Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96

• Published in: Applied and Environmental Microbiology 2004-06, Vol.70 (6), p.3213-3221
• Main Authors: Choi, Y.J, Miguez, C.B, Lee, B.H
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Amino acids; Bacteria; Base Sequence; Biological and medical sciences; cheese starters; Cloning, Molecular; enzyme activity; Enzymes; Enzymology and Protein Engineering; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; esterases; Esterases - genetics; Esterases - metabolism; food biotechnology; Fundamental and applied biological sciences. Psychology; Gene expression; gene transfer; Hydrogen-Ion Concentration; Lacticaseibacillus casei - enzymology; Lacticaseibacillus casei - genetics; Lactobacillus casei; Methylobacterium extorquens; Methylobacterium extorquens - enzymology; Methylobacterium extorquens - genetics; Microbiology; molecular cloning; Molecular Sequence Data; nucleotide sequences; Pichia - enzymology; Pichia - genetics; Pichia pastoris; recombinant proteins; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Sequence Analysis, DNA; structural genes; substrate specificity; Temperature
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/AEM.70.6.3213-3221.2004

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 microliter and 1,245 ± 42.3 S-1, respectively.