Intracellular esterase from Lactobacillus casei LILA: nucleotide sequencing, purification, and characterization.
An esterase gene (estC) was isolated from a genomic library of Lactobacillus casei LILA. The estC gene consisted of a 777 bp open reading frame encoding a putative peptide of 28.9 kDa. A recombinant EstC fusion protein containing a C-terminal six-histidine tag was constructed and purified to electrophoretic homogeneity. Characterization of EstC revealed that it was a serine-dependent dimeric enzyme. Optimum temperature, NaCl concentration, and pH for EstC were determined to be 30 degrees C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10 degrees C, 4% NaCl, and pH 5.1). Kinetic constants (KM and Vmax) were determined for EstC action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, the previously studied EstA from Lactococcus lactis MG1363 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstC and EstA.
Department of Food Science, University of Wisconsin-Madison, Madison 53706, USA.
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Fenster
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Parkin
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D000595:Amino Acid Sequence; D001483:Base Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015723:Gene Library; D006863:Hydrogen-Ion Concentration; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D012965:Sodium Chloride; D013379:Substrate Specificity; D013696:Temperature
D012965:Sodium Chloride; D004950:Esterases