High level expression of a recombinant xylanase by Pichia pastoris cultured in a bioreactor with methanol as the sole carbon source: Purification and biochemical characterization of the enzyme

The xylanase gene xyn11A from Cellulomonas uda was expressed in Pichia pastoris under the control of an inducible promoter AOX1. The recombinant xylanase was named Xyn11A_AOX1. The P. pastoris clone (C9) showing the highest xylanase activity was selected to evaluate the production of Xyn11A_AOX1 in liquid cultures in a bioreactor. The culture was carried out by fed-batch fermentation using two strategies, one-stage method using methanol, and two-stage method using glucose and methanol as carbon sources. Interestingly, after 48 h of fermentation using one-stage method, a dry cell weight of 34 g/L and total protein concentration of 1.16 g/L were obtained, where Xyn11A_AOX1 was the major enzyme secreted into the culture medium. Xyn11A_AOX1 was purified from the culture supernatant of P. pastoris/pPICZαB - xyn11A and showed an estimated molecular mass of 45 kDa. The optimal temperature and pH were 50 °C and 6.5, respectively. The K_M and V_max values were 4.5 mg/mL and 5000 U/mg protein, respectively. This is the first report on cultivating P. pastoris with methanol as the sole carbon source in a minimal salt medium in which the recombinant enzyme was obtained as the major enzyme secreted into the culture supernatant within a short fermentation time.