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, GülşahThe 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 Isolation and Molecular Characterization of Extracellular Lipase and Pectinase Producing Bacteria From Olive Oil Mills(Izmir Institute of Technology, 2004) Altan, Asena; Yenidünya, A.FazilLipases and pectinases are industrially important enzymes. These enzymes are produced by a variety of microorganisms. However there are few studies on the production of these enzymes by thermoacidophilic Bacillus species. The aim of this research was the isolation of extracellular lipase and pectinase producing thermoacidophilic Bacillus from olive oil mills and their identification by phenotypic tests, 16S-ITS rDNA RFLP and DNA sequencing. Eighty-six thermoacidophilic strains were isolated from olive, olive husk and soil contaminated with alpechin collected within different olive oil mills in Ayvalık. The strains were screened for the presence of 5 extracellular enzyme activities. These were lipase, pectinase, amylase, xylanase and cellulase. In total, 69 lipase (Tween 20 as subtrate), 32 pectinase and 68 amylase activities were detected. None of the isolates were able to produce xylanase or cellulase enzyme. All of the isolates were Gram(+) endospore forming rods, thus they were identified as Bacillus sp. Taq I was used for 16S-ITS rDNA based RFLP. The isolated strains were clustered into four groups by Taq I restriction profiles of 16S-ITS rDNA. One representative isolate among the members of each of the 16S-RFLP homology groups was chosen and used for 16S rRNA gene partial sequence analysis. Sequencing results were submitted to GenBank. So far the indicated accession numbers were obtained: AY601903 (isolate H 22 of G-3, 679 nucleotides), AY606276 (isolate S1 of G1, 330 nucleotides)