polymer Polymers 0.000 description 5
- 235000018977 lysine Nutrition 0.000 description 5
- 235000002867 manganese chloride Nutrition 0.000 description 5
- 239000011565 manganese chloride Substances 0.000 description 5
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000000862 absorption spectrum Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000019253 formic acid Nutrition 0.000 description 3
- 229940099607 manganese chloride Drugs 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 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
- 102000004157 Hydrolases Human genes 0.000 description 2
- 108090000604 Hydrolases Proteins 0.000 description 2
- WTDHULULXKLSOZ-UHFFFAOYSA-N Hydroxylamine hydrochloride Chemical compound Cl.ON WTDHULULXKLSOZ-UHFFFAOYSA-N 0.000 description 2
- 235000019766 L-Lysine Nutrition 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000222051 Papiliotrema laurentii Species 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 235000011114 ammonium hydroxide Nutrition 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- WPLOVIFNBMNBPD-ATHMIXSHSA-N subtilin Chemical compound CC1SCC(NC2=O)C(=O)NC(CC(N)=O)C(=O)NC(C(=O)NC(CCCCN)C(=O)NC(C(C)CC)C(=O)NC(=C)C(=O)NC(CCCCN)C(O)=O)CSC(C)C2NC(=O)C(CC(C)C)NC(=O)C1NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C1NC(=O)C(=C/C)/NC(=O)C(CCC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C2NC(=O)CNC(=O)C3CCCN3C(=O)C(NC(=O)C3NC(=O)C(CC(C)C)NC(=O)C(=C)NC(=O)C(CCC(O)=O)NC(=O)C(NC(=O)C(CCCCN)NC(=O)C(N)CC=4C5=CC=CC=C5NC=4)CSC3)C(C)SC2)C(C)C)C(C)SC1)CC1=CC=CC=C1 WPLOVIFNBMNBPD-ATHMIXSHSA-N 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- URDCARMUOSMFFI-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(2-hydroxyethyl)amino]acetic acid Chemical compound OCCN(CC(O)=O)CCN(CC(O)=O)CC(O)=O URDCARMUOSMFFI-UHFFFAOYSA-N 0.000 description 1
- BOWUOGIPSRVRSJ-UHFFFAOYSA-N 2-aminohexano-6-lactam Chemical compound NC1CCCCNC1=O BOWUOGIPSRVRSJ-UHFFFAOYSA-N 0.000 description 1
- 102000004092 Amidohydrolases Human genes 0.000 description 1
- 108090000531 Amidohydrolases Proteins 0.000 description 1
- 241001337994 Cryptococcus <scale insect> Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 150000008545 L-lysines Chemical class 0.000 description 1
- 102000004879 Racemases and epimerases Human genes 0.000 description 1
- 108090001066 Racemases and epimerases Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000921 elemental analysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- LJQKCYFTNDAAPC-UHFFFAOYSA-N ethanol;ethyl acetate Chemical compound CCO.CCOC(C)=O LJQKCYFTNDAAPC-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000009499 grossing Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 150000002696 manganese Chemical class 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000010926 purge Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、L−α−アミノ−ε−カプロラクタム(以下
、α−アミノ−ε−カプロラクタムをACLと略す)の
製造法に関するものである。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for producing L-α-amino-ε-caprolactam (hereinafter α-amino-ε-caprolactam is abbreviated as ACL).
L−ACLは光学分割剤、あるいはL−ACL加水分解
酵素を用いたし一リジンの製造の原料などに用いられて
いるが、その製造法はL−リジンの環化、あるいはり、
L−ACLの光学分割という複雑な方法であるため、よ
り簡便な製造方法の開発が要望されていた。L-ACL is used as an optical resolution agent or as a raw material for the production of lysine using L-ACL hydrolase, but its production method involves cyclization of L-lysine,
Since optical resolution of L-ACL is a complicated method, there has been a demand for the development of a simpler manufacturing method.
