Cloning of the glucoamylase encoding gene found in aspergillus niger genome

Glucoamylase (GA) (1,4-α-glucosidase) is an exoamylase enzyme that is involved in the hydrolysis step of all processes in which glucose is needed. Glucoamylase which is a very important industrial enzyme produces D-glucose by hydrolyzing glucosidic linkages of starch, glycogen, and oligosaccharides from their non-reducing ends. The main reason that makes GA of such importance to the industry is its role in the starch process. Starch is one of the major components of the food, textile, laundry, pharmaceutics, and paper industries. In nature, there is two forms of glucoamylase, GAI which is commonly used in industry because of its capacity of high starch hydrolyzing and GAII. Most of the bacteria, yeasts, and fungi can produce glucoamylase in various forms, but especially the fungal glucoamylase is most often used in the industry. This study was aimed that produce recombinant glucoamylase enzyme in Pichia pastoris. In this way, an industrially important enzyme, glucoamylase, could be produced as recombinant on laboratory scale from the more economical and easier-breeding yeast strain. For this purpose plasmid that containing the glaA gene encoding the glucoamylase enzyme in GAI form found in Aspergillus niger genome was designed and transferred to Pichia pastoris. The molecular cloning of the glaA gene was confirmed by sequence analysis and transfection was verified by colony PCR and determination of enzyme activity. In addition to that, the activities of the resultant recombinant enzyme under different conditions were investigated.