title: Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes.
abstract: The present work describes for the first time an elegant non-chromatographic method, the three phase partitioning for the purification and recovery of zingibain, a milk-clotting enzyme, from Zingiber officinale rhizomes. Factors affecting partitioning efficiency such as (NH4)2SO4 saturation, crude extract to t-butanol ratio and pH on zingibain partitioning were investigated. Optimal purification parameters were 50% (NH4)2SO4 saturation with 1.0:1.0 ratio of crude extract:t-butanol at pH 7.0, which gave 14.91 purification fold with 215% recovery of zingibain. The enzyme was found to be exclusively partitioned in the aqueous phase. The enzyme showed a prominent single band on SDS-PAGE. It is a monomeric protein of 33.8 kDa and its isoelectric point is 4.38. The enzyme exhibited maximal proteolytic activity at a temperature of 60 °C and pH 7.0. It was found to be stable at 40-65 °C during 2 h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca(2+), K(+) and Na(+). It was completely inhibited by heavy metal ions such as Cu(2+) and Hg(2+) and partially by Cd(+). Zingibain milk-clotting activity (MCA) was found to be highly stable when stored under freezing (-20 °C) for 30 days compared at 4 °C.
authors.affiliation: Equipe Maquav, Laboratoire Bioqual, INATAA, Université Constantine 1, Route de Ain El-Bey, 25000 Constantine, Algeria. Electronic address: mgagaoua@inataa.org.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Gagaoua
authors.affiliation: Equipe Maquav, Laboratoire Bioqual, INATAA, Université Constantine 1, Route de Ain El-Bey, 25000 Constantine, Algeria.
authors.forename: Naouel
authors.initials: N
authors.lastname: Hoggas
authors.affiliation: Equipe Maquav, Laboratoire Bioqual, INATAA, Université Constantine 1, Route de Ain El-Bey, 25000 Constantine, Algeria.
authors.forename: Kahina
authors.initials: K
authors.lastname: Hafid
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D005591:Chemical Fractionation; D057056:Cysteine Proteases / Q000737:chemistry* / Q000302:isolation & purification; D004789:Enzyme Activation; D004795:Enzyme Stability; D020939:Zingiber officinale / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007477:Ions / Q000737:chemistry; D008670:Metals / Q000737:chemistry; D053147:Plant Exudates / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D059748:Proteolysis; D027343:Rhizome / Q000737:chemistry*; D013696:Temperature
chemical_list: D007477:Ions; D008670:Metals; D053147:Plant Exudates; D010940:Plant Proteins; D057056:Cysteine Proteases; C584512:zingipain, Zingiber officinale; D000645:Ammonium Sulfate
keywords: Milk-clotting; Purification; Recovery; Three phase partitioning (TPP); Zingibain; Zingiber officinale Roscoe