Purification and Some Properties of a Protease from Sorghum Malt Variety KSV8-11
A.C. Ogbonna 1, 3, S.K.C. Obi 1, B.N. Okolo 1 and F.J.C. Odibo 2
1 Brewing Science & Technology Laboratory, Department of Microbiology, University of Nigeria, Nsukka, Nigeria.
1 A.C. Ogbonna was on study leave for the Ph.D at the above address.
2 Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria.
3 Corresponding author: Department of Brewing Science and Technology, University of Uyo, P.M.B. 1017, Uyo, Nigeria. E-mail: acogbonna@yahoo.com
J. Inst. Brew. 109(3), 179-186, 2003 | VIEW ARTICLE
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 +, 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 Km of 18 mg · mL -1 and a Vmax of 11.1 µmol · mL -1 · min -1 with casein as substrate.
Key words:
Characterization, malting, protease, purification, sorghum
Publication no. G-2003-0918-0083 ©2003 The Institute & Guild of Brewing