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Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8–11
Author(s) -
Ogbonna A.C.,
Obi S.K.C.,
Okolo B.N.,
Odibo F.J.C.
Publication year - 2003
Publication title -
journal of the institute of brewing
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.523
H-Index - 51
eISSN - 2050-0416
pISSN - 0046-9750
DOI - 10.1002/j.2050-0416.2003.tb00157.x
Subject(s) - chemistry , iodoacetic acid , chromatography , pmsf , protease , casein , ethylenediaminetetraacetic acid , size exclusion chromatography , substrate (aquarium) , sephadex , enzyme assay , enzyme , phenylmethylsulfonyl fluoride , sepharose , biochemistry , biology , organic chemistry , ecology , chelation
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 + , Ca 2+ , Co 2+ , Fe 2+ , Mg 2+ , iodoacetic acid (IAA) and p‐chloromercuribenzoate (p‐CMB), stimulated by Cu 2+ , Sr 2+ , phenylmethylsulfonyl‐fluoride (PMSF) and 2‐mercaptoethanol (2‐ME) while Mn 2+ and ethylenediaminetetraacetic acid (EDTA) had no effect. The purified enzyme had a K m of 18 mg·mL −1 and a V max of 11.1 μmol · mL −1 · min −1 with casein as substrate.