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Applied and Environmental Microbiology, June 2004, p. 3213-3221, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3213-3221.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Characterization and Heterologous Gene Expression of a Novel Esterase from Lactobacillus casei CL96

Department of Food Science and Agricultural Chemistry, McGill University, Ste-Anne-de-Bellevue, Quebec H9X 3V9,¹ Microbial and Enzymatic Technology Group, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2,² Agriculture and Agri-Food Canada, Food R & D Centre, St. Hyacinthe, Quebec J2S 8E3, Canada³

Received 17 November 2003/ Accepted 19 February 2004

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P_mxaF), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S · tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37°C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C₈), with K_m and k_cat values of 14 ± 1.08 µM and 1,245 ± 42.3 S^–1, respectively.

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