Study On The Enzymatic Properties Of Proteases From Aspergillus Oryzae And The Application In Flavor Seasoning

Proteases which catalyze the cleavage of peptide bonds in proteins to producepeptides and amino acids can improve the quality and nutrition of protein used in foodand flavoring. Microbial protease, a major source of protease, has been the focus ofresearch at home and abroad. As food safety has attracted increasing attention due toits importance, it is particularly important to investigate on the protease from safemicroorganism. The research on the enzymatic properties of different safe proteasehas a great significance for the deep application of proteases in food industry.To obtain safe strains which produce protease for the food and seasoning,3Aspergillus oryzae,3Aspergillus niger and3Bacillus subtilis had been comparedrespectively. When used chicken protein as a substrate, effects of hydrolyzation bycrude enzyme from different strains were tested. Under the same hydrolysis degree,the crude enzyme from Aspergillus oryzae was the best. The content of peptides andamino acids after hydrolyzation of crude enzyme from Aspergillus oryzae were31.74mg/mL and2.37mg/mL, respectively.For further study of the proteases of Aspergillus oryzae, ammonium sulfateprecipitation, DEAE-Sepharose FF anion exchange chromatography,Phenyl-Sepharose HP hydrophobic chromatography and Superdex-G75/200gelfiltration chromatography were used to purify the proteases from Aspergillus oryzae.Two proteases were obtained from Aspergillus oryzae and their molecular weightswere approximately37kDa (P1) and45kDa (P2), respectively. The specific activity,purification fold and recovery of P1were2469.03U/mg,6.33and10.61%,respectively, and P2was up to1427.96U/mg,3.67and1.06%, respectively.The purified P1and P2have some differences in enzymatic properties.Usingcasein as a substrate, the optimum conditions of P1and P2were pH8.0,45℃and pH7.0,45℃, respectively. The Km of P1and P2were8.36g/L and4.11g/L,respectively. Moreover, the Vm of P1and P2were12.95μg/(mL·min) and4.86μg/(mL·min), respectively. P1in pH5-10and P1in pH5-8both have good stability.They both are stable in lower than40℃, but are extremely unstable over60℃.The peptide selectivity of P1and P2were tested with insulin chinB oxidizedfrom bovine pancreas as model substrate. The results showed that P1had strongcutting ability on-Cys-Gly-,-Phe-Tyr-, while P2had very strong cutting ability on-Gly-Ser-,-Gly-Phe-.Effects of hydrolyzation by P1and P2were tested with SPI and CP as substrates, respectively. The peptide proportion of SPI hydrolyzation which molecular weight aregreater than10kDa were to9.51%(P1) and7.90%(P2), while CP hydrolyzate were58.66%to0.029%(P1) and0.48%(P2). After hydrolyzation, the total contents of freeamino acid were increased in different degrees. The proportion of delicious aminoacids (Asp, Glu, Gly, Ala) of SPI and CP hydrolyzates from P1were40.9%and63.09%, respectively, while they were35.26%and25.1%after P2hydrolyzation,respectively. The results show that P1and P2can reduce the bitterness in proteinhydrolysates in some extent.