Screening of New Psychrophiles Producing Useful Enzymes

筛选产生有用酶的新嗜冷菌

• 批准号: 07041108
• 负责人: ESAKI Nobuyoshi
• 金额: $ 5.38万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07041108/
• 关键词: psychrophilic enzyme; psychrotrophic bacteria; psychrophilic bacteria; Pseudomonas; Acinetobacter; cloning; lipase; beta-ketoadipate enol lactone hydrolase; エステル分解; エステル合成

Enzymes showing the high activities at low temperature are applicable to the additives for the detergent, food processing, enzymatic synthesis of the thermolabile materials, and so on. We tried to find these enzymes from the psychrotrophic and psychrophilic bacteria grown under the extreme conditions. Two lipase-like genes were cloned to E.coli cells from Pseudomonas sp.B11-1 and Acinetobacter sp.No.6 cells which were isolated from the cold environment. The former gene encodes the 676-aminoacid protein with the molecular weight of 72,900. This lipase contains no GxSyG motif which is considered to be common in all lipases so far studied. The gene from Acinetobacter sp.No.6 encodes the 258-amino-acid protein with the molecular weight of 28,423. The primary structure of the protein is similar to that o^f beta-ketoadipate enol lactone hydrolase of Acinetobacter calcoaceticus. The activation energies in the rho-nitrophenyl-butyl-ester hydrolysis catalyzed by both enzymes were obtained from the Arrhenius plots of the reactions. The activation energies were calculated to be 11.2 and 11.3 (Kcal/mol) with the Pseudomonas enzyme and Acinetobacter enzyme, respectively. These values are lower that that in the reaction with the lipase from the mesophile, Pseudomonas aeruginosa (25Kcal/mol).

在低温下表现出高活性的酶适用于洗涤剂添加剂、食品加工、不耐热物质的酶促合成等，我们试图从极端条件下生长的嗜冷菌和耐冷菌中寻找这些酶。从低温环境中分离的假单胞菌B11 -1和不动杆菌6中克隆了两个脂肪酶样基因。前一个基因编码676个氨基酸的蛋白质，分子量为72，900。该脂肪酶不含GxSyG基序，这被认为是迄今为止研究的所有脂肪酶中常见的。来自不动杆菌6号的基因编码258个氨基酸的蛋白质，分子量为28，423。该蛋白的一级结构与乙酸钙不动杆菌的β-酮己二酸烯醇内酯水解酶的一级结构相似。由Arrhenius图求得了两种酶催化邻硝基苯丁酯水解反应的活化能。假单胞菌酶和不动杆菌酶的活化能分别为11.2和11.3（千卡/摩尔）。这些值低于与来自嗜温菌铜绿假单胞菌的脂肪酶的反应中的值（25 Kcal/mol）。

项目成果

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T.Hisano：“来自假单胞菌属 sp.YL 的 L-2-卤酸脱卤酶的结晶和初步 X 射线晶体学研究”蛋白质：结构、功能和遗传学。

J.-Q.Liu: "Paracatalytic Inactivation of L-2-Haloacid Dehalogenase from Pseudomonas sp.YL by Hydroxylamine" J.Biol.Chem.272 (6). 3363-3368 (1997)

J.-Q.Liu：“羟胺对假单胞菌属 SP.YL 的 L-2-卤代酸脱卤酶的副催化失活”J.Biol.Chem.272 (6)。

K.-H.Jhee: "Stereospecificity of Thermotable Ornithine 5-Aminotransferase for the L-and D-Ornithine Transamination" Biochemistry. 35(30). 9792-9796 (1996)

K.-H.Jhee：“用于 L-和 D-鸟氨酸转氨作用的耐热鸟氨酸 5-氨基转移酶的立体特异性”生物化学。

K.-H.Jhee: "Stereospecificity of Thermotable Ornithine 5-Aminotransferase for the L- and D-Orinithine Transamination" Biochemistry. 35 (30). 9792-9796 (1996)

K.-H.Jhee：“用于 L- 和 D-鸟氨酸转氨作用的耐热鸟氨酸 5-氨基转移酶的立体特异性”生物化学。

H.-S.Ro: "Site-directed Mutageneisis of the Amino Acid Residues in beta-strand III [Val^<30>-Val^<36>] of D-amino Acid Aminotransferase of Bacillus sp.YM-119GC04 : FEBS Letters" 398. 141-145 (1996)

H.-S.Ro：“芽孢杆菌属 sp.YM-119GC04 的 D-氨基酸转氨酶 β-链 III [Val^<30>-Val^<36>] 中氨基酸残基的定点诱变：FEBS