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 The Rational Design of a Novel Biocatalyst Using the Heme-Nitric Oxide/Oxygen Binding Protein(Izmir Institute of Technology, 2017) Meşe Özçivici, Gülistan; Meşe Özçivici, Gülistan; 04.03. Department of Molecular Biology and Genetics; 04. Faculty of Science; 01. Izmir Institute of TechnologyRecent advances in recombinant DNA technology and protein design have led to the application of biocatalysis as an alternative to chemical catalysis in the synthesis of enantiopure products due to high regio- and enantioselectivity. Hemeproteins are proteins with a heme prosthetic group that play diverse roles in biological systems, making them good candidates for biocatalysis. The Heme-nitric oxide/oxygen binding (H-NOX) protein was identified by homology to the soluble guanylate cyclases. Here, the H-NOX domain from the methyl-accepting chemotaxis protein, Thermoanaerobacter tencogenesis (TtH-NOX), was tuned into a biocatalyst using rational design. Four variants of TtH-NOX were cloned, purified and characterized. Each variant was then tested for their catalase and peroxidase activities. The wild type TtH-NOX inefficiently catalyzed the hydrogen peroxide decomposition (catalase activity) and 2,2’-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) oxidation (peroxidase activity). However, the Y140H mutant exhibited an efficient five-fold increase in catalase and peroxidase activities as compared to the wild type. The other mutants, H102Y, H102C and Y140A TtH-NOX, were not good catalysts for both reactions. Therefore, the mutations resulted in changes in reaction rates and electronic properties of the heme group. The mutations affected the molecular mechanism of the hemeprotein, showing that both the proximal and distal pocket residues are vital for catalysis. However, the mutation of the distal tyrosine to histidine of TtH-NOX has significantly improved its catalytic activities. These observations contribute to the understanding of the physiological roles of hemeproteins. This project could also lead to discovery of novel biocatalysts and aid in the design of future biocatalysts.