Abstract: To obtain a new extracellular xylanase in high yield, the xynA (GenBank number:U57819) can be expressed in different ways. A xynA was early expressed in prokaryotic microbiology. Subsequently, we cloned the partial xynA encoding mature xylanase into expression vector pPICZαA, and then transformed into Pichia pastoris X33, so eukaryotic expression system was successfully constructed to produce heat-resistant xylanase without signal peptide. 0.5% (V/V)methanol was added in batch to induce the engineering Pichia pastoris which produced extracellular xylanase. The extracellular xylanase was analyzed by the xylan-degrading activity, SDS-PAGE and Western Blot. The results showed that the XynA contained typical catalytic structure domain of glycosidic hydrolyzyme 11 (GH11), and the predicted molecular weight of mature protein was 38.6 ku, and the isoelectric point was 6.86. Xylanase activities secreted by the seven genetic-engineering Pichia pastoris X33/pPICZαA-xynA were more than 300 U/mL, and the highest xylanase activity was up to 487.2 U/mL;the molecular weights of extracellular xylanases on SDS-PAGE and Western Blot were about 45 ku by the characteristic strips, bigger than the predicted molecular weight (38.6 ku). Therefore, a novel extracellular xylanase has been effectively expressed and may lay a foundation for its separation and purification to study the property, structure and function.

Key words: Xylanase gene, Pichia pastoris, Eukaryotic expression, Western Blot

CLC Number: 

• S330

• Q78