Optimization Of Xylanase-Producing Condition By Aspergill Us Niger And Cloning,Expression Of XYNB Gene

Xylan, as the main component of semicellulose, is a kind of polysaccharide which is only less than cellulose. Xylanase was widely, used in areas such as feedstuff, food, papermaking, alcohol production, and so on. It consists of a series of enzymes that can produce xylo-oligosaccharide and xylose utilizing xylan as substrate.Aspergillus niger (CICC40616) was selected as experimental material in this study. Xylan and yeast extract servered as the optimized carbon source and nitrogen source respectively, and the optimized proportion of CaCO3was1%, the optimized pH of medium was4.0, revealed by the single factor experiment. Orthogonal experiment with three factors and three levels was designed for the optimization of xylanase-producing condition, medium with1%xylan,1%soybean power and incubation temperature33℃was proved to be the optimized condition, and the xylanase yield increased by71.2%.XynB gene from Aspergillus niger strain CICC40616was cloned and transformed to express in heterogenous host(Saccharomyces cerevisiae INVSC1), and the result showed that xynB gene expressed successfully. Compared with a signal peptide of Saccharomyces cerevisiae, the original signal peptide of xylanase B contributed to the secretion of xylanase B more effectively. The xylanase yield of recombinant with original signal peptide was6.71U/mL after inducation for48h, and the highest yield of up to7.59U/mL appeared after inducation for72h; while the highest xylanse yield of up to3.97U/ml of the recombinant with a signal peptide turned up48h later, the activity level decreased to3.47U/ml72h later. Xylanase B was salted out with80%ammonium sulfate, and then purified by means of ultrafiltration and nickel ion affinity chromatography, a band with the size of about23kDa was detected by SDS-PAGE. The optimum reaction temperature and pH of recombinant xylanase B was measured to be50℃and5.0respectively. After incubation for30min at40℃, the enzyme activtity had hardly lost, at50℃, nearly half of the enzyme activtity got lost, at60℃, no enzyme activity remained. There was almost no enzyme activity loss after it was incubated for3hours at4℃in buffer with pH ranging from3to11. Mn2+, Zn2+, Ca2+showed inhibiting effect on the xylanase B, and Mn2+inhibited the activtity of xylanase B strongly, contributed to about30%loss of activity, Cu2+showed a certain stimulative effect on xylanase B, leading to the improvement on enzyme activity by7.9%, however, Fe, Ni2+, Co2+, Mg2+showed no obvious effect on activity of xylanase B. The Km value, specific activity, Vmax value of recombinant xylanase B was measured to be 11.25mg/mL,9110U/mg and0.218mg/min,respectively.