Characterization of a novel thermostable carboxylesterase from Geobacillus kaustophilus HTA426 shows the existence of a new carboxylesterase family

Abstract

The gene GK3045 (741 bp) from Geobacillus kaustophilus HTA426 was cloned, sequenced, and overexpressed into Escherichia coli Rosetta (DE3). The deduced protein was a 30-kDa monomeric esterase with high homology to carboxylesterases from Geobacillus thermoleovorans NY (99% identity) and Geobacillus stearothermophilus (97% identity). This protein suffered a proteolytic cut in E. coli, and the problem was overcome by introducing a mutation in the gene (K212R) without affecting the activity. The resulting Est30 showed remarkable thermostability at 65 degrees C, above the optimum growth temperature of G. kaustophilus HTA426. The optimum pH of the enzyme was 8.0. In addition, the purified enzyme exhibited stability against denaturing agents, like organic solvents, detergents, and urea. The protein catalyzed the hydrolysis of p-nitrophenyl esters of different acyl chain lengths, confirming the esterase activity. The sequence analysis showed that the protein contains a catalytic triad formed by Ser93, Asp192, and His222, and the Ser of the active site is located in the conserved motif Gly91-X-Ser93-X-Gly95 included in most esterases and lipases. However, this carboxylesterase showed no more than 17% sequence identity with the closest members in the eight families of microbial carboxylesterases. The three-dimensional structure was modeled by sequence alignment and compared with others carboxylesterases. The topological differences suggested the classification of this enzyme and other Geobacillus-related carboxylesterases in a new alpha/beta hydrolase family different from IV and VI.

FIG. 1.

Multiple sequence alignment for CE from G. kaustophilus HTA426 (GkCE) and related Geobacillus CEs. EsPript outputs (11) obtained with the sequences from the SWISSPROT databank and aligned with CLUSTAL W (43). Sequences are grouped according to similarity. The enzyme showed 97% sequence identity with Est30_Gs, 99% with EstA from G. thermoleovorans (GtEstA) NY, and 96% with Est30_Gt. Residues strictly conserved have a solid background. Symbols above blocks of sequences represent the secondary structure, springs represent helices, and arrows represent β-strands. The residues forming the hydrophobic specificity pocket are indicated by small black asterisks. Triangles represent the location of the active site. K212 represents the protease cut.

FIG. 2.

SDS-PAGE of the GK3045 gene product after 6 h of IPTG induction. Each lane contained 40 μg of protein. M, molecular size standards (P7708S; New England Biolabs). Lane 1, Rosetta (DE3) transformed with pET28a and induced; lane 2, the cell extract containing cloned CE_Gk after tangential ultrafiltration; lane 3, CE_Gk obtained by site-directed mutagenesis (K212R) without the proteolytic cut; lane 4, smaller and inactive CE_Gk obtained by site-directed mutagenesis (K212Stop); lane 5, His-Trap FF column pooled fraction in which a purified CE_Gk (of about 30 kDa) and another protein (of about 27 kDa) are shown; the latter is the result of a proteolytic cut.

FIG. 3.

pH stability profiles of the purified CE_Gk. Samples were analyzed after 24 h of incubation in media with different pH values (50 mM sodium acetate, pH 4.0 to 5.5; 50 mM potassium phosphate, pH 6 to 8; 50 mM sodium bicarbonate, pH 8 to 9; and 50 mM boric acid, pH 10.0 to 11.0) to determine residual activity under the standard reaction conditions for the enzyme assay, using p-NP caprylate as a substrate.

FIG. 4.

Temperature stability profiles of the purified CE_Gk at pH 8.0. Enzyme was incubated for different periods of time at 60°C (•), 65°C (▪), 70°C (▴), 75°C (⧫), and 80°C (○), and then activity was measured under the standard reaction conditions at 25°C using p-NP caprylate as the substrate. (Inset) Expanded study of thermostability of the enzyme at the physiological temperature of G. kaustophilus (60°C) under the experimental conditions described above.

FIG. 5.

Schematic representation of tertiary structures. (A) Modeled structure of the monomeric CE_Gk; the possible proteolytic site (K212) is highlighted in green. (B) A. acidocaldarius CE ([AaEst2] PDB code 1EVQ). (C) B. subtilis CE ([BsBEst] PDB code 2R11). (D) CE_Pf (PDB code 1AUO). (E) CE_Bc (PDB code 2H1I). Catalytic triads are shown in black (Ser-Asp-His). β-Strands and α-helices belonging to the canonical α/β hydrolase fold are shown in blue and red, respectively. Gray parts of the structures indicate the presence of β-turns. Framed lids formed by β-strands and α-helices are shown in cyan and green, respectively. These figures were rendered using SWISS-MODEL and Swiss-PdbViewer (36).

FIG. 6.

Topology diagrams of the members of the Geobacillus CE family and other members of the α/β hydrolase family. (A) Diagram of A. acidocaldarius CE (AaEst2; PDB code 1EVQ). (B) Diagram of B. subtilis CE (BsBEst; PDB code 2R11). (C) Diagram of Est30_Gs (PDB code 1TQH). (D) Diagram of the recombinant CE_Gk. (E) Diagram of EstA from G. thermoleovorans YN (GtEstA). (F) Diagram of Est30_Gt. (G) Diagram of CE_Pf (PDB code 1AUO). (H) Diagram of CE_Bc (PDB code 2H1I). α-Helices are represented by circles, and β-strands are represented by triangles. White circles represent 3₁₀-helices or other small helices in the structures. The framed figures represent those strands and loops that are not contained in the central β-sheet and that form the lids. The diagrams were made using the TOPS program (27).

FIG. 7.

Phylogenetic analysis of the studied esterases. Family IV, A. acidocaldarius CE (AaEst2) and B. subtilis CE (BsEst); family VI, CE_Bc and CE_Pf; new Geobacillus sp. family, CE_Gk, Est30_Gs, G. thermoleovorans YN CE (GtEstA), and Est30_Gt. The phylogenetic tree was constructed using TreeView software with the neighbor-joining method.