Abstract
Halohydrin dehalogenases (HHDHs) represent as important biocatalysts for the production of valuable chiral epoxides and β-substituted alcohols. The HHDH from A. radiobacter (HheC) showed great potential for biosynthesis of (S)-epichlorohydrin ((S)-ECH). However, two bottlenecks including self-degradation and enzyme-mediated racemization of product in aqueous solution restricted its wide-spread applications. To solve the problems, the biocatalytic process was performed in aqueous-organic two-phase system and structure-based engineered of HheC was performed. A newly found zigzag tunnel connecting the active site region and outside space, as well as a depressed cave structure in formation of an extra three-pot herringbone channel were demonstrated to play a pivotal role affecting the activity and enantioselectivity of the enzyme. By mutation of the involved amino acid residues, two variants E255 T and E255 G showed significantly improved activity, with specific activity increased from 59.9 U/mg to 101.6 U/mg and 77.5 U/mg, respectively, while one variant Y93 L exhibited high enantioselectivity, with ee value of (S)-ECH maintained at >99 %. In addition, the synergic variant Y93 L/E255 T had both improved catalytic efficiency and enantioselectivity. The results not only facilitate the applicability of HheC for efficient production of (S)-ECH, but also give new insights into the structural-functional relationship of the enzyme.
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