Construction Of Engineering Strains With High Yield Of Thermostable And Acidic Xylanase

As a kind of important industrial enzyme, xylanase (1,4-β-D-xylanase; EC3.2.1.8) shows a broad application prospects in paper industry, food, energy, feed and the environment fields, particularly, in production of xylooligosaccharides.A new thermostable xylanase-producing strain was isolated from cow dung compost and was identified as Paenibacillus sp. NF1 based on the evidence from its morphological characteristics and 16S rDNA sequences. Then it’s fermentation condition of xylanase-production was optimized with the response surface method, and the optimal finally determined as the following:corncob 20 g/L, beef extract 28.2 g/L, K2HPO4 5 g/L、 MgSO4 0.5 g/L, NaCl 7.1 g/L, initial pH 6.9, liquid loading 40%, rotate speed 160 r/min, inoculum size 2%, temperature 28℃. After 72 h shaking-flask fermentation, the xylanase-activity of the culture supernatant was 160.6 IU/mL, which was increased by 51.4 times compared to the old condition. The gene of xylanase(XynGH10,984 bp) was cloned from genomic DNA of Paenibacillus sp. NF1 and expressed in E.coli BL21.E.coli BL21(pET-XynGH10) secreted 10.58 IU/mg of extracellular xylanase and the recombinant xylanase (XynGH10) showed the optimum temperature and pH of 60℃ and 6.0.Thus the engineering strains P. pastoris GS115(pPIC9K-XynGH10) was successfully construct to expression the xylanase(XynGH10). The xylanase yield was 500 IU/mL from P. pastoris GS115 (pPIC9K-XynGH10) after 96 h of induction in shake flask cultures, which was 40 times higher than the E. coli BL21 (pET-XynGH10). High density culture in 10 L fermenter was performed using P. pastoris GS115 (pPIC9K-XynGH10) resulting in the highest activity of 2500IU/mL, cell wet weight of 360 g/L, crude protein of 7.6 g/L after 72 h-induction.XynGH10 from high density culture of P. pastoris GS115 (pPIC9K-XynGH10) showed strong specificity toward birchwood xylan and oat spelt xylan. The main products were xylobiose and xylotetraose when its action on birchwood xylan for 18 h. The acidic electrolyed water pretreated-corncob (AEWP-corncob) was hydrolyzed by XynGH10 for 18 h, the xylooligosaccharides (DP 2-4) products increased to 80% and the xylose was just increased 3%. These results indicated that XynGH10 is a promising candidate for biomass conversion and xylooligosaccharide production.