Cloning and expression of Thermobifida xylanase gene in the methylotrophic yeast Pichia pastoris

A gene (xyl11) encoding xylanase from Thermobifida fusca NTU22 was cloned, sequenced and expressed in Escherichia coli. The gene consists of 1014 base pairs and encodes a protein of 338 amino acids. The gene was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The xylanase productivity of the P. pastoris transformant (pPICαXYL) was about 67-fold higher than that of T. fusca NTU22 cultured in a 5-liter fermentor. The purified xylanase showed a single band at about 36 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H. The optimal pH and temperature of the purified xylanase were 7.0 and 70 °C, respectively. About 70% of original activity remained after heat treatment at 70 °C for 3 h.