Expression of the Acid-resistant Xylanase Gene from Bacillus velezensis P7 in Bacillus subtilis and Optimization of Its Production
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Abstract:
To obtain a xylanase protein that can adapt to acidic environments (e.g., in distiller’s grains), the endogenous xylanase gene from acid-resistant Bacillus velezensis P7 was successfully cloned using the polymerase chain reaction. The xylanase gene fragment and the pHT43-HIS shuttle plasmid were first digested with the restriction enzymes Bam HI and Xba I. After purification of the xylanase gene fragments with a gel recovery kit, they were then ligated with the vector fragments using T4 DNA ligase, and the recombinant plasmid was subsequently transfected into Bacillus subtilis WB800 cells. SDS-PAGE analysis of the purified xylanase revealed that it was a 40 ku polypeptide. To increase the extracellular production of xylanase by Bacillus subtilis WB800-P7 in high-density fermentation, the effects of temperature, pH, inoculum size, rotation speed, and other individual factors on the production process were studied, and orthogonal experiments were then used to determine the interactions among the factors and to predict the optimal fermentation conditions for promoting the secretion of the recombinant protein. The results suggested the optimal fermentation conditions of strain WB800-P7 to be as follows: inoculum OD600 of 0.8, IPTG concentration of 1.2 mmol/L, fermentation pH of 6.0, culture temperature of 35 ℃, inoculum size of 2%, and rotation speed of 160 r/min. Among these factors, the culture temperature had the most significant effect on enzyme production. Under these optimal conditions, the enzyme activity reached 4.21 IU after 16 h of fermentation, which was 64.45% higher than that before optimization. The acid resistance of the recombinant xylanase was analyzed, whereupon its residual enzymatic activity was found to be more than 80% of that of the original enzyme under strongly acidic and neutral conditions (pH 5.0~7.0). In conclusion, a modified B. subtilis strain with better protein expression capability and which produces a highly acid-resistant xylanase was successfully engineered in this study.
