Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Investigation of Alkaline and Thermal Stability of Alpha-L Produced by Directed Evolution(Izmir Institute of Technology, 2013) Sürmeli, Yusuf; Şanlı Mohamed, GülşahAlpha-L-arabinofuranosidase (Abf) is a type of glycoside hydrolase that cleaves the α-L-arabinofuranosidic bonds in the polysaccharides with arabinose. There are many ranges of biotechnological application fields of this enzyme such as pulp and paper industry. The purpose of this study is to identify Geobacillus vulcani GS90 by 16S rRNA analysis and to investigate the alkaline and thermal stability of Abf and its mutants produced by error-prone PCR. During this study, firstly, partial 16S rDNA gene was amplified by using universal primers, cloned and sequenced by Sanger method. The partial 16S rDNA sequence was analyzed by BLAST and phylogenetic tree was constructed. Secondly, abf and its mutants were cloned and 73 mutants were screened for functional analysis in terms of total proteins. After purification of Abf and its functional mutant enzymes, they were analyzed in terms of stability and activity against three different conditions. They were 70oC- pH 5.0, 71oC- pH 5.0 and 70oC- pH 9.6. It was detected that G. vulcani 3S-1 was the closest strain of G. vulcani GS90. In addition, it could be deduced that L307S and Q90H/L307S mutants were more stable than Abf at 71oC- pH 5.0 and less stable at 70oC- pH 9.6. According to SWISS MODEL analysis, the surrounding residues of 90th and 307th amino acid constructed no hydrogen bonds in Abf, two hydrogen bonds in Q90H/L307S and three hydrogen bonds in L307S. Moreover, it was detected that these mutants had both a longer β-strain and more number of β-strains than Abf. Finally, the predicted solvent accessibilities of Abf, Q90H/L307S and L307S were investigated and it was deduced that relocations of R447 and some asparagines could have affected the alkaline stability of them.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.Master Thesis Isolation and Identification of Lipase Producing Soil Fungus; Cloning, Sequencing and Partial Characterization of Its Lipase(Izmir Institute of Technology, 2010) Ergülen, Elvan; Arslanoğlu, AlperLipases are well known enzymes which catalyze the hydrolysis of long chain triglycerides. Contrary to many other enzymes, lipases show a wide range of substrate specificity and remarkable levels of activity and stability in non-aqueous environments. Therefore, they have a great potential in many industrial applications such as detergent industry, paper and food technology, as biocatalysts for the synthesis of organic intermediates. Lipases can be obtained from animals, plants as well as from natural and recombinant microorganisms in good yields. The aim of this thesis was isolation and identification of a lipolytic fungus and purification and characterization of its lipase enzyme. For this purpose, a lipolytic fungus was isolated from soil sample collected from Kula. Lipase activity of this fungus was detected rapidly by Rhodamin B - olive oil plate assay. The lipolytic fungus was identified by 28S rRNA gene sequence analysis and determined to be a strain of Rhizopus stolonifer. Because this lypolytic fungus was isolated from soil sample collected from Kula, it was named as R. stolonifer K45. This fungus showed best growth at 25°C and did not grow above 35°C. In the second part of the study, lipase enzyme of the fungus was partially purified but previously, optimum time and carbon source for lipase production was determined. According to this, optimum lipase production was obtained at 7th day of growth in the media including only olive oil as carbon source. Glucose when included in the growth media was observed to reduce the amount of lipase. In order to purify fungal lipase, aceton precipitation (30 %) and ultrafiltration methods were used. Lipase activity assay was performed spectrophotometrically. The chain length specificity of this lipase was detected and highest activity was observed towards p-NP laurate. The effect of different temperature and pH values on lipase activity and stability was also determined and optimum temperature and pH were found 45°C and pH 8, respectively. Furhermore, different organic solvents and metal ions were tested on lipase activity. The lypolytic enzyme was inhibited by n-hexane. However, methanol and DMSO were detected to enhance the lipase activity.Master Thesis Partial Purification and Characterization of Polyhenol Oxidase From Thermophilic Bacillus Sp.(Izmir Institute of Technology, 2009) Güray, Melda Zeynep; Şanlı Mohamed, GülşahPolyphenol 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.