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 + , 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.

Qualitative Zymographic Studies of Major Proteinases from Malted Sorghum (Sorghum bicolor L.) and Effects of Cultivar

“…Malts from all the sorghum lines showed more in-gel proteinase activity and bands than their respective ungerminated grains, confirming earlier reports (Evans and Taylor 1990;Ogbonna et al 2003) of substantial increases in proteinase activity during germination. Increase in proteinase activity during malting could have been due both to the emergence of new isozyme forms as well as increase in the activity of isozymes already present in the ungerminated seeds.…”

“…Indeed in an earlier study, Ogbonna et al (2003) had purified a cysteinetype proteinase from malts of an improved Nigerian sorghum cultivar that was stimulated 11-41% by the divalent cations Zn 2+ , Cu 2+ , and Sr 2+ . Indeed in an earlier study, Ogbonna et al (2003) had purified a cysteinetype proteinase from malts of an improved Nigerian sorghum cultivar that was stimulated 11-41% by the divalent cations Zn 2+ , Cu 2+ , and Sr 2+ .…”

A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.)

“…Timotijević et al 2 purified three forms of 47, 40 and 28 kDa aspartic proteases from mature buckwheat seeds. In sorghum malt an acidic protease with an optimal pH of 5.0 was purified from variety KSV8‐11,3 while a neutral protease with an optimal pH of 6.0 was purified from variety SK5912 4. Both were inhibited by iodoacetic acid and p ‐chloromercuribenzoate.…”

Purification and Some Properties of a Metalloprotease from Sorghum Malt Variety KSV8-1

“…Sorghum malt variety KSV8 contains two distinct proteases separated by gel filtration chromatography on Sephadex G-100, purified and characterised as KSV8-11 with a lower relative molecular weight as reported in our earlier communications (Ogbonna et al 2003), while KSV8-I with a higher relative molecular weight is reported in this paper. pH % Relative activity Stability Activity Figure 6.…”

“…Sorghum variety (KSV8)/germination; enzyme extraction/assay; protein estimation; enzyme purification protocols using 4 M sucrose solution, ion-exchange chromatography on Q-and S-Sepharose, gel filtration chromatography on Sephadex G-100 and hydrophobic interaction chromatography on phenyl-Sepharose CL-4B; molecular properties using SDS-PAGE; physicochemical and catalytic properties of the purified enzyme using standard/international methods were carried out as detailed in our earlier communications (Ogbonna et al 2003(Ogbonna et al , 2004.…”