そこで本発明者らは、工業的原料として大量かつ安価に
製造されるり、L−ACLと蟻酸から、無水酢酸中で比
較的容易に合成できるN−フォルミルーD、L−α−ア
ミノ−ε−カプロラクタム(以下、N−フォルミルーα
−アミノ−ε−カプロラクタムをf−ACLと略す)に
着目し、この物質を原料とするL−ACLの製造法を開
発すべく鋭意検討した結果、効果の顕著な本発明に到達
した。Therefore, the present inventors investigated N-formyl-D, L-α-amino-ε-caprolactam, which can be produced in large quantities and at low cost as an industrial raw material, or can be synthesized relatively easily from L-ACL and formic acid in acetic anhydride. (Hereinafter, N-formyl α
-Amino-epsilon-caprolactam (abbreviated as f-ACL)), and as a result of intensive studies to develop a method for producing L-ACL using this substance as a raw material, the present invention, which has remarkable effects, was achieved.
本発明はバチルス属に属し、f−L−ACLを加水分解
し、L−ACLを生成せしめる能力を有する微生物、す
なわちf−L−ACLアミドヒドロラーゼを有する微生
物を培養し、その培養物、菌体もしくはその処理物を用
いて、f−D、L−ACLに作用させ、f−L−ACL
を加水分解してL−ACLとし、その反応液中からL−
ACLを分離・採取することから成るL−ACLの製造
方法を提供するものである。The present invention involves culturing a microorganism belonging to the genus Bacillus and having the ability to hydrolyze f-L-ACL to produce L-ACL, that is, a microorganism having f-L-ACL amidohydrolase. Or use the treated product to act on f-D, L-ACL, f-L-ACL
is hydrolyzed to produce L-ACL, and L- is extracted from the reaction solution.
The present invention provides a method for producing L-ACL, which comprises separating and collecting ACL.
本発明に用いる微生物の例としては、本発明者らが分離
したバチルス・プレビス(Bacillus brev
is )微工研菌寄第9072号が挙げられる。An example of the microorganism used in the present invention is Bacillus brev, which was isolated by the present inventors.
is) Microtechnical Research Institute No. 9072 is mentioned.
この微生物の分類学的性質は第1表に示した。The taxonomic properties of this microorganism are shown in Table 1.
この諸性質をパージエイズ・マニュアル・オブ・デイタ
ーミネイティブ・バクテリオロジー(BergeV’s
)lanual of Determinative
BaCteriOIOQV)第8版<1974)(以
下、マニュアルと略す)に照し合わせ、該微生物はバチ
ルス・プレビス(Bacillus brcvis )
に属するものと結論した。These properties are described in the Purging Aids Manual of Determinative Bacteriology (BergeV's
) random of Determinative
According to BaCteriOIOQV) 8th edition <1974) (hereinafter referred to as the manual), the microorganism is Bacillus brcvis.
It was concluded that it belongs to
以下余白
第1表
本発明の微生物の培養物を得るには、培地中にf−L−
ACLもしくはf−D、L−ACLを誘導剤として含有
させ、本発明の微生物を培養する。Margin Table 1 Below: To obtain a culture of the microorganism of the present invention, f-L-
ACL or f-D, L-ACL is contained as an inducing agent, and the microorganism of the present invention is cultured.
誘導剤の添加方法としては、培地中にあらかじめ添加し
ておく方法、または通常の培地で培養し適当な時期に添
加する方法のいずれをとることもできる。培地の形態は
液体、固体のいずれでもよく、また培養の方法は静置培
養、振どう培養、通気攪拌培養のいずれでもよいが、工
業的には通気撹拌培養による液体培養が便利である。培
養温度は10〜45℃、好ましくは30〜40℃で行な
う。The inducing agent can be added either by adding it to the medium in advance or by culturing it in a normal medium and adding it at an appropriate time. The form of the medium may be either liquid or solid, and the culture method may be static culture, shaking culture, or aerated agitation culture, but liquid culture using aerated agitation culture is industrially convenient. The culture temperature is 10 to 45°C, preferably 30 to 40°C.
