Date

2012

Author

Gündüz, Burcu

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Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl transfer reaction between a γ-carboxyamide group of a peptide bound glutaminyl residue (acyl donor) and a variety of primary amines (acyl acceptors), including the amino group lysine. Transglutaminase has a potential in obtaining proteins with novel properties, improving nutritional quality of foods with the addition of essential amino acids, preparing heat stable gels, developing rheological properties and mechanical strength of foods and reducing the applications of food additives. The aim of this study is to develop intracellular and extracellular microbial protransglutaminase (pro-MTG) producing recombinant Pichia pastoris strains by using genetic engineering techniques. In this context first,protransglutaminase gene (pro-mtg) from Streptomyces mobaraensis was amplified by PCR both for intracellular and extracellular constructs using proper primers then they were cloned into the pPICZα-A expression vectors, separately. Both intracellular (pPICZαA::pro-mtgintra) and extracellular (pPICZαA::pro-mtgextra) constructs were prepared with strong alcohol oxidase 1 promoter which is induced by methanol. Pichia pastoris X33 cells were transfected by linear pPICZαA::pro-mtgintra and pPICZαA::pro-mtgextra, separately and plasmids were integrated into the Pichia pastoris X33 genome at AOX1 locus. After constructing the recombinant P. pastoris strains, batch shaker bioreactor experiments were performed for each recombinant cell and the best producing strains were selected according to Dot blot and SDS-PAGE analyses. The selected recombinant P. pastoris strains, carrying pPICZαA::promtgextra gene and pPICZαA::pro-mtgintra gene in their genome were named as E8 and I1, respectively. Afterwards, a controlled pilot scale bioreactor experiment in a working volume of 1 L was performed with E8 clone and produced pro-MTG was activated by Dispase I. The variations in the recombinant MTG activity, cell concentration, total protease activity, AOX activity and organic acid concentrations throughout the bioprocess were analyzed and specific growth rates, specific consumption rates and yield coefficients were calculated regarding to measured data. Maximum MTG activity was obtained as 4448 U L- 1 and the maximum cell concentration was measured as 74.1 g L-1 at t=36 h of the bioprocess. In this study, an active transglutaminase enzyme was produced extracellularly by P. pastoris for the first time and the third highest extracellular MTG activity was achieved with E8 clone.

Subject Keywords

Pichia pastoris., Transglutaminases., Recombinant proteins.

URI

http://etd.lib.metu.edu.tr/upload/12614761/index.pdf
https://hdl.handle.net/11511/21927

Collections

Graduate School of Natural and Applied Sciences, Thesis