Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
Browse
3 results
Search Results
Now showing 1 - 3 of 3
Master Thesis Investigation of Heat Stress-Induced Proteins of Cold-Adapted Pseudomonas Marginals Using Proteomic Approach(Izmir Institute of Technology, 2008) Taşoğlu, Çağdaş; Yalçın, TalatTemperature alteration is known as a common environmental stress condition which all living organisms encounter and response by producing evolutionary wellconserved specific proteins called heat stress or heat shock proteins in the cell in order to adapt and survive. In the current study, the induction of heat stress proteins in a coldadapted bacterial strain of Pseudomonas marginalis cells grown under heat stress was investigated by proteomic approach. Five different temperatures, 5, 10, 15, 24, and 30C, were examined for the purpose of determining the optimum growth temperature for the bacterium. Consequently, 15°C was observed as optimum temperature for growth while 30C was established as heat stress temperature. Total proteins from Pseudomonas marginalis cells in the late exponential phase of growth at these two temperatures were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. Totally 1391 protein spots were visualized for 15C and 1384 protein spots for 30C. After comparing with 15C, 13 protein spots that were differentially expressed in the cells exposed to heat stress (30C) were cut from the gel and fragmented into their peptides by in-gel digestion method. Finally, these proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searching. Among them, ribosome recycling factor, universal stress protein family and chaperonin GroEL were established as direct sensors of heat stress. As a result, the genes encoding these two heat stress proteins can be isolated and cloned into any other useful microorganism such as bacteria used for detoxification of industrial waste or used in bioremediation but not capable of surviving at high temperatures so that they can be efficient at those temperatures, too.Master Thesis Cloning and Expression of the Pseudomonas Ke38 Extra-Cellular Lipase Gene in E. Coli(Izmir Institute of Technology, 2013) Karakaş, Fulya; Arslanoğlu, AlperLipases are serine hydrolases that catalyze both the hydrolysis and synthesis of insoluble or poorly soluble long-chain triacylglycerols with an acyl chain length ≥ 10 carbon atoms based on the presence or absence of water. Lipases are produced and secreted by all kingdoms of life that are eukaryotes including plants, animals, fungi and prokaryotes including bacteria and archaea. However, microbial lipases, especially from bacteria, more useful than their plant and animal derivatives because of several important properties. Because of their acitivities in both aqueous and nonaqueous environments, lipases have specific applications in industry and medicine. The primary goals of this thesis were to clone and express the extra-cellular lipase gene from Pseudomonas sp. KE38, isolated from soil samples of Erciyes mountain in Kayseri, in E. coli and partial purification of the gene product. To achieve this aim, genome walking technique was used to obtain lipase gene from Pseudomonas sp. KE38, that gene was then cloned into pET28a expression vector and expressed in E. coli. The lipase expression of E. coli BL21 and its activity was screened with olive oil-Rhodamin B plate assay. After expression recombinant lipase was partially purified via inclusion body isolation. Moreover the optimum lipase production time of E. coli BL21 cells were determined and analyzed with SDS-PAGE. According to SDS-PAGE analysis the recombinant lipase was approximately 64 kDa and lipase production reached to the highest level after two hours of IPTG induction. As conclusion, recombinant lipase from Pseudomonas sp. KE38 was cloned into E. coli, expressed and partially purified.Master Thesis Partieal Purification and Characterization of Lipase Enzyme From a Pseudomonas Strain(Izmir Institute of Technology, 2008) Yapaşan, Ece; Yalçın, TalatLipase is a triacylglycerol-hydrolyzing enzyme which is catalyzed the hydrolysis of water insoluble free fatty acid and glycerols and also a wide range of chemical reactions. Beside, microbial lipases show regiospecificity and enantioselectivity properties. Therefore, microbial lipases gain the great importance for industrial applications and organic synthesis. In this study, investigation, partial purification and characterization of lipase enzyme from a Pseudomonas strain was studied by using different analytical approach.Purification step was done by size-exclusion chromatography. The molecularweight of partial purified lipase was determined by SDS-PAGE. Spectrophotometric lipase assay applied to find out the enzyme characterization. Kinetic study of enzyme was also investigated varying the substrates concentrations. Specific activity staining on gel procedures applied after native gel process. After electrophoresis, lipase activity responsive protein bands were appeared on gel.After screening for the presence of lipase activity in Pseudonomas strain which was isolated from soil, it was decided to choose intracellular enzyme sample for characterization and purification studies. The enzyme gave the highest lipase activity when p-nitrophenyl laurate used as a substrate. The optimum pH range for activity of lipase was alkaline pH ranges, about pH 8.0 and 9.0. The optimum temperature was dedicated as 25oC. In the presence of metal salts and organic solvents; while some additives sharply decreased enzyme activity, some additives were not effect the enzyme activity. Approximate molecular mass of partially purified enzyme was between 29 kDa and 43 kDa.