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Intracellular Esterase from Lactobacillus casei LILA: Nucleotide Sequencing, Purification, and Characterization

Department of Food Science, University of Wisconsin-Madison, Madison 53706

Corresponding author:
J. L. Steele; e-mail:
jlsteele{at}facstaff.wisc.edu.

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°C, 0% NaCl, and pH 5.5, respectively. EstC had significant activity under conditions simulating those of ripening cheese (10°C, 4% NaCl, and pH 5.1). Kinetic constants (K_M and V_max) 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.

Key Words: Lactobacillus casei • esterase • nucleotide sequencing • purification

Abbreviation key: Ap = ampicillin, a_W = water activity, BQL = below quantifiable limits, DFP = diisopropyl fluorophosphate, FA = fatty acid, IAA = iodoacetic acid, IPTG = isopropyl-thio-ß-D-galactoside, LAB = lactic acid bacteria, LB = Luria-Bertani, MES = 2-(N-morpholino)ethansulfonic acid, , MW = molecular weight, ORF = open reading frame, PCMB = p-chloromercuribenzoic acid, , PGL = pregastric lingual lipase, PMSF = phenylmethylsulfonyl fluoride, pNP = p-nitrophenyl, X-Gal = 5-bromo-4-chloro-3-indoyl-ß-D-galactoside

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