培養の際の培地のpHは、中性付近に保つことが望まし
い。It is desirable to maintain the pH of the medium during culture near neutrality.
本発明方法を実施する際には、バチルス属に属し、f−
L−ACLを加水分解し、L−ACLを生成せしめる能
力を有する微生物を前述の方法で1〜3日培養し、その
培養液をそのまま、またはそれから菌体を遠心分離、ろ
過等で集め、これを水またはM%液に懸濁したものをf
−D、L−ACLの水溶液に加えてL−ACLの生成反
応を行なう。反応にさきだって、菌体を物理的に破砕し
たり、界面活性剤等を用い溶菌するなどの処理をすると
反祐の進行を円滑にする効果がある。When carrying out the method of the present invention, f-
Microorganisms that have the ability to hydrolyze L-ACL and produce L-ACL are cultured for 1 to 3 days using the method described above, and the culture solution is used as is or the bacterial cells are collected from it by centrifugation, filtration, etc. f suspended in water or M% solution
-D, in addition to an aqueous solution of L-ACL, a reaction for producing L-ACL is performed. Prior to the reaction, treatment such as physically crushing the bacterial cells or lysing them using a surfactant or the like has the effect of smoothing the progress of the reaction.
本発明方法において使用するf−D、L−ACLは、D
、L−ACLを蟻酸中に溶解し、無水酢酸を滴下した後
、水を加え減圧乾固させ取得できる。f-D and L-ACL used in the method of the present invention are D
, L-ACL can be obtained by dissolving L-ACL in formic acid, adding acetic anhydride dropwise thereto, adding water and drying under reduced pressure.
本発明における反応液中での原料f−D、L−ACLの
濃度は、反応開始時で1〜30%だが5〜15%が好ま
しい。反応の進行に応じ、さらに原料を一括または分割
しそ添加し、最終L−ACL濃度を高めることもできる
。菌体中にACLラセマーゼ活性が認められる場合は、
使用する菌体量に応じた濃度の塩酸ヒドロキシルアミン
を酵素反応溶液に加えることにより、D−ACLの副生
を抑えることができる。The concentration of raw materials f-D and L-ACL in the reaction solution in the present invention is 1 to 30% at the start of the reaction, but preferably 5 to 15%. Depending on the progress of the reaction, raw materials can be added all at once or in portions to increase the final L-ACL concentration. If ACL racemase activity is observed in the bacterial cells,
By adding hydroxylamine hydrochloride to the enzyme reaction solution at a concentration depending on the amount of bacterial cells used, the by-product of D-ACL can be suppressed.
反応のpHは通常6〜11、特に好ましくは8〜9に保
たれる。反応温度は通常70℃以下で所要の時間に応じ
適宜選択される。The pH of the reaction is usually kept at 6-11, particularly preferably at 8-9. The reaction temperature is usually 70° C. or lower and is appropriately selected depending on the required time.
また、反応溶液にマンガン塩、特に好ましくは塩化マン
ガンを存在せしめることにより、酵素活性を増大させる
ことができる。Furthermore, the enzyme activity can be increased by including a manganese salt, particularly preferably manganese chloride, in the reaction solution.
採取には、先ず例えば遠心分離機などを用いて除菌した
反応液を、酸性イオン交換樹脂のカラムに通してL−A
CLを吸着させ、洗浄後アンモニア等で溶出し、溶出液
を濃縮し、L−ACLを結晶化させるか、完全にアンモ
ニアを除去した後、塩酸を加えて中和し、減圧濃縮して
L−ACL塩酸塩として結晶化することもできる。多少
のリジンが混入している場合は、例えば塩基性イオン交
換樹脂などを用いて、結晶化の前にリジンを除去してお
けばよい。For collection, first, the reaction solution is sterilized using, for example, a centrifuge, and then passed through an acidic ion exchange resin column.
