PRODUCTION AND CHARACTERIZATION OF RECOMBINANT ?-AMYLASE SACCHAROMYCOPSIS FIBULIGERA R64 EXPRESSED IN PICHIA PASTORIS

• Main Author: Sanawati, Anna
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35799
• Institution: Institut Teknologi Bandung

?-Amylase is an endoacting enzyme which hydrolyses ?-1,4 glycosidic linkages of the polysaccharides into soluble maltooligosaccaride, maltodextrin, and small quantity of glucose. ?-Amylase has been used in various industries, such as starch processing, textile, bakery, and pharmacy. A commercial production of ?-amylase in industry requires an efficient cell factory capable of producing ?-amylase at very high level. Pichia pastoris expression system offers several advantages, such as high secretion efficiency, high cell density and inexpensive culture media, which will result in the cost effective ?-amylase production. This research describes heterologous expression of Saccharomycopsis fibuligera R64 ?-amylase gene (ALP1) in P. pastoris, including determination of optimal production condition and influence of mutation to ALP1 stability. ALP1 and alp1 gene had been cloned into pPICZ?A and P. pastoris KM71H had been transformed by the resulted recombinant plasmid. ALP1 and alp1 fused with AOX1 promoter, which can induced by methanol. The ?- amylase activity was monitored after 24, 48, 72, 96, 120, and 144 hours of growth in the media containing 0.5% (v/v) of methanol and it was found that the optimal induction time was 96 hours. ?-Amylase secretion increased dramatically with activity of 8.2 x 105 U/ml upon the addition of methanol to a final concentration of 4% (v/v) for 96 hours. Characterization of ALP1 and alp1 showed that pH and temperature optimal for ALP1 and alp1 are 5.5 and 50ºC.