Master Degree / Yüksek Lisans Tezleri

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

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Access Right: Open AccessSubject: search.filters.subject.Thermophilic bacteria

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Now showing 1 - 4 of 4
  • 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
    Genotypic Characterization of Extracellular Enzyme Producing Thermophilic Bacteria in Balçova Geothermal Region
    (Izmir Institute of Technology, 2003) Yavuz, Elif; Yenidünya, Ali Fazıl; Yavuz, Elif; Yenidünya, Ali Fazıl; 04.03. Department of Molecular Biology and Genetics; 01. Izmir Institute of Technology; 04. Faculty of Science
    Thermophiles are the organisms which are adapted to live at high temperatures. The enzymes from thermophiles find a number of commercial applications because of their thermostability and thermoactivity. Therefore, the isolation of thermophilic bacteria from natural sources and their identification are very important in terms of discovering new industrial enzymes.In keeping with this view, Balçova Geothermal Region could serve as a good source for new thermophilic microorganisms with novel industrially important properties.The aim of this research was therefore the isolation of industrially important extracellular enzyme producing thermophilic bacteria from Balçova Geothermal Region and their identification by genetical means. 16S-ITS rDNA RFLP, plasmid profiling and pulsed field gel electrophoresis studies were performed for this purpose.112 thermophilic strains were isolated from various environmental samples collected within Balçova Geothermal Region. These strains were screened for the existence of 6 extracellular enzyme activities. These were, lipases, amylases, proteases, xylanases, cellulases and pectinases. In total, 110 lipase (tween 20 as substrate), 106 amylase, 55 protease, 28 xylanase, 10 cellulase and 3 pectinase activities were detected.Some other phenotypic tests were also performed for these isolated strains. Since all the isolated strains were Gram (+), endospore forming rods, they were identified as Bacillus sp.16S-ITS rDNA RFLP and plasmid RFLP profiles were produced by using two restriction endonucleases Taq I and Hae III . The isolated strains were clustered into eleven groups by Taq I restriction profiles of 16S-ITS rDNA while nine groups were obtained by Hae III digestion profiles. When these groups were compared, it was concluded that 17 genotypically different strains existed in total 112 isolates. Two of the isolated strains yielded similar RFLP profiles to those of Bacillus stearothermophilus (CECT 43) reference strain.Plasmid profiling was also performed. It was found that 23 of the isolated strains contained plasmid DNA. Hae III restriction profiles indicated the existence of three different types of plasmids.PFGE optimization studies by Sma I restriction endonuclease for thermophilic Bacilli were also performed. A new method for preparation of agarose plugs was developed.
  • 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.