CL is adsorbed, washed, eluted with ammonia, etc., the eluate is concentrated, and L-ACL is crystallized, or after ammonia is completely removed, hydrochloric acid is added to neutralize it, and L-ACL is concentrated under reduced pressure. It can also be crystallized as ACL hydrochloride. If some lysine is mixed in, the lysine may be removed using, for example, a basic ion exchange resin before crystallization.
反応液を酸性イオン交換樹脂カラムに通液した後、カラ
ムに吸着せずに流出した反応液に再び本発明の微生物を
作用させ反応を行ない、同様の方法でL−ACLを採取
すると収率を上げることができる。このように1回のみ
でなく複数にわたり反応を行なうと、L−ACLを収率
良く製造することができる。After passing the reaction solution through an acidic ion-exchange resin column, the microorganism of the present invention is applied again to the reaction solution that flows out without being adsorbed to the column, and L-ACL is collected in the same manner. can be raised. By performing the reaction not only once but multiple times in this manner, L-ACL can be produced with good yield.
以下、実施例を挙げて本発明をさらに具体的に説明する
。Hereinafter, the present invention will be explained in more detail with reference to Examples.
実施例
1)f−D、L−ACLの合成
市販のり、L−ACL12.8gを、200m1の98
%HCOOH中に氷水内で攪拌しながら溶解した後、5
1gの無水酢酸を約30分かけて滴加した。室温にてさ
らに1時間撹拌した後、200m1の水を混入させた。Example 1) Synthesis of f-D, L-ACL 12.8 g of commercially available glue, L-ACL, was added to 200 ml of 98
After dissolving in ice water with stirring in % HCOOH, 5%
1 g of acetic anhydride was added dropwise over about 30 minutes. After stirring for a further hour at room temperature, 200 ml of water were mixed in.
この反応液を減圧濃縮し、固化した残留物を酢酸エチル
−エタノール(1:1)を用いて再結晶した。収率は7
0〜85%であった。This reaction solution was concentrated under reduced pressure, and the solidified residue was recrystallized using ethyl acetate-ethanol (1:1). Yield is 7
It was 0-85%.
2)菌の培養
バチルス・プレビス(Bacillus brevis
) (微工研菌寄第9072号)を、0.5%f−
D、L−ACL、0.5%グルコース、0.2%NaH
2PO4・2H20,0,3%に2 HP 04.0−
05%M g S 0.4 ・7 H20,0,002
%MnCl2 ・4H20,0,05%酵母エキス(p
H7゜2)の培地6mlを121℃、15分滅菌したも
のに植菌し、30℃、8時間振どう培養した。菌体を遠
心分離で集め、水洗後−20℃で凍結後室温で融解した
。2) Culture of bacteria Bacillus brevis
) (Feikoken Bibori No. 9072) with 0.5% f-
D, L-ACL, 0.5% glucose, 0.2% NaH
2PO4・2H20,0,3% to 2 HP 04.0-
05%M g S 0.4 ・7 H20,0,002
%MnCl2・4H20,0,05% yeast extract (p
6 ml of H7゜2) medium was sterilized at 121°C for 15 minutes and then cultured with shaking at 30°C for 8 hours. The bacterial cells were collected by centrifugation, washed with water, frozen at -20°C, and then thawed at room temperature.
3)酵素反応
この菌体に10%f−D、 L−ACL−50mMリン
酸緩衝液(pH8,0) −1mMヒドロキシルアミン
を加え、計0.5mlとし、30℃、4時間インキュベ
ートし反応させた。第1図に反応の経過を示す。3) Enzyme reaction Add 10% f-D, L-ACL-50mM phosphate buffer (pH 8,0)-1mM hydroxylamine to the bacterial cells to make a total of 0.5ml, and incubate at 30°C for 4 hours to react. Ta. Figure 1 shows the course of the reaction.
