Abstract
BackgroundXylanases have been successfully used in food, paper, and pulp industries and are considered to be a key player in the biodegradation of xylan to valuable end products. However, most of the natural xylanases present poor activity in high-temperature and high-alkali environment. Therefore, it is necessary to modify the enzymes to meet the increasing demands of industries.ResultsDirected evolution was used to improve the specific activity and pH stability of the xylanase (XynHBN188A) that originated from Bacillus pumilus HBP8. The xylanase XynHBN188A was mutated by error-prone PCR. The mutant, XynHBN188A217, was screened from the mutant library by functional screening. The specific activity of XynHBN188A217 was 3986.7 U/mg, which was 2.8-fold higher than that of wild type. The optimum temperature of XynHBN188A and XynHBN188A217 was 50 °C and 55 °C, respectively. The optimum pH of XynHBN188A and XynHBN188A217 was pH 8.0 and pH 7.5, respectively. The half-life at 60 °C of XynHBN188A217 was 20 min. Moreover, the pH stability of XynHBN188A217 was significantly better than that of XynHBN188A. Finally, homology models and molecular docking were used to identify the location of mutation sites and to explore the mechanism of the improved properties.ConclusionThe xylanase XynHBN188A has been improved in the specific activity and pH stability by directed evolution. Also, the enlarged catalytic channel of mutant is beneficial for the substrates access and products release. It may contribute to the improved activity. The mutant XynHBN188A217 will be a potential candidate to be used for industrial application.
Highlights
Xylanases have been successfully used in food, paper, and pulp industries and are considered to be a key player in the biodegradation of xylan to valuable end products
Screening of xylanase mutant libraries The transformants in the library were spread on the remazol brilliant blue (RBB)-xylan substrate plates with IPTG induction for preliminary functional screening (Fig. 1), by 96-well cell culture plate
The 46 mutants were rescreened through shaking flask to expand culture
Summary
Directed evolution was used to improve the specific activity and pH stability of the xylanase (XynHBN188A) that originated from Bacillus pumilus HBP8. The xylanase XynHBN188A was mutated by error-prone PCR. The mutant, XynHBN188A217, was screened from the mutant library by functional screening. The specific activity of XynHBN188A217 was 3986.7 U/mg, which was 2.8-fold higher than that of wild type. The optimum temperature of XynHBN188A and XynHBN188A217 was 50 °C and 55 °C, respectively. The optimum pH of XynHBN188A and XynHBN188A217 was pH 8.0 and pH 7.5, respectively. The half-life at 60 °C of XynHBN188A217 was 20 min. The pH stability of XynHBN188A217 was significantly better than that of XynHBN188A. Homology models and molecular docking were used to identify the location of mutation sites and to explore the mechanism of the improved properties
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