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

Background: beta-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 beta-xylosidase with high specific activity for bioconversion of xylooligosaccharides has a great potential in the fields as above. Results: A beta-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 beta-xylosidases were constructed using Neighbor-Joining (NJ) and Maximum-Parsimony (MP) methods. The phylogeny and amino acid analysis indicated that the Tth xynB3 beta-xylosidase was a novel beta-xylosidase of GH3. The optimal activity of the Tth xynB3 beta-xylosidase was obtained at pH 6.0 and 95 degrees 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 V-max values for p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-alpha-L-arabinofuranoside were 0.27 mM and 223.3 U/mg, 0.21 mM and 75 U/mg, respectively. The k(cat)/K-m values for p-nitrophenyl-beta-D-xylopyranoside and p-nitrophenyl-a-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 K-i value approximately 1000 mM. It was stimulated by xylose at higher concentration up to 500 mM, above which the enzyme activity of Tth xynB3 beta-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 beta-xylosidase exhibited a high hydrolytic activity on xylooligosaccharides. When 5% substrate was incubated with 0.3 U Tth xynB3 beta-xylosidase in 200 mu L reaction system for 3 h, almost all the substrate was biodegraded into xylose. Conclusions: The article provides a useful and novel beta-xylosidase displaying extraordinary and desirable properties: high xylose tolerance and catalytic activity at temperatures above 75 degrees C, thermally stable and excellent hydrolytic activity on xylooligosaccharides.