| Ginkgo biloba extract (EGb761) contains 24% ginkgo flavone lactone(Ginkgo-flavone glycosides) and 6% terpenoids(Terjpenoids), which is expected to become the effective medicine on treatment of Alzheimer′s disease(AD). However its preclinical tests including metabolism, drug metabolism and so on can′t be conducted by modern standards of drugs. The metabolism of Ginkgo biloba extract is mianly carried out by the secretion of hydrolytic enzymes from intestinal microflora. And for that this article selects Enterococcus as a model strain. A series of studies not only clarified the enzymology and molecular biology mechanism of the inirial absorption of Ginkgo biloba extract on the main active ingredient, but also prepared and provided a theoretical basis for researching and developing ginkgo products on the good way in accordance with standards of modern medicine. So it has the theoretical study and practical application of a double significance. At present, it has not seen such successful research reported.In this study, firstly, the best composition of medium was gained through response surface method. The composition is as follows glucose 1.87896%, yeast extract 0.77474%, beef extract 0.3%, peptone 1%, KNO3 6.25mmol/L, NaCl 0.5%. And its optimal culture conditions as follows: liquid volume 60 mL (250mL), initial pH 7.0, inoculation volume 3%, temperature 36℃,after inoculation placed in constant temperature shaker 170 r/min and culture 20 h. The absorbance which was measured at OD560 value of 0.748 comparing to before optimization of 0.682 increased 9.7%.Secondly, this work identified the determination ofβ-D-glucosidase from Enterococcus as following: in the 10 mL colorimetric tube with Cypriot accessed 1.5 mL 0.02mol/L pH 5.0 citric acid-disodium hydrogen phosphate buffer solution, and then by the appropriate scale drew 0.1 mL pipette gun to join the crude enzyme extract, at 45℃water bath in warm period of time, and then incorporated the preheated 5～10 min of 0.4 mL 1 mmol/L substrate pNPG solution into them, using a stopwatch precision timing 14 min ,and then removed immediately adding 2 mL 1 mol/L of Na2CO3 solution to terminate the reaction, and last absorbanced at 400nm at metering when it was cooled to room temperature.Again with a single factor and orthogonal experiment, the fermentation conditions of Enterococcus producingβ-D-glucosidase were optimized to be the best basis for enzyme production medium nutrient as following: corn meal 3%, yeast extract powder 1.2%, peptone 1%, glucose 2%, K2HPO4 0.1%, MgSO4 7.5mmol/L; the corresponding best fermentation conditions of Enterococcus enzyme production were as following: initial pH 7.0, fermentation temperature 37℃,fermentation time 168 h, rotate speed 180 r/min, liquid volume level 50 mL (250 mL flask), inoculation 3%. In this conditions Enterococcusβ-D-glucosidase activity can reach the highest 19.1242 U/(mL·min).Enterococcus producingβ-D-glucosidase fermentation liquid after 40% to 80% ammonium sulfate fractionation, DEAE-cellulose 52 anion-exchange chromatography, Sephadex G-100 gel filtration chromatography steps and so on purified pure products ofβ-D-glucosidase, and the enzyme specific activity from the 3.37 U/mg to 33 U/mg, whose purification factor of 9.79 times, and the enzyme recovery was 5.33%. Purified enzyme protein through NATURE-PAGE and SDS-PAGE electrophoresis, it determined the relative molecular weight of approximately 121KD ofβ-D-glucosidase from Enterococcus, which consists of two subunits(65KD and 54KD). And with the enzymatic properties study found that enzyme acid was stronger, which with a strong thermal stability. The enzyme activity synergistically increased by the addition of metal cations such as 5 mmol/L of Mg2+,Ba2+,Ca2+, and Cu2+,Zn2+,Fe3+,Ag+ on the enzyme was significantly inhibited. In addition,the enzyme has a strong right pNPG substrate specificity. Kinetic studies also have shown that the Michaelis constant Km of 0.05 mmol/L, Vmax of 18.0375 U/L for the Enterococcusβ-D-glucosidase.
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