生成したL−ACLの定量は、クリプトコツカス・ラウ
レンティのアセトン乾燥菌体1■を反応液中に加えてL
−ACL全量をL−リジンに転換し、このL−リジンを
アミノ酸自動分析機で定量してL−ACL量をした。To quantify the produced L-ACL, add 1 μm of acetone-dried bacterial cells of Cryptococcus laurentii to the reaction solution and
-The total amount of ACL was converted to L-lysine, and this L-lysine was quantified using an automatic amino acid analyzer to determine the amount of L-ACL.
ここで、アセトン乾燥菌体は次の方法により製造した。Here, acetone-dried bacterial cells were produced by the following method.
L−ACL加水加水分解酵素生産高るクリプトコツカス
・ラウレンティ(cryptococcus +aur
entii) (微工研菌寄第709号)を0.5%グ
ルコース、1%DL−ACL −HCl、0.1%KH
2PO4,0,05%MgSO4・7H20.0.00
2%MnCl2 ・4H20,0,05%酵母エキス(
pt17.2)を成分とする培地300m1中、30℃
、20時間培養した後、遠心操作により集菌、そして水
を用いた洗浄を2回行なった後アセトンで脱水し、アセ
トン乾燥菌体とした。Cryptococcus laurentii (cryptococcus +aur) increases L-ACL hydrolysis enzyme production
Entii) (Feikoken Bibori No. 709) was mixed with 0.5% glucose, 1% DL-ACL-HCl, and 0.1% KH.
2PO4,0.05%MgSO4・7H20.0.00
2%MnCl2・4H20,0,05% yeast extract (
pt17.2) in 300 ml of medium at 30°C.
After culturing for 20 hours, the cells were collected by centrifugation, washed twice with water, and then dehydrated with acetone to obtain acetone-dried cells.
反応4時間口のし一リジン含有量は21.84mg/m
lであり、f−D、L−ACLからL−ACLのモル転
換率は23.3%と計算された。After 4 hours of reaction, the lysine content was 21.84 mg/m
l, and the molar conversion of f-D, L-ACL to L-ACL was calculated to be 23.3%.
4)L−ACLの抽出
遠心分離で除菌した酵素反応液30m1を、約40m1
の強酸性イオン交換樹脂“ダイアイオンPK228″の
カラムにかけてACLを吸着させた。4) L-ACL extraction 30 ml of the enzyme reaction solution sterilized by centrifugation was added to approximately 40 ml of
ACL was adsorbed on a column of strongly acidic ion exchange resin "Diaion PK228".
さらに150m1の蒸留水を流して洗浄した後、吸着し
たACLを5%アンモニア水で溶出させた。After further washing with 150 ml of distilled water, the adsorbed ACL was eluted with 5% ammonia water.
溶出液はフラクションコレクターで2mlずつ分取し、
各両分を薄層クロマトグラフィー(n−プロパツール:
28%アンモニア水=2:1を展開溶媒とし、シリカゲ
ル薄層を使用)でACLの有無を判定し、ACLを含む
50本、計100m1を得た。さらにこの溶液を強塩基
性イオン交換樹脂(“ダイアイオンPA318”)カラ
ムにかけ、微量存在するリジンを吸着させ、溶出液のA
CLの純度を上げた。この溶出液をナス型フラスコに移
し、約40℃のウォーターバス内で減圧濃縮し、アンモ
ニアを除去した”。内容物を10m1の蒸留水に再びと
かし、IN塩酸を滴下し、中和した。The eluate was collected in 2 ml portions using a fraction collector.
