Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

Abstract

A metalloprotease from sorghum malt variety KSV8-I was purified by a combination of 4-M sucrose fractionation, ion-exchange chromatography on Q-Sepharose (Fast flow), gel-filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B. The enzyme was purified 7.9-fold to give a 13.4% yield relative to the total activity in the crude extract and a final specific activity of 2128.7 U mg−1 protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 35 kDa. The purified enzyme had optimal activity at 60 °C and maximal temperature stability between 40 and 60 °C but retained over 77% of its initial activity after incubation at 70 °C for 30 min. Both pH optimum and maximal stability were at 7.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. Using 0.2 ml of 5 mM solution of each metal ion, the purified protease was slightly (P<0.05) inhibited by Zn2+, appreciably (P<0.01) inhibited by Ca2+ and Co2+ and highly significantly (P<0.001) inhibited by Ag+, Ba2+, Hg2+, Mn2+ and Pb2+. The enzyme was equally highly significantly (P<0.001) inhibited by EDTA and hydrolysed casein to give the following kinetic constants: K m = 21.0 mg ml−1; V max = 8.2 μmol ml1 min−1 and K i = 0.390 mM.