Myrosinase (thioglucoside glucohydrolase;E.C. 3.2.1.147) is a plant enzyme of increasing interest and importance to the biomedical community. Myrosinase catalyses the formation of isothiocyanates such as sulforaphane (frombroccoli) and 4-(α-L-rhamnopyranosyloxy)benzyl isothiocyanate (from moringa), which are potent inducers of the cytoprotective phase-2 response in humans, by hydrolysis of their abundant glucosinolate (β-thioglucoside N-hydroxysulphate) precursors. Our objective is to develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase. The purification of myrosinase was performed by a high-speed CCC system equipped with a spiral tube assembly. The separation column was made from a sprial tube support (CCBiotech, Rockville, MD, USA) having 4 interwoven spiral grooves (with 12 radial grooves to connect these tube in seris) to accommodate the separtion tube. The PTFE tubing(polytetrafluoroethylene, 1.6 mm ID) was first extruded through a narrow slit followed by twisting along it own axis to form flat-twisted shape and tightly inserted into the spiral grooves using a pressing tool which exactly fits to the radial grooves. The column consisted of 10 spiral layers with a total capacity of about 80 ml. The solvent system used for separation consisted of a 1:1 (v/v)) mixture from stock solutions of (A) 32% w/w PEG-1000 in water and (B) 25% (w/w) potassium phosphate buffer (made by adding 62.5 g K2HPO4 and 62.5 g KH2PO4 to 375 g water). Both salt and PEG solutions were degassed prior to mixing and subsequent phasing out at room temperature. The two phases were separated for use as CCC mobile and stationary phase, as well as for making the crude plant extract preparations. Final pH for each phase was 6.6. Crude myrosinase extracts was as follows: Seven-day-old daikon sprouts (Raphanus sativus) and 3-day-old broccoli sprouts (Brassica oleracea var. italica) were grown commercially by Hanover Foods (Ridgely, MD, USA), freeze-dried and powdered. Fresh mustard seed (Sinapis alba) powder was obtained commercially (Ground Mustard,Gourmet Collection, McCormick &Co., Inc., Sparks, MD, USA). Fresh leaves of the moringa, or horseradish tree (Moringa oleifera), were collected in California (Moringa Farms, Sherman Oaks, CA, USA), shipped at 4C to Baltimore, and freeze-dried immediately and powdered. Just before use, powdered sprouts (250 mg), ground mustard seed (250 mg)or powdered moringa leaves (400 mg), were added to a mixture containing 5 mL of the pre-equilibrated ATPS lower phase and 5 mL of the pre-equilibrated ATPS upper phase (7 mL of each were used for the moringa). The extract was vortexed for 2 min then centrifuged for 10 min using a Centrific Model (Fisher Scientific, Pittsburgh, PA, USA) at 900 g. After centrifugation, the clear lower phase containing the myrosinase activity was collected and used for injection onto the CCC. Injection volumes were 2 mL for the daikon, broccoli and mustard samples, and 3 mL for the moringa samples. An aliquot of the yellowish to greenish upper phase was examined for myrosinase activity and run on SDS-PAGE for comparison purposes. The pelleted plant material was discarded. The HSCCC separation of myrosinase was performed as follows: The spiral column was first completely filled with the upper stationary phase followed by injection of the sample solution. Then the column was rotated at 800 rpm and eluted with the mobile phase at a flow rate of 0.3 - 0.5 ml/min in the tail to head mode. The effluent from the head side of the column was continuously mobitered with a UV detecter at 280 nm and fractionated into test tubes using a fraction collector. Each collected fraction was analysed by SDS PAGE and enzymatic activity according to the method described by Shikita et al. 1999. The result showed that the myrosnase was efficiently purified aat 14.8-61.5 folds with the enzymtic activity of 58.5-79.2%.
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