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

Protein Expr Purif. 2019 Oct;162:67-71. doi: 10.1016/j.pep.2019.06.002. Epub 2019 Jun 8.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspergillus niger / chemistry
  • Aspergillus niger / enzymology*
  • Aspergillus niger / genetics
  • Calcium / metabolism
  • Cloning, Molecular
  • Enzyme Stability
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics*
  • Fungal Proteins / metabolism
  • Hydrogen-Ion Concentration
  • Peptide Hydrolases / chemistry*
  • Peptide Hydrolases / genetics*
  • Peptide Hydrolases / metabolism
  • Pichia / genetics
  • Pichia / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Temperature


  • Fungal Proteins
  • Recombinant Proteins
  • Peptide Hydrolases
  • Calcium