PDF(Pubmed)
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 Km and kcat 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.
摘要：
酯酶是手性化合物合成中的关键生物催化剂。在这里，通过基因组挖掘和系统发育分析，从巧克力微杆菌SIT101中鉴定出属于V家族的新型酯酶EstSIT01。EstSIT01在不对称水解内消旋二甲酯[顺式-1,3-二苄基-2-咪唑烷-4,5-二羧酸二甲酯]中表现出卓越的效率，产生超过99%的产率和99%的对映体过量(e.e.)(4S，5R)-单甲酯，d-生物素合成过程中的关键手性中间体。值得注意的是,表达EstSIT01的重组大肠杆菌表现出比野生菌株高40倍以上的活性。EstSIT01显示对短链p-NP酯的偏好。最佳温度和pH为45°C和10.0，Km和kcat值分别为0.147mmol/L和5.808s-1。分子对接和MD模拟表明，内消旋二酯的高立体选择性可能归因于狭窄的入口通道和独特的结合袋结构。总的来说，EstSIT01具有制备手性羧酸和酯的巨大潜力。
