Characterization And Application Of Protease From Aspergillus Oryzae

Microbial protease have been widely applied in the traditional processing. There has been litter research that microbial protease were used in functional food based on its cleavage sites. In this study, we obtained two kinds of proteases from Aspergillus oryzae, and made them apply to the preparation of casein phosphopeptides based on their cleavage specificities.An Aspergillus oryzae was choosed as the experimental strains. Its enzyme yield reached 1846 U·g-1 by single factor experiment. Ammonium sulfate precipitation, anion exchange chromatography, hydrophobic chromatography and gel filtration chromatography were used to purify the protease from Aspergillus oryzae. Two proteases named PA and PB were obtained. The molecular weights of protease PA and PB are 27 k D and 58 k D, respectively.In order to use the proteases conveniently, we analysised the basic enzymatic properties of two proteases. The optimum conditions of PA and PB are 50 °C, p H 9.0 and 55 °C, p H 8.0, respectively. Both PA and PB have a good stability at p H 5-10 and 40 °C. Both Protease PA and PB are activitied by Mn2+, while protease PB is inhibited by Fe3+ and Ni2+. The inhibitor test showed that, protease PA was serine protease, and protease PB was metal protease with Mn2+ as ligand. With casein as the substrate, Km of protease PA and PB were 1.23 g·L-1 and 0.36 g·L-1, respectively.In order to use the proteases more rationally, we studied the cleavage ability of the proteases by MALDI-TOF-MS. Protease PA has strongly cleavage ability between-Cys-Gly-,-Glu-Ala- and-Arg-Gly- residues. Protease PB has cleavage ability between-Leu-Cys-,-Val-Glu-,-Tyr-Leu- and-Arg-Gly- residues, and shows a little selectivity in peptide bonds formed between-Cys-Gly-、-Leu-Val-、-Leu-Tyr- and-Gly-Glu-. We found it was suitable for the proteases using in preparation of CPPs by analyzed the cleavage specificity of two proteases.Then, we used the proteases to prepare CPPs, analysised the physicochemical properties of the CPPs. Yield, r(N/P) and serine content of the CPPs prepared by protease PA were 10.67%, 8.76 and 8.9%, respectively, and delayed calcium deposit for 15 min. Yield, r(N/P) and serine content of the CPPs prepared by protease PB were 15.87%, 6.17 and 11.33%, respectively, and delayed calcium deposit for 35 min. Moreover, yield, r(N/P) and serine content of the CPPs prepared by trypsin were 18.46%, 7.35, and 9.91%, respectively, and delayed calcium deposit for 25 min. In conclusion, protease PA and PB applied in the preparation of CPPs have many advantages. At the same time, antioxidant activity were detected in two kinds of protease hydrolysates, provided a reference for by-products utilization in CPPs preparation.