Separation And Purification Of Aspergillus Oryzae Protease From Traditional Soybean Paste And Analysis Of Its Enzymatic Properties

Soybean paste was produced mainly relying on Aspergillus oryzae protease and amylase.It has been found that the secretion of Aspergillus oryzae protease played a key role in hydrolysis of raw materials to produce the flavor of miso.In this paper,Aspergillus oryzae protease was separated and purified,and the enzymatic properties was analized.Then the protease was used in hydrolysis of soybean protein and fermentation of soybean paste.By studying the enzymatic properties of the protease it will lay a good foundation for the application of proteases,and provide a theoretical basis for further research for the sauce industrial manufacturing conditions.It will be very meaningful for the enzyme market development.The protease produced by Aspergillus oryzae HDF-7 which was isolated from traditional soybean paste purified by using ammonium sulfate precipitations and Sephadex G-50 gel filtration chromatography.The purified protease showed on SDS-PAGE as a single band with an apparent molecular weight of 30 KDa.The N-terminal sequence of the first 12 amino acid residues showed high similarity to the neutral protease(ACCESSION:AAT68480)from Aspergillus oryzae RIB40.The optimum temperature was 50? and the optimum pH was 7.0;It was found that the protease was stable at 20? and at pH 6.0-8.0;Protease activity was activated by Mn2+and inhibited by Cu2+ strongly.The Km values of the protease for casein at 40? was 78?g/mL with Vmax 6.29?g/min.After the hydrolysis of soybean protein by the protease the total amino acid content had an increase of over 18.2%.and in the process cysteine,alanine,valine,arginine had been significantly improved;Instead of koji,with the method of using protease into raw materials directly,the flavor and shape of soybean paste obtained had a little less difference in comparison with the traditional soybean paste.According to the results,it was found that the protease stability was a bit weak,so it should be kept at low temperature and neutral environment.It had a good effect on the hydrolysis of soybean protein and played an improtant role in the process of making soybean paste.Thus,in this paper the protease was successfully puried.It wll lay the foundation for future production and application.