Isolation of novel psychrophilic microorganisms and exploitation of useful enzymes

1.从西伯利亚冻原土壤中分离到一株嗜冷细菌，命名为不动杆菌6号。细菌在4℃下生长，但在37℃下不生长。我们发现，细胞产生细胞外脂解酶作用于甘油三酯时，他们在低温下培养。LB培养基在4℃下培养7 d，对1%w/v的大豆油的降解率为60%，表明该菌可用于低温环境下去除废水中的油脂.从不动杆菌属6号菌株中克隆了编码对脂肪酸乙烯基酯具有底物偏好的冷活性脂肪分解酶的基因。该基因编码由258个氨基酸残基组成的蛋白质。该酶与β-酮己二酸烯醇-内酯水解酶具有很高的序列相似性。构建了该酶的高产体系，并对该酶进行了纯化和表征。该酶是单体的，并有效地催化具有短链酰基的脂肪酸乙烯基酯的水解。该酶还催化酯交换反应，例如，在4℃下，丙酸乙烯酯和丙醇之间产生丙酸丙酯。因此，该酶可用于通过使用乙烯基酯作为酰基供体的酯交换来合成酯。我们从不动杆菌属6号菌株中克隆了编码冷活性酯酶的基因。该基因编码301个氨基酸残基的蛋白质。我们发现弱的系统发育关系的酶酯酶/脂肪酶家族的EST组。该酶优先催化短链酰基酯的水解，其活化能低于中温酶.构建了不动杆菌6号菌株的表达载体，建立了耐冷菌的高效转化方法。

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.