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 K_m and V_max 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 K_m and V_max 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.

Keywords: Papiliotrema laurentii ZJU-L07; enzyme characteristics; epimedin C; icariin; optimization; α-L-rhamnosidase.

Figure 1

Neighbor-joining tree of P. laurentii ZJU-L07.

Figure 2

(a) Sephacryl S-200 gel chromatography of α-L-rhamnosidase. (b) DEAE SepharoseTM Fast Flow gel chromatography of α-L-rhamnosidase. (c) SDS-PAGE analysis of the purified enzyme. Lane 1, the protein marker; lane 2, the purified enzyme. (d) Purification of α-L-rhamnosidase from P. laurentii ZJU-L07.

Figure 3

Properties of α-L-rhamnosidase. (a) Optimum temperature, (b) thermal stability, (c) optimum pH, (d) pH stability, (e) effects of metal ions, (f) kinetic parameters with pNPR as substrate, and (g) kinetic parameters with epimedin C as substrate.

Figure 4

The effects of different purities of epimedin C on the yield of icariin (a) and epimedin C remaining (b).

Figure 5

(a) The amount of α-L-rhamnosidase on icariin yield at 50 °C under pH 4.0 with 3.95 U of enzyme. (b) The amount of α-L-rhamnosidase on icariin yield at 50 °C under pH 4.0 with 500 mg·L⁻¹ epimedin C. (c) Temperature influence with 3.95 U of enzyme in the reaction system containing 500 mg·L⁻¹ epimedin C at pH 7.0. (d) pH influence with 3.95 U of enzyme in the reaction system containing 500 mg·L⁻¹ epimedin C at 50 °C.

Figure 6

Response surface maps of the interaction of enzyme activity (X₁) and pH (X₂) (a), enzyme activity (X₁) and hydrolysis time (X₃) (b), and pH (X₂) and hydrolysis time (X₃) (c) on icariin yield (Y₁). The coordinate X represents the code value, not the actual value.