SOLUBILISASI BADAN INKLUSI DAN PELIPATAN ULANG ENZIM CGTASE REKOMBINAN DARI BACILLUS SP. A2-5A HASIL OVERPRODUKSI DI ESCHERICIA CALL BL21(DE3) SERTA UJI AKTIVITASNYA

• Main Author: Ahmad Ramdani, Bayu
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/64315
• Institution: Institut Teknologi Bandung

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) is an enzyme which has an activity to convert starch into cyclic oligosaccharide molecule, named cyclodextrin (CD). CD is widely use in pharmacy, food, and cosmetic industries as a multifunctional excipients. The previous research in Pharmaceutical Biotechlonogy Laboratorium ITB, recombinant CGTase (rCGTase) was produced in two forms, soluble protein and inclusion bodies (IBs). This research was focused on solubilizing the rCGTase IBs from Bacillus sp. A2-5a which was overproduced in Eschericia coli BL21(DE3), then refolded the solubilized protein into an active form. IBs was produced from recombinant E. coli BL21(DE3) with the induction of 0.1 mM IPTG at 37 °C for 3 hours. IBs was washed in 1% of Triton X-100 and solubilized in 8 M urea solution. rCGTase was purified and refolded using Ni-NTA resin. Refolding done using gradual urea method (8; 6; 4; 2; 1; 0,5M) and was done by ascending imidazole concentration. The IBs and refolded protein then was characterized by Sodium Dodecyl Sulphate Polyacrilamide Gel Electrophoresis (SDS-PAGE). rCGTase was succesfully solubilized and refolded in this research. The refolded protein showed starch hydrolisis and 13-cyclication activity in zymografi assay. The recovery for the purified rCGTase was 2.77% w/w from the total rCGTase IBs.