Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarum

Abstract

Background: β-Xylosidase is an important constituent of the hemicellulase system and it plays an important role in hydrolyzing xylooligosaccharides to xylose. Xylose, a useful monose, has been utilized in a wide range of applications such as food, light, chemical as well as energy industry. Therefore, the xylose-tolerant β-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above.

Results: A β-xylosidase gene (Tth xynB3) of 2,322 bp was cloned from the extremely thermophilic bacterium Thermotoga thermarum DSM 5069 that encodes a protein containing 774 amino acid residues, and was expressed in Escherichia coli BL21 (DE3). The phylogenetic trees of β-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 β-xylosidase was a novel β-xylosidase of GH3. The optimal activity of the Tth xynB3 β-xylosidase was obtained at pH 6.0 and 95°C and was stable over a pH range of 5.0-7.5 and exhibited 2 h half-life at 85°C. The kinetic parameters Km and Vmax values for p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The kcat/Km values for p-nitrophenyl-β-D-xylopyranoside and p-nitrophenyl-α-L-arabinofuranoside were 1,173.4 mM-1 s-1 and 505.9 mM-1 s-1, respectively. It displayed high tolerance to xylose, with Ki value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 β-xylosidase was gradually decreased. However, it still remained approximately 50% of its original activity even if the concentration of xylose was as high as 1000 mM. It was also discovered that the Tth xynB3 β-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 β-xylosidase in 200 μL reaction system for 3 h, almost all the substrate was biodegraded into xylose.

Conclusions: The article provides a useful and novel β-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75°C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.

Figure 1

SDS-PAGE analysis of recombinant Tth xynB3β-xylosidase in E. coli BL21 (DE3). Lane M: protein marker, lane 1: purified Tth xynB3 β-xylosidase.

Figure 2

Effects of pH and temperature on the activity and stability of the recombinant Tth xynB3β-xylosidase. a) Effect of pH on Tth xynB3 β-xylosidase activity. b) Effect of temperature on Tth xynB3 β-xylosidase activity. c) The thermostability of the Tth xynB3 β-xylosidase. The residual activity was monitored while the enzyme was incubated at 75°C (filled left triangles), 80°C (filled down triangles), 85°C (filled up triangles), 90°C (filled circles) and 95°C (filled squares). The maximum activity was defined as 100% (a, b) or initial activity was defined as 100% (c).

Figure 3

Effects of xylose on Tth xynB3β-xylosidase activity. The reaction was conducted with p-nitrophenyl-β-D-xylopyranoside as the substrate. The values were the mean of three separate experiments, and the variations about the mean were all below 5%.

Figure 4

Analysis of xylooligosaccharides hydrolyzed by Tth xynB3 β-xylosidase. The products of the reaction were determined using thin layer chromatography. M, mixture of xylose, xylobiose, xylotriose and xylotetraose (2.5% each, wt/vol). a Lane 1, 2, 3, 4: samples of xylobiose, xylotriose and xylotetraose (5%, wt/vol) incubated with Tth xynB3 β-xylosidase (0.3 U) for 0.5h, 1 h, 2 h, 3h, respectively. b Lane 1: samples of XOs obtained from cornstalk without hydrolysis using Tth xynB3 β-xylosidase, lane 2, 3, 4: samples of XOs obtained from cornstalk incubated with Tth xynB3 β-xylosidase (0.3 U) for 1 h, 2 h, 3h, respectively.

Figure 5

Maximum-Parsimony (MP) tree results from analysis of Tth xynB3β-xylosidases of 55 amino acid sequences. Numbers on nodes correspond to percentage bootstrap values for 1000 replicates.