Rahman Mohammad Asadur, Culsum Umma, Tang Wenhao, Zhang Shao Wei, Wu Gaobing, Liu Ziduo
State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
Hubei Vocational College of Bio-technology, Wuhan 430070, People's Republic of China.
Enzyme Microb Technol. 2016 Apr;85:1-11. doi: 10.1016/j.enzmictec.2015.12.013. Epub 2016 Jan 4.
A novel cold active esterase, EstLiu was cloned from the marine bacterium Zunongwangia profunda, overexpressed in E. coli BL21 (DE3) and purified by glutathione-S transferase (GST) affinity chromatography. The mature esterase EstLiu sequence encodes a protein of 273 amino acids residues, with a predicted molecular weight of 30KDa and containing the classical pentapeptidase motif from position 156 to 160 with the catalytic triad Ser158-Asp211-His243. Although, EstLiu showed 64% similarity with the hypothetical esterase from Chryseobacterium sp. StRB126 (WP_045498424), phylogenetic analysis showed it had no similarity with any of the established family of lipases/esterases, suggesting that it could be considered as a new family. The purified enzyme showed broad substrate specificity with the highest hydrolytic activity against p-nitrophenyl butyrate (C4). EstLiu showed remarkable activity (75%) at 0°Cand the optimal activity at pH 8.0 and 30°C with good thermostability and quickened inactivation above 60°C. EstLiu retained 81, 103, 67 and 78% of its original activity at 50% (v/v) in ethanol, isopropanol, DMSO and ethylene glycol, respectively. In the presence of Tween 20, Tween 80 and Triton X-100, EstLiu showed 88, 100 and 117% of relative activity. It is also co-factor independent. The high activity at low temperature and desirable stability in organic solvents and salts of this novel family esterase represents a good evidence of novel biocatalyst. Overall, this novel enzyme showed better activity than previously reported esterases in extreme reaction conditions and could promote the reaction in both aqueous and non-aqueous conditions, indicating its great potential for industrial applications.
从海洋细菌深海尊农氏菌中克隆出一种新型冷活性酯酶EstLiu，在大肠杆菌BL21(DE3)中进行过表达，并通过谷胱甘肽-S转移酶(GST)亲和层析进行纯化。成熟的酯酶EstLiu序列编码一个由273个氨基酸残基组成的蛋白质，预测分子量为30 kDa，包含从第156位到160位的经典五肽酶基序，催化三联体为Ser158-Asp211-His243。尽管EstLiu与金黄杆菌属StRB126(WP_045498424)的假定酯酶有64%的相似性，但系统发育分析表明它与任何已确定的脂肪酶/酯酶家族都没有相似性，这表明它可被视为一个新家族。纯化后的酶表现出广泛的底物特异性，对丁酸对硝基苯酯(C4)的水解活性最高。EstLiu在0°C时表现出显著活性(75%)，在pH 8.0和30°C时活性最佳，具有良好的热稳定性，在60°C以上迅速失活。EstLiu在乙醇、异丙醇、二甲基亚砜和乙二醇中体积分数为50% (v/v)时，分别保留了其原始活性的81%、103%、67%和78%。在吐温20、吐温80和曲拉通X-100存在的情况下，EstLiu的相对活性分别为88%、100%和117%。它也不依赖于辅因子。这种新型家族酯酶在低温下的高活性以及在有机溶剂和盐中的良好稳定性是新型生物催化剂的有力证据。总体而言，这种新型酶在极端反应条件下比先前报道的酯酶表现出更好的活性，并且可以在水相和非水相条件下促进反应，表明其在工业应用中具有巨大潜力。
