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Characterization of an arylesterase from Lactobacillus helveticus CNRZ32
Author(s) -
Fenster K. M.,
Parkin K. L.,
Steele J. L.
Publication year - 2000
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1046/j.1365-2672.2000.00993.x
Subject(s) - lactobacillus helveticus , esterase , biochemistry , lactobacillus sakei , biology , peptide sequence , serine , escherichia coli , histidine , open reading frame , sequence analysis , amino acid , microbiology and biotechnology , chemistry , gene , enzyme , lactobacillus , fermentation
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 M r of 22·6–25·1 kDa. Optimum temperature, NaCl concentration and pH for EstA activity were determined to be 35–40 °C, 3·5% NaCl and 7·5–8·0, respectively. EstA had significant activity under conditions simulating those of ripening cheese (10 °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.