Both parts were subjected to thin layer chromatography (n-proper tool:
The presence or absence of ACL was determined using 28% ammonia water = 2:1 as a developing solvent and a thin layer of silica gel, and 50 tubes containing ACL, totaling 100 ml, were obtained. Furthermore, this solution was applied to a strongly basic ion exchange resin (“Diaion PA318”) column to adsorb trace amounts of lysine, and the eluate was
The purity of CL was increased. This eluate was transferred to an eggplant-shaped flask and concentrated under reduced pressure in a water bath at about 40°C to remove ammonia.''The contents were dissolved again in 10 ml of distilled water, and IN hydrochloric acid was added dropwise to neutralize.
この溶液から減圧乾固して得られた123■の一部を赤
外吸収分析と元素分析したところ、第2図、第3図およ
び第2表に示したようにACLであることが確認された
。第3図は市販されている標品のL−ACLの赤外吸収
スペクトルを示し、第4図は本実施例で抽出されたL−
ACLの赤外吸収スペクトルを示す。When a part of 123■ obtained by drying this solution under reduced pressure was subjected to infrared absorption analysis and elemental analysis, it was confirmed that it was ACL as shown in Figures 2 and 3 and Table 2. Ta. Figure 3 shows the infrared absorption spectrum of commercially available standard L-ACL, and Figure 4 shows the L-ACL extracted in this example.
The infrared absorption spectrum of ACL is shown.
また、L−ACL加水分解酵素により完全に分解される
ことが薄層クロマトグラフィーで確認され、一方、光学
純度は高速液体クロマトグラフィーの分析では99.4
%だった。以上の結果から抽出された化合物はL−AC
Lと結論された。Furthermore, it was confirmed by thin layer chromatography that it was completely degraded by L-ACL hydrolase, and the optical purity was 99.4 as determined by high performance liquid chromatography.
%was. The compound extracted from the above results is L-AC
It was concluded that L.
第2表 5)酵素活性 前記第2〉項の培養2mlの菌体に、1%f−D。Table 2 5) Enzyme activity Add 1% f-D to 2 ml of the cultured cells in item 2> above.
L−ACL−100mHTris−HCIII街液(p
118.0>−4mMヒドロキシルアミン−5mM塩化
マンガンを加え、計1 mlとし、40℃、3時間イン
キュベートし反応させた。f−D、L−ACLからL−
ACLへのモル転換率は43.0%であった。L-ACL-100mHTris-HCIII street solution (p
118.0>-4mM hydroxylamine-5mM manganese chloride was added to make a total of 1ml, and the mixture was incubated at 40°C for 3 hours to react. f-D, L-ACL to L-
The molar conversion to ACL was 43.0%.
一方、前記第2)項の菌体を20mHEDTAで30分
間インキユベバー、酵素反応すると活性が全くなくなる
が、水洗後、酵素反応液に50m)l塩化マンガンを添
加すると活性が復活した。On the other hand, when the bacterial cells of item 2) were incubated with 20 m HEDTA for 30 minutes and subjected to enzymatic reaction, the activity disappeared completely, but after washing with water, when 50 ml manganese chloride was added to the enzyme reaction solution, the activity was restored.
6)酵素の熱安定性
前記第2)項の培養1 mlの菌体を、50mMリン酸
綬街液(1)18.0>0.5mlに分散し、20.3
0.40.50.60.70.80.90℃で各30分
間インキュベートした後、酵素活性を測定したところ、
40℃以上で活性の減少がみられた。しかし、1%f−
D、L−ACLを含む50醋リン酸WJI液<pits
、0)0.5mlに分散し、上記各温度で30分間イン
キュベートした後、水洗し酵素活性を測定したところ、
70℃ではじめて活性の減少がみられた。6) Thermostability of enzymes Disperse 1 ml of the cultured cells in item 2) above in 18.0>0.5 ml of 50 mM phosphoric acid solution (1).
After incubating at 0.40.50.60.70.80.90°C for 30 minutes each, enzyme activity was measured.
