产低温脂肪酶菌株的筛选、纯化及其部分酶学性质研究

Screening and purification for low-temperature lipase-producing bacterium and characterization of the lipase

从大黑山（大连）和海水中筛出59株低温细菌，从中筛选出10株产低温脂肪酶的菌株，分别对其进行紫外诱变，筛出一株高酶活的低温脂肪酶菌株。通过形态学、生理生化和分子生物学鉴定，菌株CZW001属于芽孢杆菌属（BacillusCohn），与Bacillusthuringiensis（NR043403．1）进化关系近，且同源性达到100％，故将其命名为BacillusthuringiensisCZWOO1。苏云金芽孢杆菌CZW001脂肪酶发酵上清液经硫酸铵沉淀、透析、超滤离心、DEAE-纤维素-52离子交换层析和SephadexG．100凝胶过滤层析获得到电泳纯的脂肪酶，纯化倍数为75．5倍，活性回收率为37．6％，12％SDS—PAGE电泳估计其分子量约40ku。对纯化后的低温脂肪酶进行二级结构预测，结果表明其二级结构成分：α-螺旋10．6％，β-折叠40．1％，β-转角18．8％，无规则卷曲30．5％。对该菌脂肪酶的酶学性质初步研究表明，酶的最适作用温度为25℃，对热敏感，60℃处理30min仅残留30％酶活性，酶的适宜作用pH范围在7～9，最适pH值为8。

From the big Montenegro （Dalian） and sea weed out the 59 low-temperature bacteria, selected from 10 strains of producing cold lipase with respect to their UV mutagenesis screen out the strains of low-temperature lipase activity of a plant height. By morphological, physiological, biochemical and molecular biological identification, the strains CZW001 belonging to Bacillus （Bacillus Cohn）, and Bcillus thuringiensis （NR 043403.1） evolutionary relations in the past, and the homology of 100%, so named for the Bacillus thuringiensis CWZ001. A lipase from Bacillus thuringiensis CZW001 was purified to homogeneity using ammonium sulfate precipitation, dialysis, DEAE-cellulose-52 anion exchange chromatography and Sephadex G-75 gel filtration chromatography. This purification protocol resulted in a 75.5-fold purification of lipase with 37.6% final yield, and the relative molecular weight of the enzyme was determined to be approximately 40ku using SDS-PAGE. Analysis by circular dichroism （CD） spectrum of purified cold-active amylase protein revealed that a-helix 10.6%, B-sheet 40.1%, turn 18.8% and random coil 30.5%. Preliminary studies have shown that the enzymatic properties of bacteria lipase, the enzyme＇s optimum temperature was 25℃ and the enzyme was thermal lability, only 30% of its activity was remained after 30min incubation at 60℃. The enzyme showed high lipolytic from ph 7.0 to 9.0 and its optimal pH for activity was 8.0.