Preparation Of Isoquercitrin And Diosgenin Based On Biotransformation

Isoquercitrin is a kind of derhamnosylation product of rutin.Modern pharmacological research shows that the pharmacological activity of isoquercitrin is much higher than rutin,and it has high medicinal value.Diosgenin is an important precursor compound for the synthesis of steroid hormone medicine.In addition,it has many biological activities,such as anti-tumor,anti-oxidation and improving cardiovascular dysfunction.These two active components of traditional Chinese medicines have low natural content in plants,and they are often prepared by acid hydrolysis and high-pressure hydrolysis in industry.The traditional method uses more inorganic acids and discharges more waste water,which causes serious environmental pollution.The aim of this study is to establish a clean and efficient technology for the preparation of isoquercitrin and diosgenin based on biotransformation?microbial transformation and enzyme catalysis?.The research work includes the following parts:1.Expression of recombinant?-L-rhamnosidase.Firstly,the gene of?-L-rhamnosidase was synthesized by total chemical synthesis and inserted into the pPIC9K vector.Then,it was integrated into the genome of Pichia pastoris GS115 by electrotransformation,and positive transformant against G418 was obtained.Furthermore,the conditions of methanol induced enzyme production were optimized.The results showed that when the initial pH value of the medium was 6.5 and the methanol concentration was 1.5%?v/v?,the activity of the enzyme was the highest after six days of methanol induction.The substrate specificity test of the recombinant enzyme showed that the recombinant?-L-rhamnosidase could selectively hydrolyze rutin to isoquercitrin with higher biological activity,but couldn't remove the rhamnose at the end of dioscin.This study sets up a foundation for the preparation of isoquercitrin by enzymatic hydrolysis.2.The establishment of enzyme catalytic technology for the preparation of isoquercitrin from rutin.Firstly,the conversion efficiency of naringinase and recombinant?-L-rhamnosidase to rutin was compared,and the conditions of enzymatic hydrolysis were optimized with rutin conversion rate as the index.Secondly,isoquercitrin was prepared in large quantity and purified by HPD-100macroporous adsorption resin.The results showed that when the ratio of enzyme to rutin was 1:4,the time of enzyme hydrolysis was 30 mins,the reaction temperature was 60?,and the pH of buffer was 4.5,the enzyme hydrolysis efficiency was the best.When the rutin concentration increased to 20 g/L,under the optimized reaction conditions,it could be converted to isoquercitrin completely within 2 hours,and the purity of isoquercitrin purified by HPD-100 macroporous resin was 97.03%..In addition,rhamnose,another product produced in the process of enzymolysis,also has high economic value.Compared with the traditional acid hydrolysis method,the enzyme hydrolysis method has higher specificity and fewer by-products,suggesting its vast prospect in industry application.3.Diosgenin was prepared by microbial transformation.Firstly,several commercial enzymes and recombinant?-L-rhamnosidase were used to hydrolyze steroidal saponins,and the results showed that they could not be worked effectively.Then,diosgenin was prepared by microbial transformation method,and the effects of four kinds of microorganisms on the yield of diosgenin were compared.Then,the fermentation conditions were optimized with the yield of diosgenin as the index.Furthermore,the effect of fermentation system amplification on diosgenin yield was investigated.The results showed that Aspergillus fumigatus was the best strain to produce enzyme.When the fermentation time was 5 days,the substrate concentration was 30 g/L,the inoculation amount was 2.0%?v/v?,the fermentation temperature was33?,and the initial pH value of the fermentation medium was natural pH,the yield of diosgenin was 1.57±0.07%.The yield of diosgenin was 1.50±0.21%and1.46±0.05%respectively when the fermentation system was magnified 10 and 20times,which was significantly higher than that of direct acid hydrolysis,indicating that this method has a good industrial application prospect.4.Purification of saponin hydrolase produced by microorganism.Firstly,the enzyme was separated by?NH₄?₂SO₄ fractional precipitation,DEAE-52 and phenyl Sepharose HP chromatography,and then the separation effect was detected by enzyme activity tracking and SDS-PAGE respectively.Two components?E1 and E2?with?-glucosidase activity were obtained and the recovery of them were 8.83%,14.04%,respectively,while their purification multiple were 90.70 times,and 116.91 times separately.SDS-PAGE analysis showed that these two active enzymes still contain some heteroproteins,which need to be further purified in order to obtain higher purity to lay a foundation for the study of diosgenin preparation by enzymatic transformation.