Enzymatic characteristics of a recombinant protease (rPepD) from Aspergillus niger expressed in Pichia pastoris

Abstract The Aspergillus niger AS3.350 protease gene ( pepD ) was successfully cloned and expressed in Pichia pastoris KM71. The rPepD activity was 331.5 U/ml, and the optimum temperature and pH were 45 °C and 8–9 respectively. In addition, enzyme activity was significantly inhibited by PMSF, EDTA, Mg 2+ , Fe 2+ and Zn 2+ ions, and stimulated by Ca 2+ which selectively bound to the T 302 and D 323 residues. Mutation in either or both of the residues inhibited rPepD expression, indicating that binding to Ca 2+ is necessary for PepD expression and activity. The rPepD showed a wide substrate range, and was particularly selective to those with hydrophobic amino acids. The degree of rPepD-mediated hydrolysis of soy protein isolate, corn flour and gluten meal were 8.7%, 38.1% and 33.6% respectively, which was higher than that by Alcalase, indicating that rPepD has potential applications in the food processing industry.