通过分子克隆手段获得大肠杆菌来源的羧酸酯酶 BioH，采用定向进化的方法提高该酶的水解活性以提升其应用价值.通过 PCR 扩增，从大肠杆菌 K12菌株中克隆得到羧酸酯酶 BioH 基因，目的基因长度为771 bp，含256个氨基酸；将其连接到 pET30a（+）质粒上并转入 BL21（DE3）宿主细胞中，经诱导表达得到所需的目的蛋白，该蛋白分子质量约为28.2 ku.野生型的 BioH 水解对硝基苯丁酸酯（p-NPB）的活性为18 U/mg，且该酶具有良好的热稳定性.以 p-NPB 为底物进行高通量筛选，其水解底物为对硝基苯酚（p-NP），在405 nm 处有最大吸收峰.挑选出水解活性提高的突变体，并通过2轮定向进化过程，成功筛选到7个水解活性提高的优良突变体，它们的活性分别提高了20%~100%.结构分析表明，突变体的突变位点均分布在远离活性中心的位置.
关键词： 克隆 水解活性 表达 羧酸酯酶 BioH 定向进化
Cloning,expression of carboxylesterase BioH and improvement of its hydrolysis activity by directed evolution XU Hongna YU Hongwei
Institute of Bioengineering,Department of Chemical and Biological Engineering, Zhejiang University,Hangzhou 310027,China Institute of Bioengineering,Department of Chemical and Biological Engineering, Zhejiang University,Hangzhou 310027,China
Summary Carboxylesterase BioH of Escherichia coli is an important enzyme in the biotin synthesis.It contains a typical catalytic triad of Ser-His-Asp and displays significant carboxylesterase activities.The enzyme shows a broad substrate specificity with a preference for short acyl chain substrates.This property makes it a biocatalyst with a good application prospect.However,research on the use of this enzyme has been quite few to date.It shows great significance to get the protein and optimize its functions.
In this study,BioH gene from E.coli K12 was cloned into pET30a(+) using double digestion method by BamHI and HindIII and was overexpressed in E.coli BL21(DE3) induced by isopropyl-β-D-thiogalactoside (IPTG).BioH protein content was calculated by image processing software Quantity One.Its hydrolysis activity was determined by the rate of hydrolyzing p-nitrophenyl butyrate(p-NPB).And its thermostability was detected after the enzyme was incubated at 60 ℃ for 40 min.In order to improve its hydrolysis activity,a directed evolution method was carried out.p-NPB was used as the screening substrate.The product,p-nitrophenol,showed the biggest absorption value at 405 nm.The mutant library was built by error-prone PCR method with adjusted Mn2+concentration.
The complete length of the BioH gene was 771 bp and the molecular mass of the protein with 256 amino acids was about 28.2 ku.The targeted enzyme accounted for one quarter of the total expressed proteins.The enzyme??s hydrolysis activity was shown as 18 U/mg and it retained 70% of its hydrolysis activity after incubated at 60 ℃ for 40 min. The thermostability of the enzyme was the foundation in the enzyme application. The optimized concentration of Mn2+ in error-prone PCR was chosen as 0.1 mmol/L determined by agarose electrophoresis of the brightness of PCR product band.And mutant library with a mortality rate of 20% to 40% was appropriate for selection.Sequence detection showed that the mutation rate was kept as 2 3 changed bases in each gene on average.In each round of directed evolution,a library with 5 000 6 000 mutants was screened.In the first round,wild-type BioH was used as the parent and three mutants with improved hydrolysis activity were selected. These mutants were K213E,Q70L/M1 70T and V1 75A,and their hydrolysis activities were improved by 10%, 30% and 50% respectively.Subsequently,the two mutants with only one mutation site for each,K213E and V1 75A,were used as parents to undergo the next round of directed evolution.Three mutants from K213E were selected,namely K213E/M1 97L, K213E/L180M and K213E/C31Y/F50S, whose hydrolysis activities were improved by 40%,50% and 95% respectively.In the mutant library of V1 75A,one mutant,V1 75A/Q129R, was selected,which showed 100% improvement in hydrolysis activity compared to the wild type.Structure analysis by molecular 3D structure software PyMOL gave a clear view of the mutation sites.Sites 31,129,1 75, 180 were located in the α-helixes and sites 50,70,1 70,1 97,213 were located in the loops of protein structure. All of them were far away from the activity sites.
This study clones and overexpresses successfully the carboxylesterase BioH in BL21(DH3).Seven mutants were selected by directed evolution in p-NPB method.The enzyme shows a good evolvability.The positive library can be used in further application of this enzyme.