Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brassica / chemistry
  • Brassica / enzymology*
  • Chromatography
  • Glucosinolates / metabolism
  • Glycoside Hydrolases / isolation & purification*
  • Glycoside Hydrolases / metabolism
  • Hydrolysis
  • Isothiocyanates / metabolism*
  • Moringa / chemistry
  • Moringa / enzymology*
  • Mustard Plant / chemistry
  • Mustard Plant / enzymology*
  • Plant Extracts / chemistry
  • Plant Extracts / isolation & purification
  • Plant Leaves / chemistry
  • Plant Leaves / enzymology
  • Plant Proteins / isolation & purification
  • Raphanus / chemistry
  • Raphanus / enzymology*
  • Seedlings / chemistry
  • Seedlings / enzymology
  • Seeds / chemistry
  • Seeds / enzymology

Substances

  • Glucosinolates
  • Isothiocyanates
  • Plant Extracts
  • Plant Proteins
  • isothiocyanic acid
  • Glycoside Hydrolases
  • thioglucosidase