Functional expression and characterization of a novel aminopeptidase B from Aspergillus niger in Pichia pastoris.

Abstract

A novel aminopeptidase B (APB-AN) was identified from Aspergillus niger CGMCC 3.1454 for the first time and was cloned and expressed in Pichia pastoris. The mature enzyme of approximately 100kDa was purified for characterization. The optimum pH and temperature of the recombinant APB-AN were determined to be 7.0 and 40°C, respectively. The enzyme was stable below 40°C and at pH values from 5.0 to 8.0. The K m and V max values were determined to be 0.61mmol/L and 11.45mmol/L/min, respectively, using Arg-pNA as the substrate. APB-AN was inhibited by Cu2+ and Fe2+ and activated by Co2+ and Na+. Most metal chelators (Ca2+, Mg2+ and Mn2+) and aminopeptidase inhibitors (bestatin and puromycin) suppressed its activity. APB-AN was found to be active towards 13 kinds of amino acid p-nitroanilide (pNA) substrates:Arg-pNA, Lys-pNA, Tyr- pNA, Trp-pNA, Phe-pNA, His-pNA, Ala-pNA, Met-pNA, Leu-pNA, Glu-pNA, Val-pNA, Pro-pNA and Ile-pNA, and the most preferred N-terminal amino acids were arginine and lysine. APB-AN also hydrolyzed 4 natural proteins: casein, bovine serum albumin, soy protein isolate and water-soluble wheat protein. It is expected that APB-AN has potential food processing applications.