Serine Protease Engineered Bacteria To Build And Modified Soy Protein Isolate Application Research

Soy protein isolate has extensive and important application in some food systems because of its highprotein content and good functional properties.In order to deeply get its high value-added and applied morecomprehensive,it is a hot research focus around the world to modify structure of SPI and to improve itsfunctional properties. In this subject, we used genetic engineering techniques to produce high-yield strains ofheat-resistant serine protease. Found incubation time and temperature at optimal enzyme activity and purifiedand modified heat-resistant serine protease,and systematically studied the impact of modified processconditions on dispersion index of soy protein isolate.and have deeply discussed the application in milkpowder.The main contents and conclusions are as follows:(1) The primers were synthesized according to gene sequence of Bacillus subtilis genomic DNA whichcontained in GenBank. Cloned the target fragment of protease gene and successfully constructed recombinantplasmid pET-28a-Sapr that transformed into an serine protease high-yield strain BL21/pET-Sapr.By inducingand expression of fusion protein,we determined the optimal induction time of4h, optimum inductiontemperature of37℃.Qualitative analysis of the crude enzyme solution of skim milk plate proved itbiologically active.(2) The serine protease was purified from the crude enzyme solution by ammonium sulphateprecipitation, ion exchange chromatography.Analyzed by SDS-PAGE the product met the purificationrequirement.Comparing to unpurified crude enzyme,enzyme activity increased by15.1times to322U/mg,butthe recovery is just2.8%, Determined the optimal temperature serine protease of4h,optimum pH of9.0.(3) Determined the optimum reaction conditions of single factor experiments by SPI dispersionindex:substrate concentration of8%,enzymatic time of1h,enzymatic temperature of70℃，enzymatic pH of9.0，enzyme activity of4000U/g.Determined optimum enzymatic reaction condition on which dispersity is12.78:the amount of enzyme of4070U/g,substrate concentration of6.00%reaction temperature71℃,pH of9.0，analyzing by four quadratic orthogonal rotation combination and response surface with the amount ofenzyme, substrate concentration, temperature, pH value as the independent variable,dispersity as index.(4) Added different proportions of modified SPI in single factor experiments.Determined that adding3%modified SPI instead of partially demineralized whey powder,which close to the body essential amino acidpatterns,using solubility,wettability, dispersibility and the number of white dots of milk formula.Bycontrast,the indicators are in line with the testing requirements.Indicators of milk formula adding modifiedSPI are apparently higher than that adding unmodified SPI.