Purification and some properties of a cysteine proteinase from sorghum malt variety SK5912

A cysteine proteinase from sorghum malt variety SK5912 was purified by a combination of 4 M sucrose fractionation, ion-exchange chromatography on Q- and S-Sepharose (fast flow), gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on Phenyl Sepharose CL-4B. The enzyme was purified 8.4-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2057.1 U mg-1 protein. SDS - PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 55 and 62 kDa, respectively. The enzyme was optimally active at pH 6.0 and 50 °C, not influenced across a relatively broad pH range of 5.0-8.0 and retained over 60% activity at 70 °C after 30-min incubation. It was highly significantly (P (less than) 0.001) inhibited by Hg^2+, appreciably (P (less than)0.01) inhibited by Ag^+, Ba^2+ and Pb^2+ but highly significantly (P (less than) 0.001) activated by Co^2+, Mn^2+ and Sr^2+. The proteinase was equally highly significantly (P (less than) 0.001) inhibited by both iodoacetate and p-chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: Km = 0.33 mg ml^-1; Vmax = 0.08 mol ml^-1 min^-1.