Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8-11

Abstract

A protease from sorghum malt variety KSV8–11 was purified by a combination of dialysis against 4 M sucrose, 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 5-fold to give a 14.1% yield relative to the total activity in the crude extract and a final specific activity of 1348.9 U mg−1 protein. SDS-PAGE revealed a single migrating protein band corresponding to a relative molecular mass of 16 KDa. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 30°C and 40°C but retained over 64% of its original activity after incubation at 60°C for 30 min. The pH optimum was 5.0 with maximum stability at pH 6.0 but 60% of the activity remained after 24 h between pH 5.0 and 8.0. The protease was inhibited by Ag+, Ca2+, Co2+, Fe2+, Mg2+, iodoacetic acid (IAA) and p-chloromercuribenzoate (p-CMB), stimulated by Cu2+, Sr2+, phenylmethylsulfonyl-fluoride (PMSF) and 2-mercaptoethanol (2-ME) while Mn2+ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a Km of 18 mg·mL−1 and a Vmax of 11.1 μmol · mL−1 · min−1 with casein as substrate.