Discovery, characterization and mechanism of a Microbacterium esterase for key d-biotin chiral intermediate synthesis

Abstract

Esterases are crucial biocatalysts in chiral compound synthesis. Herein, a novel esterase EstSIT01 belonging to family V was identified from Microbacterium chocolatum SIT101 through genome mining and phylogenetic analysis. EstSIT01 demonstrated remarkable efficiency in asymmetrically hydrolyzing meso-dimethyl ester [Dimethyl cis-1,3-Dibenzyl-2-imidazolidine-4,5-dicarboxyate], producing over 99% yield and 99% enantiomeric excess (e.e.) for (4S, 5R)-monomethyl ester, a crucial chiral intermediate during the synthesis of d-biotin. Notably, the recombinant E. coli expressing EstSIT01 exhibited over 40-fold higher activity than that of the wild strain. EstSIT01 displays a preference for short-chain p-NP esters. The optimal temperature and pH were 45 °C and 10.0, with K_m and k_cat values of 0.147 mmol/L and 5.808 s^− 1, respectively. Molecular docking and MD simulations suggest that the high stereoselectivity for meso-diester may attribute to the narrow entrance tunnel and unique binding pocket structure. Collectively, EstSIT01 holds great potential for preparing chiral carboxylic acids and esters.

Keywords

Genome mining / Esterase / Stereoselective hydrolysis / d-Biotin

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Funding

Shanghai Committee of Science and Technology(13430503400)

Project of Leading Talents in Shandong Taishan Industry(LJNY202019)

Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry(ZX2012-05)

Innovation Program of Shanghai Municipal Education Commission(11YZ227)