Optimization Of Fermentation And Enzymatic Hydrolysis Conditions Of Saponinase Produced By Endogenous Aspergillus Flavus SY_fX213.2 And Purification Of Saponinase

Diosgenin is an important precursor of many hormone steroids.Traditional methods of preparing diosgenin by acid hydrolysis may pollute the environment.In order to obtain a method that can replace the traditional acid hydrolysis method to convert plant yellow ginger into saponin,a high-yielding saponinase strain SY_fX213.2 was screened,and the saponin hydrolase was prepared by microbial fermentation.After fermentation and culture,the crude saponin enzyme solution was prepared by crushing with liquid nitrogen method,and then separated and purified by ammonium sulfate fractional salting out precipitation and secondary electrophoresis.Main research contents and results are as follows:（1）Optimization of enzyme production conditions in microbial fermentation.The optimal seed culture conditions of SY_fX213.2 were optimized by single factor method:sucrose 4.48 g/L,beef extract 1.04 g/L,Mg SO₄·7 H₂O 0.25 g/L,K₂HPO₄·3 H₂O0.75 g/L,KCl 0.25 g/L,yam rhizome powder 7.06 g/L,inoculated by spore suspension filtration method,cultured for 24 hours at 33℃.On the basis of single factor test,the optimal fermentation conditions of SY_fX213.2were optimized by multi-factor method:sucrose 17.9 g/L,beef extract 20.8 g/L,KCl 0.5g/L,Mg SO₄·7 H₂O 0.5 g/L,K₂HPO₄·3 H₂O 1.5 g/L,Fe SO₄·12 H₂O 0.01 g/L,rhizome powder 14.12 g/L.The culture temperature was 30℃,the p H value was 6.0,the inoculation quantity was 3%,the rotary speed was 110 r/min,and culture for 6 days.At this time,the biomass of thalli was 80.1 g/L,which was 23.7%higher than before optimization.The morphology of thalli was observed by optical microscope,which showed that the fungus was propagated by spore reproduction.It was white aerial mycelium at the early stage of growth,and gradually formed spherical spores at the later stage of growth,and spherical sporangium and sporangium group appeared,belonging to the genus fungiaceae.（2）Optimization of enzymatic hydrolysis conditions of saponins.The optimal enzymatic hydrolysis conditions of saponinase were obtained by single factor method with enzyme activity as the index:temperature 50℃,p H 6.8,substrate total saponin concentration 20 g/L,reaction 12 h.It was found that the efficiency of converting saponin into saponin by enzymatic hydrolysis was twice that of acid hydrolysis.The yield of enzyme preparation by freeze-drying concentrated mechanism was 0.02 g/L.The inactivation curve of saponase was drawn,and the results showed that the activity energy of saponase was retained at 80%after 3 days at-20℃and 40%after 5 days,indicating that the activity stability of saponase was good.（3）Isolation and extraction of saponinase.Compared with three methods of cell crushing of bacteria,namely liquid nitrogen grinding method,quartz sand grinding method and ultrasonic crushing method,it was found that the activity of enzyme solution prepared by liquid nitrogen grinding method was higher,about twice that of quartz sand grinding method.The lapped thalli powder was dissolved by adding the extract,and then lysozyme or SDS were added to promote cell membrane lysis.Comparison showed that both of them could assist cell membrane lysis of the bacteria to dissolve intracellular saponinase and thus improve the activity of saponinase,and SDS had a better effect.After preliminary separation of saponinase by 30%and 60%solid ammonium sulfate fractional salting out precipitation method,it was found that 90%of saponinase activity was retained after salting out,and many hybrid proteins were removed.Two saponin hydrolases with molecular weights of 212 k Da and 375 k Da were recovered by direct extraction.Two saponinases were recovered by gel cutting,and the amino acid sequence was detected by lc-ms method.The results showed that the two enzymes were new isoenzymes with the characteristics of hydrolyzing saponins and isomerases.