Kinetics of a Cloned Special Ginsenosidase Hydrolyzing 3-O-Glucoside of Multi-Protopanaxadiol-Type Ginsenosides, Named Ginsenosidase Type III

Abstract

In this paper, the kinetics of a cloned special glucosidase, named ginsenosidase type III hydrolyzing 3-O-glucoside of multi-protopanaxadiol (PPD)-type ginsenosides, were investigated. The gene (bgpA) encoding this enzyme was cloned from a Terrabacter ginsenosidimutans strain and then expressed in E. coli cells. Ginsenosidase type III was able to hydrolyze 3-O-glucoside of multi-PPD-type ginsenosides. For instance, it was able to hydrolyze the 3-O-β-D-(1-->2)-glucopyranosyl of Rb1 to gypenoside XVII, and then to further hydrolyze the 3-O-β-D-glucopyranosyl of gypenoside XVII to gypenoside LXXV. Similarly, the enzyme could hydrolyze the glucopyranosyls linked to the 3-O- position of Rb2, Rc, Rd, Rb3, and Rg3. With a larger enzyme reaction Km value, there was a slower enzyme reaction speed; and the larger the enzyme reaction Vmax value, the faster the enzyme reaction speed was. The Km values from small to large were 3.85 mM for Rc, 4.08 mM for Rb1, 8.85 mM for Rb3, 9.09 mM for Rb2, 9.70 mM for Rg3(S), 11.4 mM for Rd and 12.9 mM for F2; and Vmax value from large to small was 23.2 mM/h for Rc, 16.6 mM/h for Rb1, 14.6 mM/h for Rb3, 14.3 mM/h for Rb2, 1.81mM/h for Rg3(S), 1.40 mM/h for Rd, and 0.41 mM/h for F2. According to the Vmax and Km values of the ginsenosidase type III, the hydrolysis speed of these substrates by the enzyme was Rc>Rb1>Rb3>Rb2>Rg3(S)>Rd>F2 in order.