Mutation Breeding Of Xylanase High Production Strain

Xylanase is an important enzyme family that catalyzes the hydrolysis of xylan into xylo-oligosaccharides or xylose. Xylan is the major composition in hemicellulose. However,xylan is hardly hydrolyzed into xylo-oligosaccharides or xylose, which are widely used in industry, so xylanase has broad application prospects.In this study, Paenibacillus kribbensis 6hRe76, a xylanase-producing bacterium, was improved xylanase production by UV and ARTP mutagenesis. The mutant strains were screened using the method of kongo red dye and detected the activity of xylanase by DNS method. The media components and fermentation conditions of the highest xylanase production strain were optimized by single factor method and Response Surface Methodology(RSM).After three rounds UV mutagenesis, the xylanase-producing mutant strain90-120-150s106 was obtained. The yield of xylanase had increased to 103.08 ±0.15U/mL,which was 60.51% higher than the original strain. By two rounds ARTP mutagenesis on the mutant strain 90-120-150s106, three highly productive xylanase mutant strains 2A78s-86,2A82s-13, and 2A84s-27 were obtained, and the activity of xylanase was 135.32 ±0.48U/mL, 135.25±0.39U/mL and 133.46±0.06U/mL, respectively. Compared to the strain90-120-150s106, the output of xylanase increased by 31.27%?31.20%?29.47%, respectively.The analysis of genetic stability indicated that the mutant strain 2As82s-13 got a higher genetic stability.The media components and fermentation conditions of mutant strain 2As82s-13 were optimized by Response Surface Methodology(RSM). In the first, one factor experiment was used to screen the best fermentation time, carbon source and nitrogen source. The results showed that the best fermentation time was 72 h and the 3% wheat bran with 1% corn cob xylan and 6% yeast extract was the optimum carbon source and nitrogen source, respectively.Secondly, wheat bran with corn cob xylan, K2HPO4 and liquid volume were certified as the major factors which influenced the production of xylanase by Plackett-Burman experiment.The optimal conditions were around the 4.35% wheat bran with 1.45% corn cob xylan,0.685% K2HPO4 and liquid volume 10.5/50 mL proved by the steepest ascent experiment.According to the response surface mode constructed, the results indicated that the optimal conditions for xylanase production by mutant strain 2As82s-13 were as following: 4.428%wheat bran+1.476% corn cob xylan, 0.656% K2HPO4, 0.75% yeast extract, 0.05% MgSO4,0.075% CaCl2, 0.10% Tween 80, liquid volume 10.8 /50 mL, inoculum15%, initial pH 8.0,cultured for 72 h. The mutant strain 2AS82s-13's maximum activity of xylanase was 145.35±0.64U/mL, which has increased by 126.93% compared to the original strain 6hRe76.