High-Efficient Expression and Pilot Scale Fermentation of Streptomyces Xylanase from a Constitutive Pichia pastoris Vector

Abstract

To develop an efficient Streptomyces xylanase-expressing system necessary for large-scale industrial production, a DNA fragment encoding the endo-β-l,4-xylanase (XYL1) from Streptomyces sp. S38 was cloned into an expression vector pGAPZαA to generate a recombinant plasmid pGAPZαA-XYL1. The plasmid pGAPZαA-XYL1 was transformed into Pichia pastoris X-33. The secreted XYL1 protein was confirmed to be completely consistent to sequence as reported previously (GenBank accession number X98518.1). The secreted XYL1 protein was purified using the Q-Sepharose FF chromatography and Sephadex G-25 resin, with an estimated purity of greater than 90%. We also found that in a 50 L fermentor, the average level and enzymatic activity of secreted xylanases were up to 1.15 ± 0.09 mg/mL and 1528 ± 59 IU/mL, respectively. The pH and temperature for optimal XYL1 expression were 6.0 and 55°C, respectively. The Km and Vmax values of XYL1 were 2.0 mg/mL and 5000 μmol min−1 mg−1, respectively. In addition, the XYL1 was found to have good thermal stability and wide pH adaptability, suggesting that it may be a useful candidate for various commercial applications. The high-efficient expression and pilot scale fermentation of XYL1 in this study, together with enzymatic studies, provide a strong basis for future large-scale industrial production.