Purification of ginger proteases by DEAE-Sepharose and isoelectric focusing

Abstract

Ginger proteases in ginger rhizome (Zingiber officinale roscoe) were extrected from the ginger acetone powder and purified on DEAE-Sepharose and Sephadex G-75 columns. Before the purification, excess p-chloromercuribenzoate was added to the enzymes to prevent their autodigestion. The mercuribenzoate-proteases were further purified and fractionated by isoelectric focusing in Ampholine of pH 3–10 or pH 4–6. The proteases were fractionated into three components by the isoelectric focusing, having p I value of 4.5, 4.6 and 4.8 respectively. All these proteases had a molecular mass of 29 000 as measured by SDS- polyacrylamide gel electrophoresis and by TSK G2000SW XL gel chromatography. The Ampholine in the purified enzymes can quickly be removed by the gel chromatography of TSK G2000SW. Some divalent metal ions, such as Hg 2+, Cu 2+, Cd 2+ and Zn 2+, strongly inhibited these purified enzymes.