The Immobilization Of Thermophilic Rhamnosidase TpeRha And Its Application In The Preparation Of Icaritin

Rhamnosidase TpeRha can specifically act on the α-1 rhamnosidase bond located at C-3position of the epimedium flavonoid parent nucleus structure,which plays a crucial role in the enzymatic preparation of icaritin,an active component with various anticancer effects.In order to further improve the high temperature stability and reduce the cost of enzyme,the paper mainly studies the immobilization technology of TpeRha.Established the methods of preparing rhamnosidase cross-linked aggregates without carrier immobilization of TpeRha((PAG-R))and preparing immobilized enzyme with epoxy resin as carrier(ES-107T-R,EG-1-R and ES-103B-R),and explored the means of improving the thermal stability of immobilized enzyme,effectively solved the problem of poor thermal stability of TpeRha,and greatly improves the times of repeated use of enzyme.At the same time,the technology of transforming total flavonoids of Epimedium to icaritin was established,which laid a foundation for the preparation of icaritin.The main conclusions are as follows:1.The preparation process of cross-linked aggregates of α-L-rhamnosidase TpeRha of GH78 family was established.Using Pluronic F127(polyoxyethylene polyoxypropylene ether block copolymer F127)as protective agent,α-L-rhamnosidase cross-linked aggregate PAG-R was obtained by precipitation with 75 mg/ml ammonium sulfate and cross-linking with 40mmol/L glutaraldehyde.The highest recovery rate of enzyme activity was 60.90%,enzyme activity was 34 U/ml,and protein fixation rate was 52.15%.When the immobilized enzyme was prepared in the usual sequence of ammonium sulfate-glutaraldehyde(CLEAs),the recovery of enzyme activity was only 30.87%.The optimal temperature of PAG-R was 90 ℃ and the optimal pH was 5.0,which was similar to the enzymatic properties of the free enzyme TpeRha,but the specific activity of PAG-R is higher under different temperature and pH conditions.Under the condition of 80 ℃,pH 6.0 and 4 U/ml of enzyme,PAG-R could completely transform 1 g/L rutin to isoquercetin for 30 min.The amount of isoquercetin catalyzed by PAG-R was 2.3 times as much as that of TpeRha under the same conditions.After 10 times of repeated use of PAG-R,the relative conversion rate was about 60%.2.The technology of immobilized GH78 family α-L rhamnosidase TpeRha by epox resin was established.Based on the excellent immobilization effect,good stability and reusability of the immobilized enzyme prepared by covalent combination of epoxy carrier,11 different kinds of epoxy resin were selected to covalently immobilize TpeRha.By comparing the important indexes such as protein fixation rate,enzyme activity recovery rate and enzyme activity of immobilized enzyme,it is finally determined that the best result is to add 0.2 g ES-107 T resin and 20 U/m L TpeRha in 1 ml reaction system to react at 20 ℃ for 40 h with citric acid of 1.25mol/L and pH of 5.5 as buffer solution.The enzyme activity of immobilized enzyme reached74.61 U/g,the protein fixation rate was 99.63%,and the enzyme activity recovery rate was74.61%.3.By comparing the key indicators of enzyme immobilization,such as protein fixation rate,enzyme activity recovery rate,enzyme reuse times and recovery operation difficulty,it can be seen that the two immobilization technologies currently studied are more suitable for the immobilization of TpeRha with epoxy resin ES-107 T.4.The strategies to improve the thermal stability of epoxy resin immobilized enzyme were investigated.The thermal stability of TpeRha immobilized on epoxy resin can be effectively improved by using a certain concentration of alkaline solution and glycine solution.The suitable treatment method is: put the immobilized enzyme in the mixture of 1 mol/L pH 8phosphoric acid,20% glycerol and 10% fructose for 4 days,and then treat it with 3 mol/L glycine for 24 hours.The half-life of pd-ES-107T-R increased to 15 h at 90 ℃,which was 2.5times of ES-107T-R and 30 times of free enzyme TpeRha.ES-107T-R can completely transform 1 g/L naringin and rutin at 85 ℃,pH 5.0,enzyme dosage of 35 mg(about 2 U/m L)and 70 mg(about 4 U/m L),respectively.After repeated use for 20 times,the relative conversion rate of naringin and rutin is still 92.17% and 90.12%.5.The technology for the preparation of icaritin from total flavonoids of epimedium was established.Under the condition of pH 5.5 and temperature 80 ℃,15 U / m L PAG-R and 0.5U/m L Tpebgl3 were used to react with rhamnosidase for 2 hours,and then with glucosidase for1 hour,the total flavonoid extract of Epimedium could be transformed completely,and 0.339mmol/L icaritin was obtained.The total flavonoid extract of Epimedium can be completely transformed by the simultaneous reaction of 0.1 g ES-107T-R and 0.5 U/m L Tpebgl3 at pH 5.0and temperature 80 ℃ for 12 h.The content of icaritin is 0.312 mmol/L.6.The catalytic stability of the immobilized enzyme PAG-R and ES-107T-R in the transformation of total flavonoids of Epimedium to icraitin was compared.The results showed that the yield of rhamnosidase cross-linked aggregates was only about 60% of the initial yield after 10 cycles of reaction.And the yield of icaritin decreased about 29.95% after 10 times of repeated reaction when the reaction system was 50 m L.The catalytic stability of ES-107T-R is high.The yield of icaritin is still more than 80% of the initial yield,and the catalytic performance remains unchanged after the reaction system is expanded 50 times.Obviously,the enzyme immobilized on epoxy resin is more suitable to transform the total flavonoids of Epimedium to produce icaritin.