Gene cloning and enzymatic characterization of an endoprotease Endo-Pro-Aspergillus niger.

A novel endoprotease Endo-Pro-Aspergillus niger (endoprotease EPR) was first successfully expressed at high level in the methylotrophic yeast Pichia pastoris and the purification procedure was established. The endoprotease EPR is 95 % identity with proline specific endopeptidase from A. niger CBS513.88 (EMBL; AX458699), while sharing low identity with those from other microorganisms. The purified endoprotease EPR was a monomer of 60 kDa. Furthermore, the peptide mass fingerprinting (PMF) analysis confirmed that the purified protein was an endoprotease Endo-Pro-Aspergillus niger. A three-dimensional model revealed that the active site of the enzyme was located in Ser(179)-Asp(458)-His(491), based on template 3n2zB with sequence identity of 17.6 %. The optimum pH and temperature of the endoprotease EPR were pH 4-5 and 35 °C, and the stabilities were pH 3-7 and 15-60 °C, respectively. Furthermore, the endoprotease EPR had the ability to digest peptides with the C-terminal of proline as well as alanine, and was also capable of hydrolyzing larger peptides. The properties of the endoprotease EPR made it a highly promising candidate for future application in the field of brewing and food process.