Improved production of two expansin-like proteins in Pichia pastoris and investigation of their functional properties

Abstract

The genes encoding expanin-like proteins from Trichoderma reesei (TrSwo1) and Bacillus subtilis (BsEXLX1) were successfully expressed in Pichia pastoris. The yields of two recombinant proteins were significantly improved by the use of PMSF (phenylmethylsulfonyl fluoride) and a commercial protease inhibitor cocktail. Under the optimum culture conditions, the highest TrSwo1/BsEXLX1 expression level reached was approximately 120/860mgl−1, which was almost 2.4/86-fold as much as the highest expression level in other host cells. Purified BsEXLX1/TrSwo1 displayed synergism in cellulose hydrolysis with endoglucanase, and the maximum amount of reducing sugars released was almost 2.0/2.5-fold as high as those in reaction mixtures without expansin-like proteins. The synergistic effect reached the maximum level when 1mg of target protein per g of filter paper was loaded. Both proteins exhibited relatively high thermal stability at temperatures of 50, 70 and 90°C, and retained more than 45% residual activities after 1h of pre-incubation at 100°C, suggesting remarkable heat tolerance. They also showed resistance to denaturation by urea and SDS. Under several enzymatic hydrolysis conditions, the synergistic activity of TrSwo1 was higher than that of BsEXLX1, indicating stronger disrupting activity of TrSwo1 on cellulose than BsEXLX1. This is the first study to report high-efficient expression and unreported properties of BsEXLX1/TrSwo1.