Work

Purification of Active Myrosinase from Plants by Aqueous Two‐Phase Counter‐Current Chromatography

Year: 2014

Type: 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.

Source: Phytochemical Analysis

DOI: 10.1002/pca.2535

Authors: Kristina L. Wade, Yoichiro Ito, Aarthi Ramarathnam, W. David Holtzclaw, Jed W. Fahey

Institutions: Johns Hopkins University, Johns Hopkins Medicine, National Institutes of Health, National Heart, Lung, and Blood Institute

Language: English

Topic: Chromatography in Natural Products

Subfield: Analytical Chemistry

Field: Chemistry

Domain: Physical Sciences

Open Access status: green