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

Abstract

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of # 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5±7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase.

Molecular cloning, over expression and characterization of thermoalkalophilic esterases isolated from Geobacillus sp.

“…The recombinant esterases were very stable when held at pH 9.5-11.0 for hours, retaining 80-100% of its initial activity (data is not shown) indicating that the recombinant enzymes are extremely alkali-tolerant proteins. Similar to the results here, esterase enzyme from G. thermoleovorans and B. subtilis have been reported to be more active and stable at alkaline pH (Eggert et al 2000;Soliman et al 2007). This is in fact a desirable property for industrial applications.…”

Biochemical properties and three-dimensional structures of two extracellular lipolytic enzymes from Bacillus subtilis

“…Six extracellular lipases and two esterases have been described so far to occur in Bacillus species ( [9,12,[22][23][24][26][27][28][29][30][31][32][33][34][35][36], GenBank accession no. AJ297356) which share a conserved Ala-X-Ser -X-Gly pentapeptide containing the catalytic serine residue.…”

“…This bacterium can also efficiently utilize complex nutrient sources because it produces several extracellular hydrolytic enzymes [6]. Among them, three lipolytic enzymes have been identified: a phospholipase [7,8], a lipase LipA [9,10] and an esterase LipB [11,12].…”

Directed Evolution of an Enantioselective Bacillus subtilis Lipase

“…The specific activities of improved BSLA variants purified by phenyl sepharose chromatography (Eggert et al , 2000) were different from the wild-type enzyme. However, all improved variants still -II-2H12 58 I22V Q164H G1-II-3-H6 57 I22T L114P G1-II-2-A7 54 L124S G1-III-10-C10 56 N50S G1-III-13-G9 54 showed enzymatic activity towards the standard lipase substrate p -nitrophenyl caprylate indicating that lipolytic activity itself was retained also after the third round of random mutagenesis.…”

“…The residual enzymatic activities were determined spectrophotometrically using p -nitrophenylcaprylate as the substrate (Eggert et al ., 2000).…”