Expression Of Bacillus Pumilus Alkali Resistance Xylanase Gene In Bacillus Subtilis And Optimization Of Fermentation Conditions

Xylan accounts for the second concentration of Renewable resources as a major hemicellulose in nature. The xylan existing in grain feed is a major kind of anti-nutritional factors, it can reduce feed digestibility significantly and hinder the growth of livestock and poultry. Xylanase degradation of xylan is the most safe and effective method at present. Selecting high producing strain of xylan from the extreme environment is a common and convenient method, but such method is still limited to local local environmental and economic conditions. So transforming the xylanase at the molecular level will become the most practical technique in the future.The choice of expression vector and host strain plays an important role in expression of xylanase. As another widely used expression system following E.coli, Bacillus subtilis is not pathogenic with single-cell membrane and secreted proteins to medium directly. Therefore, Bacillus subtilis was selected as xylanase gene expression host in this study.In this study, xylanase expression vector pXN112 was constructed, then transformed into Bacillus subtilis 1A747 to obtain a new recombinant stain BXN112. The expression of xynA was induced and fermentation conditions were optimized. The experiment was divided into two parts.(1) Alkali resistance xylanase gene xynA with original signal peptide was separated from Bacillus pumilus BYG5-20, then cloned into the secreted expression vector pGJ148 to construct a intermediate plasmid pGJ148-xynA. Then the spectinomycin resistance gene specr was cloned into pGJ148-xynA to construct xylanase expression Vector pXN112 finally. The correctness of pXN112 was verify through Sequencing and restriction enzyme digestion. (2)pXN112 was transformed into Bacillus subtilis 1A747 to obtain a new recombinant stain BXN112.The expression of xynA was induced and fermentation conditions were optimized.Extracellular supernatant of BXN112 was sampled after 24h inducement at 37℃,then enzyme activity assay and SDS-PAGE analysis were carried out. The result showed that the homologous signal peptide of xynA gene acted well, the expression product was secreted to ?extracellular. extracellular supernatant xylanase enzyme activity was up to 54.13 IU / mL, and the molecular weight of expression product was 25 kDa.Fermentation temperature, pH value, culture time and conditions of carbon and nitrogen source were selected as optimized factors in the further fermentation condition optimization experiment. The optimum fermentation parameters were determined: temperature 37℃, pH value of 8.0 and incubation time 24h. Optimum fermentation medium was determined: peptone (Tryptno), 1%; yeast extract (Yeast extract), 0.5%; NaCl, 1%; sorbitol, 1%; 4% maltose as inducer. The xylanase expression level reached 93.32IU/ml after optimization.