Expression, characterization, and improvement of a newly cloned halohydrin dehalogenase from Agrobacterium tumefaciens and its application in production of epichlorohydrin.
A gene encoding halohydrin dehalogenase (HHDH) from Agrobacterium tumefaciens CCTCC M 87071 was cloned and expressed in Escherichia coli. To increase activity and stability of HHDH, 14 amino acid residues around the active site and substrate-binding pocket based on the structural analysis and molecular docking were selected as targets for site-directed mutagenesis. The studies showed that the mutant HHDH (Mut-HHDH) enzyme had a more accessible substrate-binding pocket than the wild-type HHDH (Wt-HHDH). Molecular docking revealed that the distance between the substrate and active site was closer in mutant which improved the catalytic activity. The expressed Wt-HHDH and Mut-HHDH were purified and characterized using 1,3-dichloro-2-propanol (1,3-DCP) as substrates. The specific activity of the mutant was enhanced 26-fold and the value of k cat was 18.4-fold as compared to the Wt-HHDH, respectively. The Mut-HHDH showed threefold extension of half-life at 45 °C than that of Wt-HHDH. Therefore it is possible to add 1,3-DCP concentration up to 100 mM and epichlorohydrin (ECH) was produced at a relatively high conversion and yield (59.6 %) using Mut-HHDH as catalyst. This Mut-HHDH could be a potential candidate for the upscale production of ECH.
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, Zhejiang, People's Republic of China.
Zhi-Qiang
ZQ
Liu
Ai-Cun
AC
Gao
Ya-Jun
YJ
Wang
Yu-Guo
YG
Zheng
Yin-Chu
YC
Shen
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D055162:Biocatalysis; D001711:Biotransformation; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004811:Epichlorohydrin / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008401:Gas Chromatography-Mass Spectrometry; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013696:Temperature; D000517:alpha-Chlorohydrin / Q000031:analogs & derivatives / Q000378:metabolism
D008670:Metals; D004811:Epichlorohydrin; C033489:1,3-dichloro-2-propanol; D000517:alpha-Chlorohydrin; D006867:Hydrolases; C430848:halohydrin dehalogenase