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.

ABSTRACT PRODUCTION OF TANNASE BY ASPERGILLUS NIGER &Ouml / zt&uuml / rk, Alev Deniz M.S., Department of Chemical Engineering Supervisor: Prof. Dr. Ufuk Bakir Co-Supervisor: Prof. Dr. B. Z&uuml / mr&uuml / t &Ouml / gel August 2006, 90 pages In this study, a filamentous fungus, Aspergillus niger was evaluated in terms of extracellular tannase production. The effect of tannic acid, glucose and nitrogen sources on tannase and biomass productions was investigated and their concentrations were optimized. The highest enzyme activity was recorded as 316 U/ml in the optimized medium containing 8% Tannic acid, 1% Glucose, 0.4% (NH4)2HPO4, 0.1% K2HPO4, 0.1% MgSO4.7H2O, 0.01% ZnSO4.7H2O, 0.0005% NaCl in a shake-flask bioreactor at 35oC and 175 rpm. The bioreaction profile including tannic acid, gallic acid, pyrogallol, glucose concentrations, pH, biomass and extracellular tannase production were determined under the optimized conditions. The maximum extracellular tannase activity (316 U/ml) was observed on the 4th day of cultivation. However, biomass continued to increase up to the 9th day of fermentation. Increase in biomass concentration during the first two days and after the 7th day was high. The microorganism used tannic acid and glucose during the first two days by considering the sharp decrease in tannic acid and glucose concentrations. The increase in biomass concentration after the 7th day was directly proportional to the decrease in pyrogallol concentration in this period of time. The pH of the cultivation medium decreased from 5.5 to 2.3 owing to the assimilation of glucose and the production of gallic acid. Keywords: Tannase, Aspergillus niger, Enzyme production, Cultivation profile, Tannic acid.

In this study, the novel idea of in-vivo directed evolution was applied in order to achieve variants of the enzyme galactose oxidase with increased activity. This procedure was done under starvation conditions in Escherichia coli BL21 Star (DE3). Previous studies have been carried out in order to improve the activity of this enzyme using directed evolution methods. In this study, the same idea was used in-vivo, during stationary phase adaptive mutations inside the host organism, hence called in-vivo directed evolution. This method gave variants with improved enzyme activity as compared with the wild-type enzyme, and some variants showed activities that were even higher than the variants of previous directed evolution studies, hence making this method a promising approach for the random mutagenesis of genes of interest. The above mentioned mutations are carried out by a special group of polymerases, the error-prone polymerases. Phylogenetic analysis of these error-prone polymerases was also carried out in order to investigate the relationship between the number of error-prone polymerases and the level of complexity of organisms, and both the number of error-prone polymerases and the ratio of error-prone polymerases to total DNA polymerases of six organisms were studied. It was found that as the organism gets more complex, the number of error-prone polymerases and their ratio to the total polymerases increase.