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 < 0.001) inhibited by Hg 2+ , appreciably ( P < 0.01) inhibited by Ag + , Ba 2+ and Pb 2+ but highly significantly ( P < 0.001) activated by Co 2+ , Mn 2+ and Sr 2+ . The proteinase was equally highly significantly ( P < 0.001) inhibited by both iodoacetate and p ‐chloromercuribenzoate and hydrolysed casein to give the following kinetic constants: K m = 0.33 mg ml −1 ; V max = 0.08 µmol ml −1 min −1 .