title: Purification of active myrosinase from plants by aqueous two-phase counter-current chromatography.
abstract: BACKGROUND
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Lewis B. and Dorothy Cullman Chemoprotection Center, Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205, USA.
authors.forename: Kristina L
authors.initials: KL
authors.lastname: Wade
authors.forename: Yoichiro
authors.initials: Y
authors.lastname: Ito
authors.forename: Aarthi
authors.initials: A
authors.lastname: Ramarathnam
authors.forename: W David
authors.initials: WD
authors.lastname: Holtzclaw
authors.forename: Jed W
authors.initials: JW
authors.lastname: Fahey
mesh_terms: D001937:Brassica / Q000737:chemistry / Q000201:enzymology*; D002845:Chromatography; D005961:Glucosinolates / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D006868:Hydrolysis; D017879:Isothiocyanates / Q000378:metabolism*; D029587:Moringa / Q000737:chemistry / Q000201:enzymology*; D009149:Mustard Plant / Q000737:chemistry / Q000201:enzymology*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification; D018515:Plant Leaves / Q000737:chemistry / Q000201:enzymology; D010940:Plant Proteins / Q000302:isolation & purification; D031224:Raphanus / Q000737:chemistry / Q000201:enzymology*; D036226:Seedlings / Q000737:chemistry / Q000201:enzymology; D012639:Seeds / Q000737:chemistry / Q000201:enzymology
chemical_list: D005961:Glucosinolates; D017879:Isothiocyanates; D010936:Plant Extracts; D010940:Plant Proteins; C037152:isothiocyanic acid; D006026:Glycoside Hydrolases; C022739:thioglucosidase
keywords: Counter-current chromatography; broccoli; daikon; glucosinolate; isothiocyanate; moringa; mustard