Efficient production of icariin and baohuoside I from Epimedium Folium flavonoids by fungal α-L-rhamnosidase hydrolysing regioselectively the terminal rhamnose of epimedin C.

Industrial application of icariin and baohuoside I has been hindered by the short supply to a great extent. In this work, a novel GH78 α-L-rhamnosidase AmRha catalyzed the bioconversion of low-value epimedin C in crude Epimedium Folium flavonoids (EFs) to icariin and baohuoside I was developed. Firstly, the high-level expression of AmRha in Komagataella phaffii GS115 attained an enzyme activity of 571.04 U/mL. The purified recombinant AmRha could hydrolyze α-1,2-rhamnoside bond between two rhamnoses (α-Rha(2 → 1)α-Rha) in epimedin C to produce icariin with a molar conversion rate of 92.3%, in vitro. Furtherly, the biotransformation of epimedin C to icariin by the recombinant Komagataella phaffii GS115 cells was also investigated, which elevated the EFs concentration by fivefold. In addition, biotransformation of epimedins A-C and icariin in the raw EFs to baohuoside I was fulfilled by a collaboration of AmRha and ß-glucosidase/ß-xylosidase Dth3. The results obtained here provide a new insight into the preparation of high-value products icariin and baohuoside I from cheap raw EFs.

Linker-peptide-mediated one-step purification and immobilization of α-L-rhamnosidase from Bacteroides thetaiotaomicron for direct biotransformation from epimedin C to icariin.

Icariin, the most effective bioactive component in Epimedium, is also the index component of Epimedium quality control in Pharmacopoeia. It was a very attractive approach for bioconversion from epimedin C to icariin. However, its potential was impeded by poor stability and non-recyclable properties of free enzymes. In this study, we have fused the linker (4LP) to α-L-rhamnosidase BtRha and successfully prepared the immobilized enzyme (incubated 4LP-BtRha@Na-Y) to produce icariin from epimedin C. The activity recovery of 4LP-BtRha@Na-Y was 79.6 %, and enzyme activity was 209.8 U/g, which was 1.75-fold and 1.6-fold higher than that of immobilized BtRha (BtRha@Na-Y), respectively. The optimal reaction temperature and pH of 4LP-BtRha@Na-Y was 55 °C and 6.5, respectively. The thermal stability of immobilized enzyme was significantly improved by incubation in phosphate buffer containing 20 % glycerol and 10 % fructose. The kcat/Km value of incubated 4LP-BtRha@Na-Y was 7.98 × 105 s-1M-1, which increased by 8 % compared with free BtRha. Finally, under suitable conditions, 1 g/L epimedin C was transformed into icariin with icariin yield 75.1 %, and the relative conversion rate retained 74.9 % after reused 13 cycles. This experiment provides a new idea for one-step purification and immobilization of α-L-rhamnosidase for direct biotransformation from epimedin C to icariin, which will have great prospects in food and pharmaceutical production.

Modification to increase the thermostability and catalytic efficiency of α-L-rhamnosidase from Bacteroides thetaiotaomicron and high-level expression.

The α-L-rhamnosidase BtRha from Bacteroides thetaiotao VPI-5482 is a specific enzyme that selectively hydrolyzes the α-1,2 glycosidic bond between rhamnose and rhamnose, allowing the bioconversion of epimedin C to icariin. In this study, BtRha was molecularly modified using B-factor-saturation mutagenesis strategy and the introduction of disulfide bonds, resulting in a mutant with significantly improved catalytic efficiency, S592C, and two thermally stable mutants, E39W and E39W-S592C. The results showed that the half-lives of E39W and E39W-S592C at 55 °C were 10.4 and 9.4-fold higher, respectively, than that of the original enzyme, The mutant S592C showed a 63.3% reduction in Km value and a 163.6% increase in catalytic efficiency (kcat/Km value), which improved the ability to hydrolyze epimedin C to icariin effectively. In addition, high-level expression of α-L-rhamnosidase mutant S592C was established. With 0.1 mM IPTG as an inducer, induction temperature of 32 °C, induction pH of 7.0 and induction OD600 of 50, the maximum activity of mutant S592C reached 182.0 U/mL in terrific broth medium after 22 h. This is the highest enzyme activity of α-L-rhamnosidase which can convert epimedin C to icariin to date. All the results provide a specific and cost-effective α-L-rhamnosidase mutant, which will raise its potential interest for the food and pharmaceutical applications.

Immobilization of high temperature-resistant GH3 ß-glucosidase on a magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8/zeolitic imidazolate framework.

The magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNCZ8) was used to immobilize the high-temperature resistant GH3 ß-glucosidase (Tpebgl3) from the Thermomotoga petrophila DSM 13995. The Tpebgl3 has great potential in the catalytic conversion of pharmaceutically active components. The magnetic carrier (FSNCZ8) which provided stronger adsorption capacity, magnetic and high-temperature stability than the previously discussed Fe3O4-NH2-Cellu-ZIF8 without tetraethyl silicate coated. The properties of FSNCZ8-Tpebgl3 (FSNCZ8-T) were as follows: The optimal temperature and pH were 90 °C and 5.5, respectively; the highest activity of FSNCZ8-T approached 2672 U/g; Fe2+ enabled immobilized enzyme to increase its relative activity to 194%. The main characteristics of FSNCZ8-T, including thermal stability, pH stability, and glucose tolerance, were greatly enhanced by adding Fe2+, which was also superior to free enzymes; moreover, the residual activity of FSNCZ8-T was 74% of the initial activity at the end of 10 repeated cycles.