Recombinant production of aspergillus niger a-amylase enyzme in pichia pastoris

Amylase enzyme catalyzes the breakdown of starch. a-amylase enzymes act on the α-1,4 glycosidic bond. The use of amylases is quite common in areas such as baking and fruit juice production in the food industry, detergent, textile and paper industries. Recombinant production of enzymes, which have so many uses in the industrial field, has been a subject of much study in recent years. Pichia pastoris (P. pastoris) is a methylotrophic yeast and has been successfully used in recombinant protein production as an expression system. The advantages of P. pastoris include having inducible promoters, to make post-translational modifications required for eukaryotic proteins, to grow in very cheap growth media, and to reach very high cell densities. In this study, the production of Aspergillus niger (A. niger) a-amylase enzyme was carried out for the first time in P. pastoris under the control of the ethanol-inducible SNT5 promoter. For this purpose, the A. niger a-amylase gene, which was provided in a codon-optimized form, was transformed into the P. pastoris MK115-PDI strain. Protein expression was performed with transformant Pichia cells selected from plates containing the antibiotic zeocin in shake flasks at 28 ˚C temperature, pH 6.0 and 225 rpm. As a result of the SDS-PAGE analysis, optimum protein production conditions were determined with the clone that was found to produce the 58kDa a-amylase enzyme. For this purpose, protein was produced in shake flasks under different temperature (20, 24 and 28 ˚C) and different pH (3, 4, 5, 6 and 7) conditions and the samples taken were analyzed. In the productions carried out at 24 ˚C temperature, pH 6 and pH 7, enzyme activity of 3349 U/mL and 4519 U/mL was obtained, respectively. a-amylase enzyme production was carried out with a two-stage fermentation strategy in a 5L bioreactor. Fermentation was continued for 107 hours by keeping the temperature at 24 ˚C, pH 6 and dissolved oxygen level at 20%. At the end of fermentation, 2223 mg/L protein was produced and 44062 U/mL enzyme activity was calculated. The produced enzyme was characterized and the optimum working temperature was determined as 60 ˚C and pH 7. As a result of this thesis study, A. niger a-amylase enzyme was produced in P. pastoris MK115-PDI strain under the control of the ethanol-inducible SNT5 promoter and data was obtained for its industrial production.