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. Jan-Feb 2015;26(1):47-53.
doi: 10.1002/pca.2535. Epub 2014 Aug 7.

Purification of Active Myrosinase From Plants by Aqueous Two-Phase Counter-Current Chromatography

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Free PMC article

Purification of Active Myrosinase From Plants by Aqueous Two-Phase Counter-Current Chromatography

Kristina L Wade et al. Phytochem Anal. .
Free PMC article

Abstract

Introduction: 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 (from broccoli) 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.

Objective: To develop an aqueous two-phase counter-current chromatography (CCC) system for the rapid, three-step purification of catalytically active myrosinase.

Methods: A high-concentration potassium phosphate and polyethylene glycol biphasic aqueous two-phase system (ATPS) is used with a newly developed CCC configuration that utilises spiral-wound, flat-twisted tubing (with an ovoid cross-section).

Results: Making the initial crude plant extract directly in the ATPS and injecting only the lower phase permitted highly selective partitioning of the myrosinase complex before a short chromatography on a spiral disk CCC. Optimum phase retention and separation of myrosinase from other plant proteins afforded a 60-fold purification.

Conclusion: Catalytically active myrosinase is purified from 3-day broccoli sprouts, 7-day daikon sprouts, mustard seeds and the leaves of field-grown moringa trees, in a CCC system that is predictably scalable.

Keywords: Counter-current chromatography; broccoli; daikon; glucosinolate; isothiocyanate; moringa; mustard.

Figures

Figure 1
Figure 1
Schematic view of spiral tube assembly (STA) counter-current chromatography (CCC) configuration: (A) external view of assembled spiral tubing support rotor in the 12-radial groove configuration shown in the inside view (B) and wound with flat-twisted PTFE tubing (C).
Figure 2
Figure 2
The SDS-PAGE gels for myrosinase-rich fractions obtained by CCC (STA) from (A) daikon, (B) mustard, (C) broccoli and (D) moringa. Fractions were concentrated about fivefold with centrifugal filters (3 K MWCO) then prepared as described in the ‘Experimental’ section. Lanes: (1) MW standards; (2) crude tissue extract in upper phase of solvent system (not utilised for chromatography); (3) crude tissue extract in lower phase of solvent system; (4–9) peak myrosinase-containing fractions; (10) myrosinase standard. Red rectangles highlight myrosinase bands.
Figure 3
Figure 3
The CCC (STA) chromatograms and myrosinase activity purified from four different sources: (A) daikon – freeze-dried 7-day-old daikon sprouts (Raphanus sativus); (B) mustard – commercial ground mustard seed (Sinapis alba); (C) broccoli – freeze-dried 3-day-old broccoli sprouts (Brassica oleracea var. italica); and (D) moringa – freeze-dried leaves of the horseradish tree (Moringa oleifera). Dry plant material was comminuted into equal portions of the solvent system upper and lower phases, which were vigorously mixed, then centrifuged, and only the lower phase was used for CCC with inline de-salting of the eluent prior to detection by absorbance at 280nm as described in the ‘Experimental’ section (solid line; relative scaling). Myrosinase activity (dashed red line) and specific activity (dotted blue line) are plotted against a common axis.
Figure 4
Figure 4
The SDS-PAGE gels of daikon myrosinase (0.038 U/mg protein) purified by a 160 mL mixer–settler barricaded disk (MSBD) CCC configuration (1.3 U/mg protein), then further purified by concanavalin A-Sepharose chromatography as described in the ‘Experimental’ section. Only the active fractions (f4–f7) eluted with methyl-α-d-mannopyranoside are shown (which yielded 1.9, 5.6, 12 and 10 U/mg protein, respectively).

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