Preparation Of Icaritin By Co-immobilizing Multiple Enzymes Based On Cross-linking Enzyme Aggregates

Icaritin is a high-value isoprene flavonoid compound,which has significant therapeutic activity on liver cancer and is regarded as another great breakthrough in Traditional Chinese medicine after artemisinin.Icaritin soft capsule,has been approved to be marketed in China in January 2022 as a treatment for advanced liver cancer.The preparation of icaritin by acid and alkali hydrolysis and chemical synthesis has the problems of high cost,complex operation and severe environmental pollution.With the global attention to green pollution-free manufacturing,the preparation of icaritin by an efficient,green and mild biosynthesis method based on protease catalysis is widely concerned.Immobilized enzyme can overcome the shortcomings of free enzyme such as low catalytic efficiency,difficulty in reuse and lack of stability.In this study,an α-L-rhamnosidase and a β-D-glucosidase were co-immobilized by cross-linking enzyme aggregates to construct combi-CLEAs,which successfully realized the efficient one-pot preparation of icaritin.Detailed results are as follows:(1)Screened and analyzed the activities of various epimedin C hydrolases,and identified two active glycosidases for icaritin enzymatic synthesis.The recombinant α-L-rhamnosidase Rha1(MT779021)andβ-D-glucosidase Glu4(MT779019)derived from Talaromyces Stollii CLY-6could be successfully expressed by Pichia pastoris.Rha1 had the hydrolytic activity of rhamnoside bond on the inner side of epimedin C,with specific enzyme activity up to 6.67 U/mg.Glu4 had the hydrolysis activity of glucoside bond with a specific enzyme activity of 8.43 U/mg.According to the preliminary study of enzymatic properties,the two glycosidases have the same optimal reaction p H and similar optimal reaction temperature,which could be used for one-pot preparation of icaritin.(2)α-L-rhamnosidase Rha1 and β-D-glucosidase Glu4 were immobilized by ammonium sulfate precipitation followed by glutaraldehyde crosslinking to obtain two single crosslinking enzyme aggregates: Rha1-CLEAs and Glu4-CLEAs.Based on the recovery of enzyme activity,the concentration of ammonium sulfate and the proportion of glutaraldehyde were optimized.When the volume ratio of precipitant and glutaraldehyde was 1:1 and 0.2%,the recovery of activity reached the highest.Additionally,the influence of temperature and p H,thermal stability,p H stability,kinetic parameters and reusability of CLEAs were investigated.Immobilized enzymes exhibited more outstanding heat stability and wider p H stability than free enzymes.Both Rha1-CLEAs and Glu4-CLEAs maintained over 60% catalytic activity after repeated use for 10 times.In addition,secondary structure analysis confirmed that the immobilized precipitation played a vital role in increasing the thermal stability of the enzyme.(3)Rha1 and Glu4 were co-immobilized to obtain combi-CLEAs,which were successfully used in one-pot preparation of icaritin.Epimedin C could be completely hydrolyzed to icaritin within 30 min by combi-CLEAs,and the catalytic performance of co-immobilized enzyme was also improved compared with that of two single CLEAs mixtures.The overall enzyme activity was 1.18 times of the single CLEAs mixtures and 1.45 times of free enzymes.In addition,sugar tolerance of Rha1 and Glu4 was significantly improved after immobilization.Enhanced sugar tolerance resulted in the high catalytic activity of Rha1 and Glu4 at ultra-high substrate concentrations.The yield of icaritin could reach 77.45% under the condition of 100 mg/m L epimedin C,which laid a foundation for industrial application.