Theses and Dissertations

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Browsing Theses and Dissertations by Subject "chromatography"

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    Purification and some Biochemical and Immunological Characterization of a Protease from Serratia Marcescens
    (Obafemi Awolowo University, 1986) Obiallor, Nkem Nicholas; Sonukan, Olusola
    An extra cellular protease purified 30-fold was induced in cultures of Serratia marcescens (NCIB 1377) during growth in liquid synthetic medium containing vitamin-free casein (sigma) as the inducer. Purification was by means of ammonium sulphate precipitation and chromatography on sephadex G-100 and DEAE-sephadex (A-50) columns. The molecular weight estimated by gel filtration was approximately 45,000. Optimum temperature and pH of activity, using casein as substrate were 40oc and 8.5 respectively. The protease was stable for 60 minutes at 30-40oC, losing all detectable activity at 60oC, even for 10 minutes. Ca++ and Mg++ did not affect the enzyme activity. Sulfhydryl reagents, IAA, dithiothreitol and L-cysteine could not inhibit its activity; and metal chelators, dithizone and NacN failed to be inhibitory to the protease activity. However, EDTA, at relatively high concentrations inhibited the protease activity. Inhibition of enzyme activity by 2,4-Dinitrophenol indicated need for metabolic energy in enzyme activity. The protease was well inhibited by PMSF indicating it is a serine enzyme. The protease digested a wide range of proteins but with a preference for the milk proteins. It possessed an apparent km of approximately 0.75mg/ml for casein. The sephadex G-100 fraction of the protease was used to raise antibodies in locally bred rabbit. The antibody to this protease was found to inhibit its enzymic activity. Ouchterlony double-diffusion tests revealed antigenic relatedness between all the enzyme fractions at the different stages of purification. The protease shares no antigenic.