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

Abstract

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 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.

FIG. 1.

Schematic representation of plasmids pCESTa (A), pCESTb (C), pCESTc (D), and pCESTd (E) and location of estI in a 3.3-kb inserted fragment (B). Diagrams are not drawn to scale.

FIG. 2.

Partial nucleotide sequence and deduced amino acid sequence of the estI gene from L. casei CL96. The proposed ribosomal binding site (RBS) and the −10 and −35 regions of the putative promoter are indicated. The stop codon is marked by an asterisk. A consensus amino acid sequence found in many lipolytic enzymes is boxed. The two inverted arrows at the end of the nucleotide sequence indicate a potential transcriptional termination site.

FIG. 3.

(A) Multiple alignment of the possible catalytic triad of L. casei CL96 EstI (LcaCL) (GenBank accession number AY251019) with lipase precursors from S. epidermidis (Ste) (AF090142) and S. aureus (Sta) (AAA26633), an esterase from B. stearothermophilus (Bst) (AF237623), and a tributyrin esterase from G. thermocatenulatus (Bth) (CAA64621). The residues of the catalytic triad are marked with asterisks. (B) Sequence alignment of the active-site consensus motif of EstI (LcaCL) with a tributyrin esterase from Lactococcus lactis subsp. cremoris (Lcc) (AF059739), an esterase from Lactococcus lactis (Lcl) (AF157601), an arylesterase (EstB) from L. casei LILA (LcaLILA) (AF494421), an esterase (EstC) from L. casei LILA (AF506279), a lipase precursor from S. epidermidis (Ste), esterases from D. radiodurans (Der) (AAF09912) and Arabidopsis thaliana (Ara) (AC002510), an FGH from Anabaena azollae (Ana) (AF035558), an esterase from E. coli (Eco) (AAC73458), esterase D (EstD) from Homo sapiens (Hsa) (NP001975), and FGH from Saccharomyces cerevisiae (Sac) (CAA84054). Highlighted residues are conserved in the majority of sequences aligned.

FIG. 4.

Phylogenetic tree of lipolytic and esterolytic enzymes based on the alignment of EstI or EstI-like proteins. All of these enzymes contain the active-site motif GXSXG as described in the text. The proteins, as well as their accession numbers and sources, are described in the legend to Fig. 3.

FIG. 5.

(A) Overexpression of esterase from L. casei CL96 in E. coli BL21(DE3)/pLysS. Protein samples were separated in an SDS-12% polyacrylamide gel and stained with Coomassie brilliant blue R-250. Lanes: M, molecular mass standards; 1, E. coli BL21(DE3)/pLysS bearing vector pET29a; 2 to 7, E. coli BL21(DE3)/pLysS containing pET29a-estI after 2, 4, 6, 8, 10, and 12 h of induction, respectively; P, purified esterase (3 μg). (B) Staining of a 12% nondenaturing gel for activity of esterase from L. casei CL96 in E. coli BL21(DE3)/pLysS. Lanes 1 to 4, soluble fractions of crude lysates from 10, 8, 6, and 4 h after induction, respectively.

FIG. 6.

Total esterase activity profiles of recombinant E. coli and M. extorquens (A) and recombinant P. pastoris (B).

FIG. 7.

(A) Substrate specificity assay. Rates of hydrolysis were measured with fatty acids of various acyl chain lengths, from pNP-acetate to pNP-palmitate. The highest level of activity, observed with the C₈ substrate, was taken as 100%. (B) Effects of inhibitors on recombinant esterase activity. Different concentrations of DEPC, PMSF, PAO, and PGO were incubated with the enzyme for 30 min at 37°C. The reaction was stopped by chilling on ice, and aliquots were assayed by the standard method.