Master Degree / Yüksek Lisans Tezleri
Permanent URI for this collectionhttps://hdl.handle.net/11147/3008
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Master Thesis Cloning of Novel Sericin Like Proteins and Optimization of Their Expression(Izmir Institute of Technology, 2022) Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of TechnologySericin is a protein that helps fibroin fibers link together to create the cocoon, and it forms silk with fibroin. Fibroin is used in textile production as well as in biomaterial applications. Sericin has essential biocompatibility, biodegradability, cryoprotection, antioxidant properties, and a multi-component structure. Thanks to these properties, its use in biomaterial and biomedical fields is expanding and also used in cosmetic research and wound healing, and drug delivery thanks to its high fiber structure. Sericin can be obtained from silkworm cocoons by degumming method but changes the structure of the obtained protein since the protein is exposed to high temperatures. Sericin produced in different batches is obtained in different quantities and these create inconsistencies in the quality of the biomaterials obtained from the sericin, limiting the use of the sericin as a biomaterial. Besides, obtaining protein by recombinant production provides the advantage that the repetitive chain length can adjust as desired and the protein can be standardized. In this thesis, recombinantly generated and optimized a novel sericin-like protein (Ser-12mer) with the native sericin sequence encoding twelve repeats of recombinantly conserved 38 amino acid motifs in Escherichia coli and characterized its structural properties. In addition, the effects of induction cell density and cell culture media on the expression of the previously produced sericin-like protein (Ser-4mer) was investigated and its expression and concentration were increased by optimization. Recombinant production of a sericin-like protein will provide an understanding of the sequence-structure relationships and significantly expand their applications as biomaterials.Master Thesis Cloning, Expression and Characterization of Sericin Like Octamer-Repeat Protein(01. Izmir Institute of Technology, 2021) Şahin, Alper; Sürmeli Eraltuğ, Nur Başak; 01. Izmir Institute of TechnologySilk proteins are natural polymers which are synthesized by insects like silkworms and spiders. There are two proteins in the silk obtained from the cocoon of B. mori, these are silk fibroin and silk sericin. While the silk fibroin is the main part of the cocoon with a high commercial importance, sericin is the "glue" protein that holds fibroins together. Sericin has wide variety of applications from cosmetics to biomaterial research. Because of its high fibrillar structure, it can be used in wound healing or drug delivery studies. It can be obtained from the degumming process of cocoons which includes chemical treatment like alkali treatment and boiling, or biological treatment like enzymatic digestion. However, sericin obtained by these processes are not consistent in structure, and the sequence of the protein obtained depends on the extraction methodology and conditions. However, proteins obtained by recombinant production can be standardized, and repeat-chain length can be adjusted as desired. In this thesis study, a sericin like protein which includes eight repeats of the 38 amino acid sequence of the natural sericin was cloned and expressed in E. coli. The resulting protein, Ser-8mer, was analyzed in terms of fibril structure and secondary structure. It was found out that the protein has beta sheet conformation in contrast to commercial sericin with random coils. And as a result of this conformation, it forms insoluble self-assembled fibril structures which shows a promising contribution to the biomaterial research.Master Thesis Investigation of Alkaline and Thermal Stability of Alpha-L Produced by Directed Evolution(Izmir Institute of Technology, 2013) Sürmeli, Yusuf; Şanlı Mohamed, Gülşah; Şanlı Mohamed, Gülşah; 04.01. Department of Chemistry; 04. Faculty of Science; 01. Izmir Institute of TechnologyAlpha-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.