Microbial, Biochemical and Food Biotechnology

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Browsing Microbial, Biochemical and Food Biotechnology by Subject "ABC transporter"

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    Biological synthesis of gold nanoparticles by Thermus scotoductus SA-01
    (University of the Free State, 2010-01) Van Marwijk, Jacqueline; Van Heerden, E.
    English: The usual strategy to prepare gold nanoparticles involves the reduction of a gold salt in solution by various reducing agents in the presence of a stabilizer. These particles are mostly spherical with poor monodispersity. An alternative means is to use biological material to mediate particle synthesis. Microorganisms such as fungi have demonstrated the ability to produce nanoparticles of different shapes and sizes extending beyond the scope of chemical means, and the microbial interaction with metals also supply eco-friendly methods for nanoparticle production. It has been hypothesized that the proteins involved in nanoparticle synthesis require a co-factor such as NADH / NADPH, as previous studies have indicated that NADH- and NADPH-dependent enzymes are important factors in the biosynthesis of metal nanoparticles. Thermus scotoductus SA-01, a thermophilic bacterium, isolated from an AngloGold Ashanti mine near Carletonville, Republic of South Africa, was used for purification of a gold(III) reducing and nanoparticle synthesizing protein. This bacterium has the ability to produce gold nanoparticles, and more than one pathway can be followed to produce these particles. A protein was purified to homogeneity by using a combination of several liquid chromatography resins. The N-terminal sequence was obtained by using automated Edman degradation. The protein purified is not a classical oxido-reductase and was identified as an ABC transporter peptide-binding protein (~70kDa). This discovery shows that gold nanoparticles can be produced by proteins other than oxidoreductases. The interaction of the protein extracted and purified from Thermus scotoductus SA-01, as well as the recombinant proteins, with liquid gold under varying physico-chemical conditions have been studied using TEM, EDS, and by measuring the plasmon resonance band, to illustrate the effect on particle morphology and to elucidate the protein mechanism. The size and the shape of particles could, to an extent, be manipulated by controlling the environmental parameters. The purified protein as well as the recombinant proteins was only able to produce nanoparticles in the presence of sodium dithionite and it is thus hypothesized that the donation of electrons via the disulphide bridge in the protein is involved in the reduction of the gold ions. Even though the recombinant proteins had the ability toproduce nanoparticles they were not as efficient as the native protein, but when the optimum parameters for the recombinant proteins are established they could be used in the upscale production of gold nanoparticles or gold nanosheets.
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    Characterization and expression of protein(s) involved in gold nanoparticle formations by Thermus scotoductus SA-01
    (University of the Free State, 2010-05) Erasmus, Mariana; Van Heerden, E.; Albertyn, J.
    English: Developments in the biosynthesis of nanoparticles have increased significantly during the last few years as a result of the growing interest in the unique properties displayed by nanoparticles. These particles are extremely small in size and have a large surface to volume ratio, giving them unique physical and chemical properties at this scale that differs considerably from when they are used in larger form. These exceptional properties are used in a wide variety of applications, ensuing nanotechnology to become a multidisciplinary field. Research into application structure types are extended daily and as a result, the next few years will be crucial as applications for nanomaterials in the industry are most likely to be increased. Gold nanoparticles is receiving more and more attention because of its wide variety of uses in optical, electronic, magnetic, catalytic, and biomedical applications, but even more due to them being the most stable of all the metal nanoparticles. Several methods are used to produce these metal nanoparticles, but are mostly making use of toxic chemicals in the synthesis protocol, which are harmful to the environment and human health. To overcome this problem, researchers are making use of more “greener” alternatives through the use of biological systems and microorganisms in nanoscience and nanotechnology. These microorganisms have unique potential in producing nanoparticles that are environmentally friendly and display different shapes, sizes and distributions. Among the different microorganisms used, bacteria have received the most attention in the nanoparticle production process, but have not been as successful as chemical synthesis to produce monodisperse noble metal nanoparticles. In this study, successful gold reduction and nanoparticle formation with different shapes, sizes and distribution was obtained; however, these particles were not monodisperse. This was achieved with a thermostable protein of ± 70 kDa that was identified as an ABC transporter, peptide-binding protein and which was purified from Thermus scotoductus SA-01; an extremophile and thermophilic bacterium that was isolated from groundwater samples from Mponeng (a deep South African gold mine in the Witwatersrand Supergroup operated by AngloGold Ashanti) at a depth of 3.2 km with ambient rock temperature of 60ºC. The protein was expressed in Escherichia coli and Thermus thermophilus HB27, a mesophilic and a thermophilic expression host respectively. It was found that the expression host might have an influence on the way the protein is folded and therefore influence nanoparticle formation. Expression studies was also done on the protein that either included or excluded Histidine-Tags and a leader peptide, but it was found that neither the His-Tags nor the leader peptide had any influence on the nanoparticles produced. Gold reduction and nanoparticle formation was obtained through reduction of a surface exposed disulphide bond in the ABC transporter, peptide-binding protein, using sodium dithionite as electron donor and reducing agent. In general it was found that nanoparticle formation was dependant on environmental parameters but control of this process was not complete. Chemical reduction did influence the nanoparticle formation process in some instances, but overall it could be seen that the presence of the protein played a significant role in slowing down the reaction rate, yielding a level of control over the nanoparticles produced and ensuring a more environmentally friendly, biological process for the production of gold nanoparticles.