Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Molecular Cloning, Overexpression and Characterization of Thermostable Esterase and Lipase From Thermophilic Bacillus Sp.(Izmir Institute of Technology, 2009) Tekedar, Hasan Cihad; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of TechnologyThe organisms that reside in hot places called thermophiles become very useful tool for biotechnology. The natural consequence of adapting to hot environments for thermophiles is encoding thermostable enzymes which make them a target for scientists.We have aimed to use microorganisms that were previously isolated and characterized as a Bacillus sp. from Balçova Geotermal region in İzmir for their lipase and esterase activity. In order to measure esterase and lipase activity, the strains were incubated in the media that contain the detergent tween 20 and media containing rhodamin-B, respectively. Three strains out of almost 110 bacterial strains have displayed high lipase and esterase activity at the same time. Three different esterase (Est1, Est2, Est3) and two different lipase (Lip1, Lip2) from different environmental samples were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA. The deduced amino acid sequence of the three types of esterase gene exhibited similar amino acid sequence identity with few amino acid differences. However sequenced lipase genes were complicated to explain so that characterization studies have been made for only esterases.For over expression in Escherichia coli, the esterase genes and lipase genes were sub-cloned in pET28a vector with a strong T7 promoter. A one step purification of the recombinant esterases and lipases was achieved using His-Select HF nickel affinity gel.Enzyme assays using variety of p-nitrophenyl (p-NP) esters with different acyl chain lengths (C2-C16) as the substrate have confirmed the esterase activity.All three esterase showed a very high specific activity toward all tested p-NP esters. Optimum pH and temperature, stability in terms of pH and temperature, the effect of several metal ions, inhibitors and detergents on activity were determined for purified Est1, Est2, Est3 separately and compared to each other.Master Thesis Immobilization of Thermophilic Recombinant Esterase Enzyme by Entrapment in Coated Ca-Alginate Beads(Izmir Institute of Technology, 2009) Gülay, Seçkin; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of TechnologyRecently, industrial enzymes produced by micro-organisms are being utilized widely, especially from thermophilic ones due to their ability to withstand intense heat. Esterase enzymes from thermophilic micro-organisms are special interest in a variety of biotechnological applications because of their many useful properties. Due to potential use as biocatalysts in variety of biotechnological applications, esterase enzyme isolated from Balçova (Agamemnon) geothermal site were aimed to be immobilized via a costeffective protocol, in order to be re-used over very long periods of time. Previously, the gene encoding thermophilic esterase from the thermal environmental samples, isolated from Balçova (Agamemnon) geothermal site, was cloned and respective protein was expressed in Escherichia coli in our group. In this study using that recombinant esterase enzyme, expression, purification and immobilization studies were carried out. The esterase enzyme was immobilized in the Ca-alginate beads which were coated with silica and the effects of the temperature and pH on the immobilized enzyme was determined and diameter of the beads, reuse and surface of the beads were analyzed. Immobilization yield for coated beads was determined as 71.27% and compared with non-coated ones which were 45.80%. Analysis of surface morphologies of beads was compared with Scanning Electron Microscope.