Development of New Methods on Isotope-Labeling of Amino Acid and Vitamin with Microbial Enzymes

微生物酶同位素标记氨基酸和维生素新方法的发展

• 批准号: 63880021
• 负责人: SODA Kenji
• 金额: $ 5.12万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B).
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63880021/
• 关键词: Amino acid; Vitamin; Microbial enzyme; Isotope-labeling; 同位元素標識法; 同位体元素標識法

New methods on isotope-labeling of amino acid and vitamin were established with microbial enzymes, which have high stereo-specificity of reaction. First, or ^<35>S- labeling ofS-alkylcysteine was established by beta -replacement reaction of S-alkylcysleine alpha, beta -lyase, which was newly purified from a thermophile (Bacillus sp. 41A). Second, deuterium or tritium-labeling of D-amino acid was developed Stereoselectively with D-amino acid aminotransferase. Stereoselectively-deulerated NADH and NADPH were also prepared by coupling of glutamate racemase and glutamate dehydrogenase. Further, double isotope-labeling of Lysine was carried out by L-lysine epsilon -dehydrogenase and amino acid racemase. Finally. Substitution of S- (beta -aminoethyl) - cysteine for active-site lysine of thermostable aspartate aminotransferase was established by gene technology and chemical modification. Thus, many effective isotope-labeling of amino acid, vitamin and protein were developed.

利用具有较高反应立体特异性的微生物酶建立了氨基酸和维生素的同位素标记新方法。首先，从一株嗜热菌(芽孢杆菌属)中分离纯化了一种新的裂解酶，即S标记的烷基半胱氨酸。41a)。其次，用D-氨基酸氨基转移酶立体选择性地进行了D-氨基酸的氚或氚标记。通过谷氨酸外消旋酶和谷氨酸脱氢酶的偶联，制备了立体选择性的NADH和NADPH。进一步利用L赖氨酸表观脱氢酶和氨基酸外消旋酶对赖氨酸进行双同位素标记。终于来了。通过基因工程和化学修饰，建立了S-(β-氨基乙基)-半胱氨酸取代耐热天冬氨酸氨基转移酶活性中心赖氨酸的方法。因此，许多有效的氨基酸、维生素和蛋白质的同位素标记应运而生。

项目成果

Misono Haruo: "Sterespecific Abstraction of εーproーRーHydrogen of LーLysine by LーLysine εーDehydrogenase from Agrobaclerium tumefaciens" Journal of Biochemistry. 107. 169-172 (1990)

Misono Haruo：“通过根癌农杆菌的 L-赖氨酸 ε-脱氢酶对 L-赖氨酸的 ε-pro-R-氢进行立体特异性提取”《生物化学杂志》107. 169-172 (1990)。

Alvaro Martinez del Pozo, Marcello Merola, Hiroshi Ueno, James M. Manning, Katuyuki Tanizawa, Kenji Soda and Dagmar Ringe: "Stereospecificity of Reaction Catalyzed by Bacterial D-amino Acid Transaminase." The Journal of Biological Chemistry. 264. 17784-17

Alvaro Martinez del Pozo、Marcello Merola、Hiroshi Ueno、James M. Manning、Katuyuki Tanizawa、Kenji Soda 和 Dagmar Ringe：“细菌 D-氨基酸转氨酶催化反应的立体特异性”。

Ken-ichi Ishiwata, Nobuhiro Fukuhara, Masao Shimada, Nobuyoshi Makiguchi, and Kenji Soda: "Enzymatic Production of L-Tryptophan from DL-Serinc and lidole by a Coupled Reaction of Tryptophan Synthase and Amino Acid Racemase." Biotechnology and Applied Bioc

Ken-ichi Ishiwata、Nobuhiro Fukuhara、Masao Shimada、Nobuyoshi Makiguchi 和 Kenji Soda：“通过色氨酸合酶和氨基酸消旋酶的偶联反应，从 DL-丝氨酸和 lidole 酶促生产 L-色氨酸。”

Nobuyoshi Esaki, Hiroko Shimoi, Nobuyoshi Nakajima, Toshihisa Ohshima, Hidehiko Tanaka and Kenji Soda: "Enzymatic in Situ Determination of Stereospecificity of NAD-dependent Dehydrogenasc" The Journal of Biological Chemistry. 264. 9750-9752 (1989)

Nobuyoshi Esaki、Hiroko Shimoi、Nobuyoshi Nakajima、Toshihisa Ohshima、Hidehiko Tanaka 和 Kenji Soda：“NAD 依赖性脱氢酶立体特异性的酶原位测定”《生物化学杂志》。

Nobuyoshi Nakajima: "Enzymatic In Situ Analysis by 1-H-NMR of the Hydrogen Transfer Stereospecificity of NAD(P)-Dependent Dehydrogenases." Jounal of Biochemistry. 106. 515-517 (1989)

Nobuyoshi Nakajima：“通过 1-H-NMR 对 NAD(P) 依赖性脱氢酶的氢转移立体特异性进行酶促原位分析。”