A gene encoding xylanase 2 mutant from Trichoderma reesei (T2C/T28C, named mxyn2) was cloned into the Pichia pastoris X33 strain using the vector pPICZ alpha A. Recombinant Mxyn2p was functionally expressed in P. pastoris X33 and secreted into the supernatant. Real time qPCR demonstrated that an increase in gene copy number correlated with higher levels of expression. Supernatant from methanol induced cells was concentrated by ultrafiltration with a 10 kDa cut off membrane, and purified with ion exchange chromatography using SP Sepharose Fast Flow chromatography. Recombinant Mxyn2p protein had the highest activity at 75 degrees C, while recombinant protein encoded by the "wild type" xylanase gene xyn2, also expressed in Pichia, was 20 degrees C lower. The Mxyn2p enzyme retained more than 70% of its activity after incubation at 80 degrees C for 10 min. The effects of the optimal pH and temperature for higher expression levels in P. pastoris were also determined, 6.0 and 22 degrees C, respectively. The maximum xylanase activity of Mxyn2p was 13,000 nkat/mg (9.88 g/l) in fed-batch cultivation after 168 h induction with methanol in a 501 bioreactor. (C) 2014 Elsevier Inc. All rights reserved.