SYNTHETIC GENE CGTASE BACILLUS SP. A2-5A: OPTIMIZATION OVEREXPRESSION IN ESCHERICHIA COLI, PURIFICATION AND ENZYMATIC CHARACTERIZATION OF ITS GENE PRODUCT

• Main Author: Sanna Yustiantara, Putu
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/80669
• Institution: Institut Teknologi Bandung

Cyclodextrin (CD) is a cyclic oligosaccharide molecule composed of 6, 7 and 8 sugar units referred to as ?, ?, and ?cyclodextrins, respectively. CDs can be produced through enzymatic reaction catalyzed by CD glycosyltransferase (CGTase) via ?-1,4 bond. Increased demand in industrial level of cyclodextrin causes production of recombinant CGTase. The construction of a synthetic gene encoding CGTase derived from Bacillus sp. A2-5a had been done previously to generate recombinant CGTase (rCGTase) in Escherichia coli. In current research, rCGTase expressed from synthetic gene had been successfully optimized for its production using two host cells, various IPTG concentrations as inducer and post-induction incubation temperatures. The highest yield of rCGTase was obtained in Escherichia coli host BL21 (DE3) with induction of 0.1 mM IPTG. Increase amount of soluble rCGTase was obtained by lowering incubation temperature at 25 °C. The rCGTase protein was 76 kDa in size and has been successfully purified using nickel affinity column using imidazole gradient concentration to separate it from protein impurities. The protein was shown to be active for ?-cyclization and starch hydrolysis using zymography method. The ?- cyclization activity using pregelatinazation of substrate was two times higher than that without pregelatinazation, the optimal temperature of the reaction was at 55 °C and T50 of the enzyme was 62 °C. Sagoo was the substrate that gave the highest activity. Cyclization ratio for ?, ?, and ?cyclodextrins was 5: 80 : 15, respectively. Value of Vmax, Km and Kcat were 19.23 mg ?-CD/mg CGTase/min, 7.347 mg/ml; 1105.17/sec, respectively and the efficiency (Kcat/Km) was 150.42.