Preliminary Study On Acid Protease From Rhizopus

Acid protease is an enzyme which decomposes protein in the acid conditions.Acid protease has good pH tolerance. So it has been widely applied in food, medicalindustry and animal husbandry etc. Now, Acid protease is yielded by the microbialfermentation. Marketable strains which generate acid protease has only a few ofAspergillus niger, Aspergillus usamii and Aspergillus oryzae. If a new strain isscreened out, it will efficiently work out these problem that enzyme activity is lowand strains which generate acid protease is ageing in the industrial production.A strain which yields acid protease was screened from a few strains stored in thelaboratory by decomposing caseins, named RN-11. Using ITS sequence analysis ofthe RN-11strain for molecular identification,with its morphological characters, theRN-11strain was identified for Rhizopus nigricans （Rhizopus stolonifer）.By the way of solid-state culture, single factor experiment and Response surfaceanalysis were applied to optimize solid medium of the RN-11strain. The optimumconstitute of culture medium was obtained: the weight of bran15g, H2O10.6ml, theweight of sucrose0.69g, the weight of NaNO31.0g, the weight of K2HPO40.042g inthe250ml triangular flask. the optimal enzyme activity of acid protease in theory was159.51U/g, and the experimental value of acid protease activity was160.16U/g, whichwas5.1higher than initial medium. When the initial pH of the optimize solid mediumis2.5, the weight of medium is27g, the inoculum concentration is1ml, the time whenthe strain is cultured is60h and The growth temperature of the strain is37℃, theoutput of acid protease is largest.The study investigated the extraction of acid protease from solid koji withdifferent solutions: distilled water,0.05mol/L lactate buffer （pH3.5）,0.15mol/LNaCL and0.03mol/L phosphate buffer. From this experiment,0.05mol/L lactatebuffer （pH3.5） was chosen to extract RN-11acid protease effectively. Following this,to determine the optimum extracting factors, the proteases were extracted with varioussolution pH, extracting time, the ratio of the extracting solution to the solid koji. Theresult indicated that0.05mol/L lactate buffer of pH3.0gave optimal extraction withthe temperature setted to30℃, the ratio of etracting solution to solid koji being10:1and leaching time lasting for105min.The acid protease was puried from the crude enzyme product of RN-11strainthrough ammonia sulfate fractional precipitating, dialysis and Sephadex G-75gel chromatography. The crude acid protease was purified41.79times and its recovery ofenzyme activity was24.39%.The study of the enzyme properties showed that the molecular weight of thepurified acid protease is approximate70Kda by the SDS-PAGE electrophoresis. It washigh active between pH2.0³.0with optimal activity at pH2.5and between at40⁵0℃with optimal activity at50℃. It had good stability at2.0⁴.0and themostability at30⁵0℃for60min.The acid protease was activated by Na+、K+、Mn²⁺、Cu²⁺、Ca²⁺,but inhibited by Zn²⁺、Li²⁺、Fe²⁺under the5M mol/L concentration, and it was notaffected by Mg²⁺.