Research On Isolation,Purification And Catalytic Conditions Of Saponin Hydrolase From Aspergillus Tubingensis HG57

Diosgenin is an important precursor for the synthesis of steroid hormone drugs.At present,the traditional acid hydrolysis processof diosgeninhas problems such as high pollution and high waste,which seriously affects the healthy development of the diosgeninindustry.Microbial transformation is the most promising method because of its high efficiency and environmentalprotecting capacity.In ourprevious study,the high efficient transformed strain of Aspergillus tubigensis HG57 was obtained,while had the problem of unstable conversion rate in industrial application.To solve this problem,this paper studied the key saponin hydrolase in the process of transforming saponin with HG57.The results are as follows:(1)The reducing sugar produced from the degradation of saponin by enzymes was detected by DNS(3,5-dinitrosalicylic acid)method,and the detection condition wasoptimized,which is substrate concentration 10 g/L,reaction temperature 50 °C,pH 5.5,and reaction time 4 h.Thus a method for detecting the total enzyme activity of saponin hydrolase was established.(2)The saponin hydrolase produced by HG57 was isolated and purified by ammonium sulfate fractionation,anion exchange chromatography,and SDS-polyacrylamide gel electrophoresis(SDS-PAGE)to obtain three groups of protein constituents,namely,A,B,and C.Amolecular weight of 70 kDa is a key saponin hydrolase.(3)The diosgenin of saponin hydrolyzate catalyzed by saponin hydrolase was determined by HPLC.The diosgenin yield of protein A constituent was significantly higher than that of B and C constituents.The condition for diosgeninusing A constituent was optimized,whichis reaction temperature 50 ?,enzyme concentration 34 mg/mL,and reaction time 72 h;the high-resolution liquid chromatography-mass spectrometer(HR-LC-MS)was applied to detect the saponin species before and after the conversion of the A constituent,and found thatfive water-soluble saponins were degraded todiosgenin andfive water-insoluble saponins.The above results provide an important theoretical basis for the subsequent construction of engineering bacteria for the efficient conversion of saponins,which lays a foundation for its industrial application.