Overexpression of Streptomyces olivaceoviridis A1 Xylanase with High Specific Activity and Analysis of Enzymic Properties
来源于橄榄绿链霉菌A1的高比活木聚糖酶XYNB的高效表达及性质的比较分析

High-level expression of xylanase with high specific activity is an effective way to improve xylanase fermentation potency in recombinant host and decrease its production costs. The gene xynB encoding the mature protein of xylanase with high specific activity from Streptomyces olivaceoviridis A1 was cloned into Pichia pastoris expression vector pPIC9 and introduced into the host Pichia pastoris by electroporation. The results of SDS-PAGE and activity assay of the xylanase expressed by recombinant P. pastoris showed that the xylanase gene xynB was overexpressed and secreted, and the expressed xylanase had normal bioactivity. In 3L fermentor, the expression level of xylanase protein in recombinant P. pastoris was 1 4mg/mL, and the xylanase activity exceeded 1200IU/mL. The results of SDS-PAGE showed that the expressed xylanase(XYNBa) could be modified by glycosylation, had a single apparent molecular weight of about 31kD, while the deglycosylated xylanase(XYNBb) treated with Endo H had an apparent molecular weight of about 21kD, which was as same as that of original xylanase(XYNB) from Streptomyces olivaceoviridis A1 The results of the researches on enzymatic properties revealed that there were remarkable differences on specific activity,V max and thermal stability among XYNB,XYNBa,XYNBb. The analysis of enzymatic hydrolysate for different xylans revealed that the main constitutions of enzymatic hydrolysate were xylobiose,xylotriose and xyloquaiose, which account for more than 95% of all hydrolysate.