Transglutaminases (EC 2.3.2.13) are enzymes that catalyze an acyl
transfer reaction between a &gamma / -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 pPICZ&alpha / -A expression vectors,
separately. Both intracellular (pPICZ&alpha / A::pro-mtgintra) and extracellular
(pPICZ&alpha / A::pro-mtgextra) constructs were prepared with strong alcohol oxidase
1 promoter which is induced by methanol. Pichia pastoris X33 cells were
transfected by linear pPICZ&alpha / A::pro-mtgintra and pPICZ&alpha / A::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 pPICZ&alpha / A::promtgextra
gene and pPICZ&alpha / A::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.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12614761/index.pdf |
| Date | 01 September 2012 |
| Creators | Gunduz, Burcu |
| Contributors | Calik, Pinar |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | M.S. Thesis |
| Format | text/pdf |
| Rights | Access forbidden for 1 year |
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