14-20: Cloning and expression of Thermoascus aurantiacus xylanase gene in Pichia pastoris

Expression of thermophilic Thermoascus aurantiacus xylanase gene (xynA) in yeast is a desirable process to allow other studies on protein structure and large-scale application. In this study, we carried out the cloning and expression of this gene. The gene of xylanase (xynA) from Thermoascus aurantiacus was amplified by RT-PCR from the total RNA. A recombinant plasmid pPIC9-xyn  was constructed by inserting gene xynA into Pichia pastoris secretory vector pPIC9K. Enzyme was induced by methanol to express the recombinant extracellular xylanase at 28°C. The recombinant strain was identified by Congo red selection and confirmed by PCR analysis. The best xylanase producing clone was selected to produce the enzyme for characterization. The higher enzyme production was 641,16U/mL at 96 hours and pH 5.0. The enzyme was stable under a large range of pH (3,5-10,5) for 24h. Differences were observed in the optimum temperature and thermostability. Native xylanase optimum temperature was 75°C and it was stable in range 35°C to 75°C after 1 hour of incubation. However, the recombinant enzyme shown optimum activity at 60°C. The crude recombinant enzyme was stable from 35°C to 80°C, but after purification the stability was from 35°C to 65°C. This was the first study on cloning and expression of xynA gene from T. aurantiacus in P. pastoris.