Isolation of novel psychrophilic microorganisms and exploitation of useful enzymes

新型嗜冷微生物的分离和有用酶的开发

基本信息

批准号：
12575019
负责人：
ESAKI Nobuyoshi
金额：
$ 8.06万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

1. A psychrotrophic bacterium, Acinetobacter sp. strain no. 6, was isolated from Siberian tundra soil. The bacterium grew at 4℃ but did not grow at 37℃. We found that the cells produce extracellular lipolytic enzymes acting on triglycerides when they are cultivated at low temperatures. When cultivation was carried out at 4℃ with the LB medium, 60% of soybean oil (initial concentration, 1% w/v) was degraded in 7 days, indicating that the bacterium is useful in removing fats from waste water under cold environments.2. The gene coding for a cold-active lipolytic enzyme with a substrate preference for fatty acid vinyl esters was cloned from Acinetobacter sp. strain no. 6. The gene encoded a protein consisting of 258 amino acid residues. The enzyme showed a high sequence similarity to β-ketoadipate enol-lactone hydrolase. An overproduction system for the enzyme was constructed, and the enzyme was purified to homogeneity and characterized. The enzyme was monomeric and efficiently catalyzed hydrolysis of fatty acid vinyl esters with a short-chain acyl group. The enzyme also catalyzed transesterification, for example, between vinyl propionate and propanol yielding propyl propionate at 4℃. Thus the enzyme is useful for the synthesis of esters by transesterification using vinyl esters as an acyl donor.3. We cloned a gene coding for a cold-active esterase from Acinetobacter sp. strain no. 6. The gene encoded a protein of 301 amino acid residues. We found weak phylogenetic relationship of the enzyme to the EST group of the esterase/lipase family. It preferentially catalyzed the hydrolysis of esters with short-chain acyl group and showed lower activation energy than those of mesophilic enzymes.4. A novel vector for Acinetobacter sp. strain no. 6 was constructed, and an efficient transformation method for the psychrotrophic bacterium was established.

1。一种精神营养细菌，acinetobacter sp。应变号。 6，从西伯利亚苔原土壤中分离出来。细菌在4℃中生长，但在37°时没有生长。我们发现，当细胞在低温下培养时，细胞会产生作用于甘油三酸酯上的细胞外脂肪酶。当用LB培养基在4℃进行栽培时，60％的大豆油（初始浓度为1％w/v）在7天内降解，表明该细菌可用于在寒冷环境下从废水中去除脂肪。2。用底物偏爱脂肪酸乙烯基酯的冷活性脂肪酶编码的基因是从杆菌杆菌中克隆的。应变号。 6。该基因编码了由258个氨基酸残基组成的蛋白质。该酶显示出与β-酮磷酸烯醇 - 内乳酮水解酶的高序列相似性。构建了酶的过量生产系统，并将酶纯化为均匀性并进行了特征。该酶是单体的，并有效地用短链酰基基团催化脂肪酸乙烯基酯的水解。该酶还催化了丙酸乙酯和丙醇之间的持续酯化，在4°处产生丙酸酯。该酶可用于通过使用乙烯基酯作为酰基供体的酯交换酯的合成。3。我们克隆了一个基因，该基因编码来自acinetobacter sp的冷活动酯酶。应变号。 6。该基因编码了301个氨基酸残留物的蛋白质。我们发现酶与酯酶/脂肪酶家族的EST组的系统发育关系弱。它优先用短链酰基基团催化酯的水解，并显示出比中嗜酶的活化能更低的活化能。4。 acinetobacter sp。的新型矢量。应变号。建立了6，并建立了一种有效的基质细菌转化方法。