Purification and characterization of proteases from Aspergillus oryzae

Abstract:

[Objective] This study aimed to purify and characterize proteases from Aspergillus oryzae. [Methods] Ammonium sulfate precipitation, DEAE-Sepharose FF anion exchange chromatography, Phenyl-Sepharose HP hydrophobic chromatography and Superdex-G75/200 gel filtration chromatography were used to purify the proteases from A. oryzae. The molecular weight and purity were determined by SDS-PAGE, and the enzymatic hydrolyzates of soy protein isolated were assayed by HPLC. [Results] Two proteases (P1 and P2) were obtained from Aspergillus oryzae and their molecular weights were approximately 37 kD and 45 kD, respectively. Using casein as a substrate, the optimum conditions of P1 and P2 were pH 8.0, 45 °C and pH 7.0, 45 °C, respectively. The Km of P1 and P2 were 8.36 g/L and 4.11 g/L, respectively. Moreover, the Vm of P1 and P2 were 12.95 μg/(mL·min) and 4.86 μg/(mL·min), respectively. Both P1 and P2 had the highest hydrolytic activity to casein while lowest activity to BSA (Bull Serum Albumin). Meanwhile, the molecular weight of peptides in soy protein isolated hydrolyzates had different distributions. [Conclusion] Purified P1 and P2 have some differences in enzymatic properties. And they both have strong cutting capacity towards the hydrophobic peptide bond, but there are also differences in the specificity of bond groups. These results have a great significance for the application of proteases from Aspergillus oryzae in the food industry.