| Ginsenoside compound-K [20(S)- β -D-glucopyranosyl protopanaxadiol, for short, C-K], a kind of non-naturally occurring ginseng saponin, is a metabolite came from naturally occurring protopanaxadiol-type ginsenosides such as Rb1 and Rb2 by biotransformation of the intestinal bacteria. C-K, which is named IH901 and has been on the clinical trial (Phase III) in Korea, has been reported to show various pharmacological actions including chemopreventive effects, inhibitions of cancer metastasis and proliferation, the induction of cancer cellular apoptosis, the reversal of cancer multidrug resistance and the obstruction of tumor-angiogenesis. Therefore, a large scale of preparation and production for C-K is becoming a key point of industrialization and attracting a great deal of attention.At present, there are many useful methods including acidic hydrolysis, basic hydrolysis, Smith degradation, and heat processing, which are totally belong to chemical processing, for preparing low-polar ginsenoside derivatives. Since all chemical methods attack and hydrolyze the glycosidic bond at the C2o position of ginsenoside, through which are unable to obtain C-K. Therefore, biotransformation is an only processing way for C-K preparation. Among various bioprocesses enzymatic degradation has certain advantages duo to a mild condition, a feasible operation, a high selectivity and fewer by-products. A research group (group 1806) of Dalian Institute of Chemical Physics in Chinese Academy of Sciences has screened different hydrolytic enzymes and developed an enzyme extract, named SE enzymes (SEE), which has been shown a highly hydrolytic activity toward ginsenoside Rb1 (Rb1), from a kind of mollusks. The ultimate goal of our research will separate and purify the Rb1 hydrolasefrom SEE to understand its function and characterization.Beginning with β -glucosidase assays, the present paper compared the quantitative and qualitative analysis for the original enzyme. For the quantitative analysis, the different reaction conditions with a substrate as p-nitrophenyl β-D-glucopyranoside (pNPG) from a substrate as Rbi were compared. The results are shown as follows: 1) Polyacrylamide gel electrophoresis is the main method for the qualitative analysis with a applied capacity of the sample around 7-10ug; 2) In the purification, the p-nitrophenol released by pNPG hydrolysis and the ginsenoside Rd (Rd) released by Rb1 hydrolysis can act as the analytic objectives, respectively; 3) For the pNPG assay, the analytic range is from 0.0067-0.04 mM, the reaction is assayed in pH 5.2 and 37℃ for 30min incubation; 4) For the Rbl hydrolysis, the analytic range is from 0.059umoyml~1.884umol/ml. The reaction is incubated in pH 4.5 and 40℃ for 6min. In order to determine a procedure of the enzyme purification, several methods were compared as follows: 1) A large scale of SEE was treated in Ammonium sulfate and ethanol precipitation methods, but with unsatisfied recovery and purity; 2) The better separation was found in DEAE-sepharose ion exchange (DSIE), hydroxylapatite (HA)and gel filtration chromatography, respectively; 3) There are at least seven β -glucosidase isoenzymes in the SEE displayed by the combined results of DSIE and the activity staining. HA chromatography showed that the β -glucosidase is most likely an acidic or neutral protein. On the basis of the above results, a feasible procedure was designed and applied for separation and purification as a combination of DSIE stepwise chromatography and DSIE gradient chromatography with gel filtration chromatography. Therefore, a Rbl hydrolase was purified to homogeneity on SDS-PAGE and validated with or without the activity staining. The enzyme on SDS-PAGE and gel filtration (size exclusion) chromatography was estimated to be most likely a tetramer (approximate Mr 460 kDa) with potential subunits of Mr 110-115 kDa.At last, we studied the characterization of the purified β -glucosidase as follows: 1) The optimum pH is 5.6 and the optimum temperature is 80℃. The enzyme was sta... |
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