A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was ide ntified in the Bacillus subtilis genomic DNA sequence. We have cloned and o verexpressed lipB in B. subtilis and Escherichia coli and have also purifie d the enzyme from a B. subtilis culture supernatant to electrophoretic homo geneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular fil m technique. LipB preferentially hydrolysed triacylglycerol-esters and p-ni trophenyl-esters of fatty acids with short chain lengths of less than or eq ual to 10 carbon atoms. Triolein, which is a typical substrate for true lip ases, was not hydrolysed at all. These results led us to classify LipB as a n esterase rather than a lipase. The catalytic triad of LipB consists of re sidues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence ali gnments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bac illus lipases. All other bacterial lipases contain a glycine residue instea d of the alanine at position-2 with respect to the catalytic serine. We hav e investigated the role of this alanine residue by constructing LipB varian t A76G, thereby restoring the lipase-specific consensus motif. When compare d with LipB this variant showed a markedly reduced thermostability but an i ncreased stability at pH 5-7. Determination of the specific activities of w ild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had convert ed the esterase LipB into a monoacylglycerol hydrolase.