Wang Xiong, Lin Hao, Zheng Yu, Feng Juan, Yang Zujun, Tang Lixia
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu 610054, China.
J Biotechnol. 2015 Jul 20;206:1-7. doi: 10.1016/j.jbiotec.2015.04.002. Epub 2015 Apr 17.
Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) displays a broad substrate range with high regio- and enantioselectivity of both ring-closure and ring-opening reactions, making the enzyme a useful catalyst for the production of optically pure epoxides and β-substituted alcohols. In this study, we report a novel method using an MDC-Analyzer-facilitated combinatorial strategy to improve the activity and stability of HheC by simultaneously randomizing multiple contiguous residues. Six contiguous active-site residues, which are the hotspots for improving the activity of HheC, were simultaneously selected and randomized using the MDC-Analyzer-facilitated combinatorial strategy, resulting in a high-quality mutagenesis library. After screening a total of 1152 clones, three positive mutants were obtained, which exhibited approximately 3.5-5.9-fold higher kcat values than the wild-type HheC toward 1,3-dichloro-2-propanol (1,3-DCP). However, the inactivation half-life of the best mutant (DG9) at 55 °C decreased 9-fold compared with that of the wild-type HheC. To improve the stability of mutant DG9, seven contiguous potential surface amino acids were revealed by using the B-FITTER tool. Two charged amino acids, Glu and Lys, which are more abundant in thermophilic proteins than in their mesophilic counterparts, were selected to substitute those seven amino acids and were combined together via an MDC-Analyzer-facilitated combinatorial strategy. Two mutants displaying 1.6- and 2.3-fold higher half-life τ1/2 (55 °C) values than their DG9 template were obtained after screening only 384 clones. The results indicated that an MDC-Analyzer-facilitated combinatorial strategy represents an efficient tool for the directed evolution of functional enzymes with multiple contiguous targeting sites.
来自放射形土壤杆菌AD1的卤代醇脱卤酶（HheC）具有广泛的底物范围，在闭环和开环反应中均具有高区域选择性和对映选择性，这使得该酶成为生产光学纯环氧化物和β-取代醇的有用催化剂。在本研究中，我们报告了一种使用MDC分析仪辅助组合策略的新方法，通过同时随机化多个相邻残基来提高HheC的活性和稳定性。使用MDC分析仪辅助组合策略同时选择并随机化了六个相邻的活性位点残基，这些残基是提高HheC活性的热点，从而产生了一个高质量的诱变文库。在总共筛选了1152个克隆后，获得了三个阳性突变体，它们对1,3-二氯-2-丙醇（1,3-DCP）的kcat值比野生型HheC高约3.5-5.9倍。然而，最佳突变体（DG9）在55°C下的失活半衰期与野生型HheC相比缩短了9倍。为了提高突变体DG9的稳定性，使用B-FITTER工具揭示了七个相邻的潜在表面氨基酸。选择了两个在嗜热蛋白中比在中温蛋白中更丰富的带电荷氨基酸Glu和Lys来取代这七个氨基酸，并通过MDC分析仪辅助组合策略将它们组合在一起。仅筛选384个克隆后，就获得了两个半衰期τ1/2（55°C）值比其DG9模板高1.6倍和2.3倍的突变体。结果表明，MDC分析仪辅助组合策略是一种用于具有多个相邻靶向位点的功能酶定向进化 的有效工具。
