Browsing by Subject "Biochemistry, Molecular Biology and Biophysics"

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    Biodegradation of atrazine by atrazine chlorohydrolase: characterization of mutant enzyme and immobilization system for water purification.
    (2012-01) Aggarwal, Amit
    Atrazine is the most widely used herbicide by corn and sorghum growers. Atrazine chlorohydrolase (AtzA) dechlorinates atrazine, producing non-toxic and non-phytotoxic hydroxyatrazine. In this study, we report the cloning and initial experiments for partial characterization of mutant AtzA (Colin Scott, Colin J. Jackson, Chris W. Coppin, et al. 2009. Catalytic Improvement and Evolution of Atrazine Chlorohydrolase. Appl. Environ. Microbiol. 75(7):2184-2191). A plasmid vector was designed for ease of purification of the enzyme with the help of a his-tag and to ensure high expression of soluble protein in recombinant E.coli. The specific activity and substrate specificity of the purified mutant AtzA was then compared to the wild type enzyme. It was observed from these initial studies that the mutant enzyme had somewhat similar characteristics to the wild type enzyme. Further, the current study also describes immobilization recombinant E. coli cells expressing the wild type atrazine chlorohydrolase in a silica/polymer porous gel. This novel recombinant enzyme-based method utilizes both adsorption and degradation to remove atrazine from water. A combination of silica nanoparticles (Ludox TM40), alkoxides, and an organic polymer were used to synthesize a porous gel. Gel curing temperatures of 23°C or 45°C were used to either maintain cell viability or to render the cells non-viable, respectively. The enzymatic activity of the encapsulated viable and nonviable cells was high and extremely stable over the time period analyzed. At room temperature, the encapsulated non-viable cells maintained a specific activity between (0.44 ± 0.06) μmol/g-min and (0.66 ± 0.12) μmol/g-min for up to 4 months, comparing well with free, viable cells specific activities (0.61 ± 0.04 μmol/g-min). Gels cured at 45°C had excellent structural rigidity and contained few viable cells, making these gels potentially compatible with water treatment facility applications.
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    Developing a Disulfide Replacement Picture of APOBEC3G
    (2010-04-21) Biermann, Mitch
    The human protein APOBEC3G (A3G) interferes with HIV infection by acting as a cytidine deaminase, an enzyme that induces numerous mutations in HIV’s genetic material that ultimately destroy it. But A3G is only successful at this for a time. The HIV protein viral infectivity factor (Vif) destroys A3G. Developing a way to mask A3G from Vif is a major therapeutic goal. Uncovering the three-dimensional structure of A3G is crucial to rational drug design. The catalytic C-terminal domain of A3G has been solved, but the crucial Vif-interacting N-terminal domain remains invisible to medicinal chemists. A major obstacle toward this goal is the N-terminal domain’s poor solubility. Here we explore a novel technique, disulfi de replacement, in which pairs of cysteine residues are incorporated into the protein at hypothetically close positions and checked for disulfi de bonding. We isolated a model peptide containing a disulfi de bond from its reduced form, and we observed an engineered disulfi de from the Ctd of A3G at two residues known to be spatially close. However, the sensitivity of the approach in digested peptide samples must be improved. We would like to acknowledge Yongjian Lu, Takahide Kono, the Chemistry Department Mass Spectrometry Facility, and all the other members of the Matsuo lab for their support.