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- Title
Improving the thermostability of GH11 xylanase XynXM from Aspergillus brunneoviolaceus CBS 621.78 by the design of cord region.
- Authors
Tongbiao Li; Jiaze Tang; Siqi Li; Hui Peng; YanYan Zhu; Jinjin Zhu; Yue Li; Chaoying Liu; Enzhong Li
- Abstract
Xylanases which hydrolyze ß-1,4-glycosidic bonds of xylans to yield xylooligosaccharides have a potential to be used in pulp bleaching, food processing, and feed processing. However, the poor thermostability of xylanases is a critical limiting factor in industrial applications. The thermostability of xylanases can be improved by protein engineering and computer-assisted design. In the present study, the thermostabilizing mutation G143W locating at the cord region of xylanase XynXM from Aspergillus brunneoviolaceus CBS 621.78 was predicted by the FireProt web server and constructed by site-directed mutagenesis. Heat tolerance experiments were performed in mutant G143W and wildtype XynXM. G143W exhibited a substantially improved thermostability with half-life of inactivation at 45°C enhanced from 41.5 min to 65.3 min, which was approximately 1.6-fold increase over that of wildtype. Furthermore, the optimum temperature was 50°C, which has the best reactive activity, and 5°C higher than that of XynXM (45°C). The reactive optimal pH of G143W increased from pH 5.0 to pH7.0. The pH stability span (4.0-9.0) of G143W displayed a significant improvement comparing to that of XynXM. Comparing to the wildtype, the half-life of G143W at 35°C in simulated intestinal fluid (pH 6.8, 10 mg/mL trypsin solution) was increased by 35.8 min. Novel hydrophobic contacts and hydrogen bonds were discovered in the model of G143W, which explained the reason of enhanced thermal stability. The key residues Trp57, Asn84, Val86, Tyr114, Gln117, Trp120, Glu127, Tyr137, Pro139, Ser141, Thr144, Ala161, Pro167, and Glu218 were identified in protein-ligand interaction by the molecular docking analysis using AutoDock Vina, which contributed to the catalytic activity and stability of enzyme. This study developed a thermostabilizing mutant G143W locating in the cord region of xylanase by the prediction of FireProt web server, which laid a foundation for exploring the functional relationship between cord region and xylanase XynXM.
- Subjects
ENZYME stability; SITE-specific mutagenesis; PROTEIN-ligand interactions; MOLECULAR interactions; PROTEIN engineering; XYLANASES
- Publication
Journal of Biotech Research, 2022, Vol 13, p130
- ISSN
1944-3285
- Publication type
Article