Optimize The Fermentation Conditions Of Endophytes Producing Saponin Hydrolase From Dioscrea Zingiberensis And Preliminary Isolation And Purification Of Enzyme

Diosgenin is an important precursor for synthesis of more than 200 steroidal hormone medicines,which itself also have antioxidant,fall hematic fat,inducing cancer cell apoptosis various biological activity.The traditional production method producing diosgenin was acidic hydrolysis that hydrolyzing the steroid saponin form dioscorea zingiberensis C.H.Wright,which would generate a lot of waste water and the low utilization rate of yam dioscorea and it caused great environment pollution.Enzymatic hydrolysis mild,environmental protection,is ideal method to address the issue of water pollution and improve the diosgenin yield.This paper used endophytic fungi SYfx213.2 fermentation to produce saponin glycosidase to obtain diosgenin,is a new way of production of diosgenin.This paper from the perspective of molecular biology to identify the endophytic fungi SYfx213.2,the saponin glycosidase form fermentation production as research object,which used response surface method to optimize the fermentation conditions produce saponin glycosidase of endophytic fungi SYfx213.2,and verification the best fermentation conditions to produce enzyme,and preliminary separation and purification saponin glycosidase,determine its molecular weight.the main results were as follows:(1)The strains(SYfx213.2)efficient produce enzyme was identified:aspergillus aspergillus flavus.(2)Optimization of the fermentation conditions for endophytic fungi SYfx213.2 by single factor optimization,and the results of single factor optimization were: 8 g/L sugar,2.0 g/L beef extract,1.5 g/L potassium hydrogen phosphate,0.5 g/Lmagnesium sulfate,0.5 g/L potassium chloride,Fermentation was carried out at temperatures33°C,the inoculum size to 15%,speed at100 rpm and initial pH at5.0.(3)Optimization of the fermentation conditions by response surface,and the response surface analysis the best conditions of 8.95 gl-1 sucrose,2.08 gl-1 beef extract paste,and14.12 gl-1 inducer of PDZW,under the condition the fermentation produce diosgenin yield was 19.73 mg/L.the product yield increased about 6.8 ? as compared to the diosgenin yield in not optimized condition,and diosgenin conversion rate was reached 73.67%.(4)The saponin glycosidase used acetone precipitation to enrichment,to optimize the acetone precipitation experiment,and the effect of saponin glycosidase crude enzyme liquid in the following conditions for acetone precipitation was best,with four times the volume of acetone solution processing saponin glycosidase crude enzyme liquid,precipitation time was 1 hour,and acetone precipitation frequency for once.(5)The saponin glycosidase was obtained by twice PAGE for separation and purification,and the molecular weigh was 64.60 kDa which was preliminary determinated by SDS-PAGE.