title: Exploring the thermostable properties of halohydrin dehalogenase from Agrobacterium radiobacter AD1 by a combinatorial directed evolution strategy.
abstract: As a crucial factor for biocatalysts, protein thermostability often arises from a combination of factors that are often difficult to rationalize. In this work, the thermostable nature of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) was systematically explored using a combinatorial directed evolution approach. For this, a mutagenesis library of HheC mutants was first constructed using error-prone PCR with low mutagenesis frequency. After screening approximately 2000 colonies, six mutants with eight mutation sites were obtained. Those mutation sites were subsequently combined by adopting several rounds of iterative saturation mutagenesis (ISM) approach. After four rounds of saturation mutagenesis, one best mutant ISM-4 with a 3400-fold improvement in half-life (t1/2) inactivation at 65 °C, 18 °C increase in apparent Tmvalue, and 20 °C increase in optimum temperature was obtained, compared to wild-type HheC. To the best of our knowledge, the mutant represents the most thermostable HheC variant reported up to now. Moreover, the mutant was as active as wild-type enzyme for the substrate 1,3-dichloro-2-propanol, and they remained most enantioselectivity of wild-type enzyme in the kinetic resolution of rac-2-chloro-1-phenolethanol, exhibiting a great potential for industrial applications. Our structural investigation highlights that surface loop regions are hot spots for modulating the thermostability of HheC, the residues located at these regions contribute to the thermostability of HheC in a cooperative way, and protein rigidity and oligomeric interface connections contribute to the thermostability of HheC. All of these essential factors could be used for further design of an even more thermostable HheC, which, in turn, could greatly facilitate the application of the enzyme as a biocatalyst.
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China.
authors.forename: Zhiyun
authors.initials: Z
authors.lastname: Wu
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Deng
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China.
authors.forename: Yapei
authors.initials: Y
authors.lastname: Tong
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Liao
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Xin
authors.affiliation: Research and Development Center, Angel Yeast Co., Ltd., Yichang, China.
authors.forename: Huashun
authors.initials: H
authors.lastname: Yu
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China.|Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, 610054, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Feng
authors.affiliation: School of Life Science and Technology, University of Electronic Science and Technology of China, No. 4, Section 2, North Jianshe Road, Chengdu, 610054, China. lixiatang@uestc.edu.cn.|Center for Informational Biology, University of Electronic Science and Technology of China, Chengdu, 610054, China. lixiatang@uestc.edu.cn.
authors.forename: Lixia
authors.initials: L
authors.lastname: Tang
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology / Q000235:genetics*; D055162:Biocatalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D015723:Gene Library; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D016133:Polymerase Chain Reaction; D013696:Temperature; D000517:alpha-Chlorohydrin / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: C033489:1,3-dichloro-2-propanol; D000517:alpha-Chlorohydrin; D006867:Hydrolases; C430848:halohydrin dehalogenase
keywords: Biocatalysis; Combinatorial directed evolution; Halohydrin dehalogenase; Optically pure compounds; Thermostability