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

• Published in: Journal of the Institute of Brewing 2003, Vol.109 (3), p.179-186
• Main Authors: Ogbonna, A.C., Obi, S.K.C., Okolo, B.N., Odibo, F.J.C.
• Format: Article
• Language: English
• Subjects: Beers; Biological and medical sciences; Cereal and baking product industries; Characterization; Fermented food industries; Food industries; Fundamental and applied biological sciences. Psychology; malting; protease; purification; sorghum
• ISSN: 0046-9750; 2050-0416
• DOI: 10.1002/j.2050-0416.2003.tb00157.x

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.