As　a　chiral　precursor　for　the　important　anticoagulant　Edoxaban,　enantioselective　synthesis　of　(S)-3-cyclohexene-1-carboxylic　acid　is　of　great　significance.　The　complicated　procedures　and　generation　of　massive　solid　waste　discourage　its　chemical　synthesis,　and　the　alternative　biocatalysis　route　calls　for　an　enzyme　capable　of　asymmetric　hydrolysis　of　racemic　methyl-　3-cyclohexene-1-carboxylate.　To　this　end,　we　engineered　the　E.　coli　esterase　BioH　for　improved　S-enantioselectivity　via　rational　design.　By　combinatorial　modulation　of　steric　and　aromatic　interactions,　a　positive　mutant　Mu3　(L24A/W81A/L209A)　with　relatively　high　S-selectivity　in　hydrolyzing　racemic　methyl-3-cyclohexene-1-carboxylate　was　obtained,　improving　the　enantiomeric　excess　from　32.3%　(the　wild　type)　to　70.9%.　Molecular　dynamics　simulation　was　conducted　for　both　(R)-　or　(S)-　complexes　of　the　wild　type　and　Mu3　to　provide　hints　for　the　mechanism　behind　the　increased　S-selectivity.　Moreover,　the　reaction　conditions　of　Mu3　in　methyl-3-cyclohexene-1-carboxylate　hydrolysis　was　optimized　to　improve　the　conversion　rate　to　2　folds.　©　2019　Japan　Society　for　Bioscience,　Biotechnology,　and　Agrochemistry.