Purification and Characterization of a Novel α-L-Rhamnosidase from Papiliotrema laurentii ZJU-L07 and Its Application in Production of Icariin from Epimedin C

Abstract

Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, Papiliotrema laurentii ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specific activity of α-L-rhamnosidase was 29.89 U·mg−1 through purification, and the molecular mass of the enzyme was 100 kDa, as assayed by SDS-PAGE. The characterization of the purified enzyme was determined. The optimal temperature and pH were 55 °C and 7.0, respectively. The enzyme was stable in the pH range 5.5–9.0 for 2 h over 80% and the temperature range 30–40 °C for 2 h more than 70%. The enzyme activity was inhibited by Ca2+, Fe2+, Cu2+, and Mg2+, especially Fe2+. The kinetic parameters of Km and Vmax were 1.38 mM and 24.64 μmol·mg−1·min−1 using pNPR as the substrate, respectively. When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of Km and Vmax were 3.28 mM and 0.01 μmol·mg−1·min−1, respectively. The conditions of enzymatic hydrolysis were optimized through single factor experiments and response surface methodology. The icariin yield increased from 61% to over 83% after optimization. The enzymatic hydrolysis method could be used for the industrialized production of icariin. At the same time, this enzyme could also cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside in naringin and neohesperidin, which could be applicable in other biotechnological processes.

Advancements in the Biotransformation and Biosynthesis of the Primary Active Flavonoids Derived from Epimedium

“…Icariin, baohuoside I, and icaritin are the most effective components in Epimedium [31,118,119,122], and considered as high-value Epimedium flavonoids. Enzyme transformation is the preferred method for the preparation of icariin, baohuoside I, and icaritin, due to its high selectivity and catalytic efficiency, fewer by-products, mild reaction conditions, and convenient purification of the products [114,130].…”

MICROBIAL Α-L-Rhamnosidases: CLASSIFICATION, DISTRIBUTION, PROPERTIES AND PRACTICAL APPLICATION

“…hesuelica, Cryptococcus victoriae, C . terricola, Pichia angusta, Saccharomyces cerevisiae and Papiliotrema laurentii [23,[47][48][49][50]. Although α-L-rhamnosidases are widely represented in various groups of microorganisms, α-L-rhamnosidases of fungal origin are the most studied.…”

“…laurentii and T . bacterium [47,63], Mg 2+ -in A . nidulans [30], cations Co 2+ , Сu 2+ , and Ni 2+ -in A. alternata [65], 0.2 mM Zn 2+ completely inactivated the α-L-rhamnosidase of A .…”