A decrease in activity was observed at temperatures above 40°C. However, 1%f-
D, 50% phosphoric acid WJI solution containing L-ACL < pits
, 0) After dispersing in 0.5 ml and incubating at each of the above temperatures for 30 minutes, washing with water and measuring enzyme activity,
A decrease in activity was first observed at 70°C.
〔発明の効果〕
本発明方法によれば、簡便でかつ効率良<L−ACLを
製造することができる。[Effects of the Invention] According to the method of the present invention, L-ACL can be produced simply and efficiently.
第1図は反応時間とL−リジンの生成量との関係を示す
ものである。第2図は標品のL−α−アミノ−ε−カプ
ロラクタムの赤外吸収スペクトルを、第3図は本発明で
得られたし一α−アミノーε−カプロラクタムの赤外吸
収スペクトルを示す。
特許出願人 東 し 株 式 会 社第1図
0 0 0 0 0 0 0 0 0
o 0’:。FIG. 1 shows the relationship between the reaction time and the amount of L-lysine produced. FIG. 2 shows the infrared absorption spectrum of standard L-α-amino-ε-caprolactam, and FIG. 3 shows the infrared absorption spectrum of L-α-amino-ε-caprolactam obtained in the present invention. Patent applicant Toshi Co., Ltd. Figure 1 0 0 0 0 0 0 0 0
o 0':.
Claims (1)
ミノ−ε−カプロラクタムを加水分解し、L−α−アミ
ノ−ε−カプロラクタムを生成せしめる能力を有する微
生物の培養物、菌体もしくはその処理物を、N−フオル
ミル−D,L−α−アミノ−ε−カプロラクタムに作用
せしめることを特徴とするL−α−アミノ−ε−カプロ
ラクタムの製造法。(1) A culture, bacterial cells, or the like of a microorganism that belongs to the genus Bacillus and has the ability to hydrolyze N-formyl-L-α-amino-ε-caprolactam to produce L-α-amino-ε-caprolactam. A method for producing L-α-amino-ε-caprolactam, which comprises allowing a treated product to act on N-formyl-D,L-α-amino-ε-caprolactam.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP7446887A JPS63283591A (en) | 1986-12-17 | 1987-03-30 | Production of l-alpha-amino-epsilon-caprolactam |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP29892386 | 1986-12-17 | ||
JP61-298923 | 1986-12-17 | ||
JP7446887A JPS63283591A (en) | 1986-12-17 | 1987-03-30 | Production of l-alpha-amino-epsilon-caprolactam |
Publications (1)
Publication Number | Publication Date |
---|---|
JPS63283591A true JPS63283591A (en) | 1988-11-21 |
Family
ID=26415624
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP7446887A Pending JPS63283591A (en) | 1986-12-17 | 1987-03-30 | Production of l-alpha-amino-epsilon-caprolactam |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS63283591A (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0462948A2 (en) * | 1990-06-21 | 1991-12-27 | Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. | Pharmaceutical compositions comprising 3-amino-epsilon-caprolactames for enhancing learning and memory |
US5118806A (en) * | 1988-07-22 | 1992-06-02 | Takeda Chemical Industries, Ltd. | Thiazolo[5,4-b]azepine compounds |
-
1987
- 1987-03-30 JP JP7446887A patent/JPS63283591A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5118806A (en) * | 1988-07-22 | 1992-06-02 | Takeda Chemical Industries, Ltd. | Thiazolo[5,4-b]azepine compounds |
US5300639A (en) * | 1988-07-22 | 1994-04-05 | Takeda Chemical Industries, Ltd. | Thiazolo [5,4-b]azepine compounds |
EP0462948A2 (en) * | 1990-06-21 | 1991-12-27 | Sigma-Tau Industrie Farmaceutiche Riunite S.p.A. | Pharmaceutical compositions comprising 3-amino-epsilon-caprolactames for enhancing learning and memory |
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