A new bacterial carboxylesterase (CarEst3) was identified by genome mining and found to efficiently hydrolyze racemic methyl 3-cyclohexene-1-carboxylate (rac-CHCM) with a nearly symmetric structure for the synthesis of (S)-CHCM. CarEst3 displayed a high substrate tolerance and a stable catalytic performance. The enantioselective hydrolysis of 4.0 M (560 g•L −1 ) rac-CHCM was accomplished, yielding (S)-CHCM with a >99% ee, a substrate to catalyst ratio of 1400 g•g −1 , and a space-time yield of 538 g•L −1 •d −1 .
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