Three phase partitioning of zingibain, a milk-clotting enzyme from Zingiber officinale Roscoe rhizomes
2015
Gagaoua, Mohammed | Hoggas, Naouel | Hafid, Kahina
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 33.8kDa 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 2h. The enzyme was found to be highly stable against numerous metal ions and its activity was enhanced by Ca2+, K+ and Na+. It was completely inhibited by heavy metal ions such as Cu2+ and Hg2+ 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.
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