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Nucleotide Sequencing, Purification, and Biochemical Properties of an Arylesterase from Lactobacillus casei LILA

^* 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, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motif identified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 ± 2.5 kDa, while the subunit molecular weight was 36.7 ± 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0, 50 to 55°C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10°C, and 4% NaCl). Kinetic constants (K_M and V_max) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.

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

Abbreviation key: Ap = ampicillin, a_w = water activity, DFP = diisopropyl fluorophosphate, IAA = iodoacetic acid, IPTG = isopropyl-thio-ß-D-galactoside, LAB = lactic acid bacteria, LB = Luria-Bertani, MES = 2-(N-morpholino) ethanesulfonic acid, 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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