Characterization of a novel beta-xylosidase, XylC, from Thermoanaerobacterium saccharolyticum JW/SL-YS485

Abstract

The 1,914-bp open reading frame of xylC from Thermoanaerobacterium saccharolyticum JW/SL-YS485 encodes a calculated 73-kDa β-xylosidase, XylC, different from any glycosyl hydrolase in the database and representing a novel glycohydrolase family. Hydrolysis occurred under retention of the anomeric configuration, and transglycosylation occurred in the presence of alcohols as acceptors. With the use of vector pHsh, expression of XylC, the third β-xylosidase in this bacterium, increased approximately 4-fold when a loop within the translational initiation region in the mRNA was removed by site-directed mutagenesis. The increased expression of xylC(m) is due to removal of a stem-loop structure without a change of the amino acid sequence of the heterologously expressed enzyme (XylC(rec)). When gel filtration was applied, purified XylC had molecular masses of 210 kDa and 265 kDa using native gradient gel electrophoresis. The protein consisted of 78-kDa subunits based on SDS gel electrophoresis and contained 6% carbohydrates. XylC and XylC(rec) exhibited maximum activity at 65°C and pH(65°C) 6.0, a 1-h half-life at 67°C, a K(m) for p-nitrophenyl-β-D-xyloside of 28 mM, and a V(max) of 276 U/mg and retained 70% activity in the presence of 200 mM xylose, suggesting potential for industrial applications.

FIG. 1.

Nondenaturing gradient (4 to 30%) polyacrylamide gel analysis of purified native XylA and C. Lanes: M, molecular mass markers (in kDa); 1, XylC (5 μg); 2, XylA (2 μg). Enzymes were purified by Sepharose column chromatography and gel filtration as presented in Table 2. XylA and XylC were separated during phenyl-Sepharose chromatography.

FIG. 2.

Secondary structure of the translation initiation region (TIR) of the target gene. Predicted secondary structure of the 70 nt of TIR of pHsh-xylC (parental gene structure) (a) and pHsh-xylC^m (modified gene structure) (b) calculated with the Mfold algorithm of Zuker (38). The elimination of the loop in pHsh-xylC^m did not lead to a change of the amino acid sequence in the heterologously expressed (recombinant) enzyme XylC^rec.

FIG. 3.

Gene sequence for the xylC^m insert in plasmid pHsh-xylC^m (a) and SDS-PAGE analysis of total soluble proteins in recombinant E. coli JM109 cells harboring pHsh-xylC or pHsh-xylC^m (b). (a) Restriction sites, including the 3′ HindIII cloning sites, Hsh promoter sites (−10 and −35 regions), translation initiation region (70 nt), and transcription terminator sites, are illustrated. Oversized italic bases represent mutant nucleotides. rbs, ribosome binding site. (b) Lane M, low-molecular-weight marker; lane 1, E. coli JM109 containing the plasmid pHsh; lane 2, E. coli JM109 containing the plasmid pHsh-xylC; lane 3, E. coli JM109 containing the plasmid pHsh-xylC^m; lane 4, purified recombinant XylC^rec.

FIG. 4.

(a and b) Effects of pH (a) and temperature (b) on activity of purified recombinant XylC^rec. The optimum pH for xylosidase activity was determined by incubation at 65°C for 2 min in the pH^65°C range from 4.5 to 7.5 (pH was measured at 65°C; see the work of Wiegel [32] for explanation and calibration) in 100 mM potassium hydrogen phthalate-imidazole buffer (▴).The optimum temperature for enzyme activity was assayed in 100 mM potassium hydrogen phthalate-imidazole buffer at pH^65°C 6.0 at 45 to 75°C for 5 min (⧫). Each assay was performed with 1.3 μg of protein per ml. (c) Effects of pH on the purified recombinant XylC^m activity. Residual activities were assayed in 100 mM potassium hydrogen phthalate-imidazole buffer (pH 6.0) at 65°C for 5 min after preincubation of the enzyme in 50 mM potassium hydrogen phthalate-imidazole buffer at pH^65°C 4.5 to 8 at 67°C for 2 h (▪) or 65°C for 1 h (⧫). (d and e) Thermostability of purified recombinant XylC^m. Residual activities were assayed in 100 mM potassium hydrogen phthalate-imidazole buffer (pH^65°C 6.0) at 65°C for 5 min after preincubation of the enzyme in 50 mM potassium hydrogen phthalate-imidazole buffer (pH^65°C 6.0) at 50°C (▴), 65°C (▪), and 70°C (⧫) for varied times (d) and at 66°C, 67°C, 68°C, and 69°C for 1 h (e).

FIG. 5.

Effects of the substrate pNP-xyloside concentration on the activity of XylC (○) and XylA (•), both purified from T. saccharolyticum JW/SL-YS485. The variations in the duplicate assays are smaller than the symbols.

FIG. 6.

(a) Stereochemical analysis of reaction mixtures containing pNP-xyloside and XylC from T. saccharolyticum JW/SL-YS485. The enzymatic reaction was carried out at 40°C for 1, 2, 5, and 10 min. The migration times of β-xylose and β-xylopyranoside were 6.58 and 6.88 min, respectively. (b) Thin-layer chromatography of the transglycosylation products from pNP-xyloside (5 mM) and various alcohols in 50 mM morpholineethanesulfonic acid (MES) buffer (pH^65°C 6.0). Lane M, pNP-xyloside; lane 0, control (reaction mixture without alcohol); lane 1, plus methanol; lane 2, plus ethanol; lane 3, plus 1-propanol; lane X, standard xylose.