Expression Of Xylanase Gene XynB From Streptomyces Olivaceoviridis A1 In Pichia Pastoris

Xylanase refers to a type of enzyme which can hydrolyze xylans into xylooligosaccharides and D-xylose.It broadly exists in microorganism and has wide commerical application in industrial processes,such as feed, paper, foodstuff, medicine and energy industries.The xylanase gene xynB encoding the native protein of xylanase from Streptomyces olivaceoviridis A1 was cloned into Pichia pastoris expression vector pPIC9 a. The recombinants were constructed by transforming pPIC9 a -xynB into P. pastoris GS115.The assay results revealed that the xylanase gene xynB was overexpressed and secreted effectually in P. pastoris.In 3L fermentor the expression level of xylanase XYNBa exceeded 1200IU/ml and the expressed xylanase had normal bioactivity.The molecule weight of XYNBa was determined as about 31kD which is higher than 23kD of original enzyme XYNB from Streptomyces olivaceoviridis A1.XYNBb was gotten by deglycasylation of XYNBa,whose molecule weight returned to 23kD.We comparised the enzymatic properties of XYNBa expressed in P. pastoris, XYNBb deglycasylated from XYNBa and XYNB produced from Streptomyces olivaceoviridis Al :There was little difference among the three enzymes on optimal pH,the optimal pH of XYNB and XYNBa were both 5.2,the optimal pH of XYNBb was 5.0;The optimal temperature of XYNB and XYNBa were both 60 C,while the optimal temperature of XYNBb was 50癈;Because of glycosylation the thermal stability of XYNBa was better than XYNB and XYNBb;The specific activity of XYNBa and XYNBb were 883.88IU/mg and 832.5HU/mg respectively,which were both lower than 2814.45IU/mg of XYNB;The Km values of XYNB and XYNBa were similar to each other which were 21.56(g/kg) and 20.87(g/kg),while the Km value of XYNBb was 27.10(g/kg);The Fmax of XYNBa and XYNBb were 4568umol/mg.min and 5329umol/mg.min respectively which were lower than 27623 umol/mg.min of XYNB; Additionally all of the three enzymes did not display cellulase activity.They all had well resistance to pepsion and trypsin,and were not sensitive to metal iron, surface active agent and chelating agent.The analysis of different xylans enzymatic hydrolysate revealed:By XYNBa,that the main constitutions of enzymatic hydrolysate of birch wood xylans were xylotriose and xyloquaiose,which account for 68.43% and 16.50% respectively,additionally there was 11.79% of xylobiose; The main constitutionsof enzymatic hydrolysate of corncobs xylans were xylobiose and xylotriose, which account for 81.78% and 11.55%.The result indicated that this xylanase was a kind of 1,4- b -D-xylanohydrolase and was fit to used in industrial procession of xylooligosacc harides.