Isolation of novel psychrophilic microorganisms and exploitation of useful enzymes

1.嗜冷菌--不动杆菌属。菌株6号是从西伯利亚冻土带土壤中分离到的。该菌在4℃时生长，37℃时不生长。我们发现，当细胞在低温下培养时，会产生作用于甘油三酯的胞外脂解酶。结果表明：1.该菌在4℃的条件下，在7天内可降解60%的大豆油(初始浓度为1%w/v)，表明该菌对低温环境下废水中的脂肪有较好的去除效果。从不动杆菌中克隆了一种对脂肪酸乙烯基酯具有底物选择性的冷活性脂解酶基因。该基因编码由258个氨基酸残基组成的蛋白质。该酶与β-酮基己二酸烯醇内酯水解酶具有较高的序列相似性。构建了该酶的高产体系，并对该酶进行了纯化和性质鉴定。该酶是一种单体酶，能有效催化含有短链酰基的脂肪酸乙烯酯的水解。该酶还催化丙酸乙酯和丙醇之间的酯交换反应，在4℃时生成丙酸丙酯。因此，该酶在以乙烯基酯为酰基给体的酯交换反应中是有用的。从不动杆菌中克隆了一个编码冷活性酯酶的基因。该基因编码一个301个氨基酸残基的蛋白质。我们发现该酶与酯酶/脂肪酶家族的EST基团有较弱的亲缘关系。它优先催化含有短链酰基的酯类的水解，表现出比中温酶更低的活化能。一种新型不动杆菌载体。构建了6号菌株，建立了一种高效的嗜冷菌转化方法。

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.