Effect of introducing disulfide bridges in C-terminal structure on the thermostability of xylanase XynZF-2 from Aspergillus niger

In this study, a mutant xylanase of high thermostability was obtained by site-directed mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The optimum temperature of the variant XynZFTA Was improved from 45 degrees C to 60 degrees C, and XynZFTA retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50 degrees C for 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH = 5.0). The pH stability span (5.0 similar to 7.0) of the mutant xylanase Was increased to 3.0 similar to 9.0. Overall, the results implied that the introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH stability of XynZF-2.