Klebsiella pneumoniae, a mercury-resistant bacterial strain able to reduce ionic mercury to metallic mercury, was isolated from wastewater of Casablanca. This strain exhibits high minimal inhibition concentrations for heavy metals such as mercury 2400 microM, lead 8000 microM, silver 2400 microM, and cadmium 1000 microM. This bacterium was immobilized in alginate, polyacrylamide, vermiculite, and cooper beech and was used for removing mercury from a synthetic water polluted by mercury by using a fluidized bead bioreactor. Immobilized bacterial cells of Klebsiella pneumoniae could effectively volatilize mercury and detoxify mercury compounds. Moreover, the efficiency of mercury volatilization was much greater than with the native cells. The highest cleanup and volatilization rates were obtained when Klebsiella pneumoniae was entrapped in alginate beads, with a cleanup rate of 100% and a volatilization rate of 89%. Immobilized cells in alginate continuously volatilized mercury even after 10 days without loss of activity.
A new procedure utilizing immunoaffinity column chromatography has been used for the purification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from human erythrocytes. The comparison between this rapid method (one step) and the traditional procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography shows that the new method gives a highest specific activity with a highest yield in a short time. The characterization of the purified GAPDH reveals that the native enzyme is a homotetramer of ~150 kDa with an absolute specificity for the oxidized form of nicotinamide adenine dinucleotide (NAD(+)). Western blot analysis using purified monospecific polyclonal antibodies raised against the purified GAPDH showed a single 36 kDa band corresponding to the enzyme subunit. Studies on the effect of temperature and pH on enzyme activity revealed optimal values of about 43 degrees C and 8.5, respectively. The kinetic parameters were also calculated: the Vmax was 4.3 U/mg and the Km values against G3P and NAD(+) were 20.7 and 17.8 muM, respectively. The new protocol described represents a simple, economic, and reproducible tool for the purification of GAPDH and can be used for other proteins.
Immobilized cells of Enterobacter agglomerans, able to reduce azo dyes enzymatically, were used as a biocatalyst for the decolorization of synthetic medium containing the toxic azo dye methyl red (MR). This bacterial strain exhibits high ability to completely decolorize 100 mg/L of MR after only 6 h of incubation under aerobic conditions. Cells of E. agglomerans were immobilized in calcium alginate, polyacylamide, cooper beech, and vermiculite, and were used for the decolorization of MR from synthetic water by using a fluidized bed bioreactor. The highest specific decolorization rate was obtained when E. agglomerans was entrapped in calcium alginate beads and was of about 3.04 mg MR/g cell/h with a 50% conversion time ( t(1/2)) of about 1.6 h. Moreover, immobilized cells in calcium alginate continuously decolorized MR even after seven repeated experiments without significant loss of activity, while polyacrylamide-, cooper beech-, and vermiculite-immobilized cells retained only 62, 15, and 13% of their original activity, respectively.
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