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Nigerian Food Journal
Nigerian Institute of Food Science and Technology
ISSN: 0189-7241
Vol. 25, No. 2, 2007, pp. 1-18
Bioline Code: nf07021
Full paper language: English
Document type: Research Article
Document available free of charge

Nigerian Food Journal, Vol. 25, No. 2, 2007, pp. 1-18

 en Purification and some properties of a serine protease from sorghum malt variety ICSV400
Ogbonna, A.C


A serine protease from sorghum malt variety ICSV400 was purified by a combination of sucrose fractionation, ionexchange 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 6.2-fold to give a 9.5% yield relative to the total activity in the crude extract and a final specific activity of 1667.5 U mg-1 protein. SDS-PAGE revealed two migrating protein bands corresponding to apparent relative molecular masses of 59 and 63 KDa, respectively. Using casein as substrate, the purified protease had optimal activity at 50°C and maximal temperature stability between 50°C and 60°C but retained over 50% of its original activity after incubation at 70°C for 30 min. Both pH optimum and maximum was 5.0 while 55% of the activity remained after 24 h between pH 4.0 and 8.0. The enzyme was highly significantly (p<0.001) inhibited by Ag+, Mn2+ and Pb2+, highly significantly (p<0.001) activated by Cu2+, Fe2+, Sr2+ and Zn2+ but unaffected by both Ca2+ and Co2+. The protease was equally highly significantly (p<0.001) inhibited by phenylmethylsulphonylfluoride (PMSF), unaffected by iodoacetic acid (IAA), p-chloromercuribenzoate (p-CMB) and ethylenediaminetetraacetic acid (EDTA) but highly significantly (P<0.001) stimulated by 2-mercaptoethanol (2-ME). The purified enzyme hydrolysed casein to give the following kinetic constants: Km = 0.56; Vmax = 0.111mmol. ml-1.min-1.

Characterization, germination, kinetics, serine protease, purification, sorghum

© Copyright 2007 - Annals of African Medicine.

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