title: Characterization of an arylesterase from Lactobacillus helveticus CNRZ32.
abstract: An esterase gene (estA) was isolated from a previously constructed genomic library of Lactobacillus helveticus CNRZ32. The estA gene consisted of a 558 bp open reading frame encoding a putative peptide of 21.3 kDa. Protein sequence homology searches using BLAST revealed that EstA had low amino acid sequence identity with the serine-dependent arylesterases TesI (24%) and EtpA (26%) from Escherichia coli and Vibrio mimicus, respectively. A recombinant EstA fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstA revealed that it was a serine-dependent enzyme having a monomeric Mr of 22.6-25.1 kDa. Optimum temperature, NaCl concentration and pH for EstA activity were determined to be 35-40 degrees C, 3.5% NaCl and 7.5-8.0, respectively. EstA had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, pH 5.1). EstA hydrolysed a variety of ester compounds and preferred those with substituted phenyl alcohol and short-chain fatty acid groups. Site-directed mutagenesis suggested that the S10 and H164 residues were essential for EstA activity.
authors.affiliation: Department of Food Science, University of Wisconsin-Madison, WI 53706, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Fenster
authors.forename: K L
authors.initials: KL
authors.lastname: Parkin
authors.forename: J L
authors.initials: JL
authors.lastname: Steele
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004269:DNA, Bacterial; D004926:Escherichia coli; D015870:Gene Expression; D007778:Lactobacillus / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D002265:Carboxylic Ester Hydrolases; C098953:EstA protein, bacteria; C019734:arylesterase