Rekombinant mikrobiyal transglutaminaz enziminin Pichia pastoris'te hücre dışı üretimi

While biotechnological developments are advancing rapidly, enzymes are produced and used in the food industry and many other fields for improving production, increasing efficiency and quality. Transglutaminase enzyme is an enzyme that has been used as a food additive in the field of biotechnology for a long time and can be synthesized by many organisms. Pichia pastoris expression system, a methylotrophic yeast, has been successfully used in the production of many recombinant proteins for both research and industrial purposes in recent years and is one of the most effective heterologous protein systems. In this study, extracellular expression of Streptomyces mobaraensis microbial transglutaminase (MTGase) enzyme was performed in P. pastoris under the control of alcohol dehidrogenase (ADH) promoter. For this purpose, firstly the pADH2α-proMTGase expression vector was constructed and transformed into P. pastoris KM71 strain. Protein expression study was performed with transformant Pichia clones selected on zeocin antibiotic agar plates under standard Pichia growth conditions (28°C temperature, pH 6.0) in shake flask, and the study was continued with the protein producing clone which was confirmed by SDS-PAGE. It was determined in the previous study by our laboratory that low temperature is suitable for the production of MTGase protein. Therefore, the production was repeated under three different pHs (3.0, 5.0 and 7.0), while keeping the temperature constant at 20°C. As a result of 96 hours of protein expression in shake flask, the best production was obtained at pH 7.0 and the amount of protein reached was calculated as 396 ng/mL. The enzyme activity was determined as 10200 U/mL for pH 7.0. Within the scope of this thesis, the S. mobaraensis MTGase enzyme was produced recombinantly under the control of ADH promoter in P. pastoris for the first time. It has been shown by the study that the ADH is a suitable promoter for the production of the MTGase enzyme.