Paralogous gene analysis reveals a highly enantioselective 1,2-O-isopropylideneglycerol caprylate esterase of Bacillus subtilis

Carboxylesterase NP of Bacillus subtilis Thai 1-8, characterized in 1992 as a very enantioselective (S)-naproxen esterase, was found to show no enanti opreference towards (S)-1,2-O-isopropylideneglycerol (IPG) esters. The ybfK gene was identified by the B. subtilis genome project as an unknown gene w ith homology to carboxylesterase NP. The purpose of the present study was t o characterize the ybfK gene product in order to determine whether this par alogue of carboxylesterase NP had an altered or enhanced stereospecificity. The ybfK gene was cloned and expressed in B. subtilis using a combination o f two strong promoters in a multicopy vector. The enzyme was purified from the cytoplasm of B. subtilis by means of anion exchange and hydrophobic int eraction chromatography. The purified YbfK is an enzyme of 296 amino acids and shows an apparent molecular mass of 32 kDa (SDS/PAGE). Comparison of th e activities of YbfK and carboxylesterase NP towards caprylate esters of IP G revealed that YbfK produces ((S)-IPG with 99.9% enantioselectivity. Therefore, we conclude that we have isolated a paralogue of carboxylesteras e NP that can be used for the enantioselective production of (S)-IPG.