Master Degree / Yüksek Lisans Tezleri

Permanent URI for this collectionhttps://hdl.handle.net/11147/3008

Browse

Filters

2009 - 200922010 - 20111

Settings

Author: search.filters.author.Şanlı Mohamed, GülşahSubject: search.filters.subject.Thermophilic bacteria

Search Results

Now showing 1 - 3 of 3
  • 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 Technology
    The 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 Microencapsulation in Alginate-chitosan/Caci2 Polyelectrolyte Beads
    (Izmir Institute of Technology, 2011) Tercan, Cisem; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of Technology
    In recent years, enzyme immobilization has gained importance for design of artificial organs, drug delivery systems, and several biosensors. Polysaccharide based natural biopolymers used in enzyme or cell immobilization represent a major class of biomaterials which includes agarose, alginate, dextran, and chitosan. Especially, chitosan has used many biomedical applications, including tissue engineering, because of its biodegradability and biocompatibility, non-toxicity and degradation in the body. In this research, Recombinant esterase enzyme was purified from Thermophilic Bacillus sp. That was isolated from Balçova (Agamemnon) Geothermal region in İzmir by using one-step affinity purification chromatography. In the second step, purified enzyme encapsulated in alginate-chitosan/CaCl2 polyelectrolyte beads that were prepared by adding dropwise a protein-containing sodium alginate mixture into a chitosan-CaCl2 crosslinker solution. And then the polyelectrolyte beads were stabilized in at the same crosslinker solution 30 minutes more. In the third step, the effect of different conditions were tested such as temperature and pH, bead diameter, reuse of beads. Also the effects of inhibition of CaCl2, ZnCl2, MgCl2, CuSO4, MgSO4, Sodium dodecyl sulfate (SDS) and Triton X-100 onto the immobilized and free enzyme activity were studied. In the last step, analysis of surface morphologies of polyelectrolyte beads were determined and examined by means of Scanning Electron Microscope.
  • Master Thesis
    Partial Purification and Characterization of Polyhenol Oxidase From Thermophilic Bacillus Sp.
    (Izmir Institute of Technology, 2009) Güray, Melda Zeynep; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of Technology
    Polyphenol oxidases are enzymes that catalyze the oxidation of phenolic compounds using molecular oxygen. The ability of polyphenol oxidases to act on phenolic compounds makes them highly useful biocatalysts for various biotechnological applications. They are commonly found in animals, plants and fungi. Recent genome analysis have shown that polyphenol oxidases are also widespread in bacterial species. In this study, detection, partial purification and characterization of polyphenol oxidase from thermophilic Bacillus sp., which was isolated from a geothermal region was achieved. The samples from bacterial culture were boiled and compared with not boiled ones in order to prove the existence of enzyme in bacterium. The existence was also supported with the appearance of dark bands on polyacrylamide gel after staining with catechol solution. Results of activity staining and activity measurements of samples from intracellular and extracellular extract revealed that the enzyme was intracellular. Partial purification was performed by acetone precipitation and gel filtration chromatography with 35% yield and 1.24 purification fold. Characterization studies indicated that the enzyme showed highest activity at pH 7.0 and 60C, was stable at temperatures between 30 and 60C and more than 80% of activity was retained in the pH range of 5-8. The results of agent and metal ion effect on enzyme activity revealed that the enzyme was totally inhibited in the presence of DTT and sodium diethyldithiocarbamate and highly activated with copper ions whereas other agents or metal ions did not have significant effect on activity. Km and Vmax values for the enzyme were determined as 91mM and 2.25 .abs/min/ml, respectively.