Kim Young-Ok, Park In-Suk, Nam Bo-Hye, Kim Dong-Gyun, Jee Young-Ju, Lee Sang-Jun, An Cheul-Min
Biotechnology Research Division, National Fisheries Research and Development Institute, Busan 619-705, Republic of Korea.
J Microbiol Biotechnol. 2014 Sep;24(9):1260-8. doi: 10.4014/jmb.1405.05043.
Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative β-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Serx- x-Lys motif that is conserved among all β-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme is a serine protein and was active against p-nitrophenyl esters of C2, C4, C8, and C10. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity of 55% remained at up to 5°C with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by Cd(2+), Cu(2+), and Hg(2+) ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.
对在大肠杆菌中构建的芽孢杆菌属PBS - 2基因文库进行筛选，鉴定出一个具有脂解活性的克隆。序列分析显示一个开放阅读框，编码一个由378个氨基酸残基组成的多肽，预测分子量为42 kDa。该酯酶与芽孢杆菌属JDR - 2和梭菌属BNL1100的推定β - 内酰胺酶分别具有69%和42%的同一性。该酯酶含有一个Serx - x - Lys基序，在迄今发现的所有β - 内酰胺酶中保守。当携带该基因的大肠杆菌细胞在18°C培养时，蛋白质PBS - 2以可溶和不可溶两种形式产生。该酶是一种丝氨酸蛋白，对C2、C4、C8和C10的对硝基苯酯有活性。酶活性的最适pH和温度分别为pH 9.0和30°C。在高达5°C时仍保留55%的相对活性，活化能为5.84 kcal/mol，这表明该酶是冷适应性的。酶活性受到Cd(2+)、Cu(2+)和Hg(2+)离子的抑制。正如丝氨酸酯酶所预期的那样，活性受到苯甲基磺酰氟的抑制。该酶在商业洗涤剂和有机溶剂存在下具有显著的活性和稳定性。这种冷适应性酯酶有潜力作为低温下使用的生物催化剂和洗涤剂添加剂。
