Cloning, expression and characterization of a new 2-Cl-propionic acid ester hydrolase from B. subtilis

Applying an agar plate assay, a novel gene encoding an esterase from Bacillus subtilis Est4B was cloned and highly overexpressed in E. coli. The protein sequence revealed high homology to the srfD gene, which encodes the fourth open reading frame, a putative thioesterase gene from the surfactin synthetase gene cluster. It was almost identical to an unpublished sequence from a putative surfactin synthetase cluster from B. subtilis A13. The enzyme (Est4B1) was produced in E. coli, purified to homogeneity and crystallized. ational prediction of the protein fold showed high structural similarity of Est4B1 to haloperoxidases and dehalogenases and much lower similarity to lipases and esterases. However, by primary sequence analysis we found a typical esterase/thioesterase/lipase motif. Biocatalytic activity on several model esterase substrates was detected and quantified. This is the first time enzymatic activity could be shown for this type of independent putative thioesterases and this enzyme may serve as a model protein to solve the structure of this type of hydrolytic enzymes, which is commonly found in gene clusters of non-ribosomal peptide synthetases.