1. Biochemical properties of a novel thermostable and highly xylose-tolerant β-xylosidase/α-arabinosidase from Thermotoga thermarum
- Author
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Yingjuan Huang, Liangliang Wang, Fei Wang, Yu Zhang, Huaxiang Gu, Xun Li, and Hao Shi
- Subjects
Bioconversion ,Xylose tolerant ,Management, Monitoring, Policy and Law ,Xylose ,Applied Microbiology and Biotechnology ,Thermotoga thermarum ,chemistry.chemical_compound ,Hydrolysis ,Xylooligosaccharides ,Hemicellulose ,Thermostability ,biology ,Renewable Energy, Sustainability and the Environment ,Thermophile ,Research ,Substrate (chemistry) ,β-xylosidase ,Enzyme assay ,General Energy ,chemistry ,Biochemistry ,biology.protein ,α-arabinosidase ,Biotechnology - 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 K m and V max 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 k cat /K m 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 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 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.
- Published
- 2012