Cloning And Characterization Of Gene Encoding ?-L-arabinofuranosidase And Bifunctional ?-xylosidase/?-L-arabinopyranosidase

Ginsenosides,belonging to triterpenoid saponins,are one of the prime active ingredients of Panax species.Ginsenosides have many pharmacological effects,such as anti-tumor,anti-oxidation and immune regulation.Among them,ginsenoside Rd is one of the main metabolites of protopanaxadiol(PPD)-type ginsenosides in the human intestine,which has a distinct effect on cardio-cerebral vascular system,nervous system and immune system,and it's also one of the precursors that could be converted into the highly active rare ginsenoside Compound-K.For the content is relatively low,ginsenoside Rd could not meet the requirements by the way of extracting it from plants.However,ginsenoside Rd can be prepared by the hydrolysis of sugar moieties from the major ginsenosides(Rb1,Rb2,Rb3 and Rc)with specific glycoside hydrolases.Here,we cloned,expressed and characterized an ?-L-arabinofuranosidase(EC3.2.1.55)and a bifunctional ?-xylanase(3.2.1.37)/?-L-arabinopyranosidase genes from a Cellulosimicrobium strain(Cellulosimicrobium aquatile Lyp51)that was previously isolated from our lab.The recombinant ?-L-arabinofuranosidase(CaAraf51)fused a histidine tag was purified by nickel affinity chromatography with a specific activity of 15 U/mg.CaAraf51 could hydrolyze the outer C-20 arabinofuranoside moieties of ginsenoside Rc into ginsenoside Rd.Under optimal pH5.0 and 40?,the Michaelis parameters Km and Vmax for pNP?Araf and ginsenoside Rc were 1.1 mM and 25?mol/min/mg and 0.57 mM and 6.25 ?mol/min/mg,respectively.When 0.87 mg/mL ginsenoside Rc incubated with 9.6 U/mL purified enzymes in phosphate buffer of pH 7.5 at 30? for 1 hour,the yield of ginsenoside Rd was 0.71mg/mL/h,and the conversion efficiency was 90%.The specific activity of the recombinant bifunctional enzyme(Xylarap51)was 15.45 U/mg.Xylarap51 could hydrolyze the outer C-20 xylose and arabinopyranoside moieties of ginsenoside Rb3 and Rb2 into ginsenoside Rd.Finally,we fused CaAraf51 and Xylarap51 into one multifunctional enzyme(Afxyap51),which could convert ginsenoside Rc?Rb3 and Rb2 into Rd,and the hydrolysis order was Rc>Rb3>Rb2.This experiment not only explored the saponin hydrolase which can transform ginsenoside Rb2,Rb3 and Rc,but also laid a foundation for the application of enzyme protein.