Characterization of a protease from Aspergillus oryzae and application in casein phosphopeptides preparation

Abstract:

[Objective] This study aimed to purify and characterize protease from Aspergillus oryzae, and to apply in casein phosphopeptides (CPPs) preparation. [Methods] Ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography and Butyl-Sepharose HP hydrophobic chromatography were performed to purify the enzyme. The molecular weight and purity were determined by SDS-PAGE, and cleavage sites were detected by MALDI-TOF-MS. [Results] A protease named PE with the weight about 58 kD was obtained from Aspergillus oryzae. Protease PE shows maximal activity at pH 8.0 and 55 °C. It was inhibited by Fe3+, and activitied by Mn2+. With casein as the substrate, Km and Vm of the protease were 0.36 g/L and 18.18 mg/(L?min), respectively. Protease PE has cleavage ability in -Leu-Cys-, -Val-Glu-, -Tyr-Leu- and -Arg-Gly- residues of bovine insulin chain B, showing a wide range of residue specificity. CPPs were obtained after proteolysis of casein using PE by barium-ethanol precipitation. Yield and r (N/P) of the CPPs were 15.87% and 6.17, respectively, and delayed calcium deposit for 35 min. [Conclusion] The research provided a reference for Aspergillus oryzae protease using in the functional food industry.

Key words:Aspergillus oryzae, Protease, Purification, Enzymatic properties, Cleavage site, Casein phosphopeptides