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, Mg²⁺, Fe²⁺ and Zn²⁺ ions, and stimulated by Ca²⁺ which selectively bound to the T³⁰² and D³²³ residues. Mutation in either or both of the residues inhibited rPepD expression, indicating that binding to Ca²⁺ 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.

Keywords: Aspergillus niger; Cloning and expression; Enzymatic characteristics; Hydrolysis characteristics; Protease.