ISOLASI DAN PEMURNIAN SIKLODEKSTRIN-P HASIL PRODUKSI ENZIM SIKLODEKSTRIN GLIKOSILTRANSFERASE REKOMBINAN DARI BACILLUS SP. A2-5A DENGAN SUBSTRAT PATI SAGU MASAK

• Main Author: Mutia Meirani, Mira
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/64318
• Institution: Institut Teknologi Bandung

Cyclodextrin (CD) is a cyclic oligosaccharide with cylindrical shaped, has hydrophobic inner cavity and hydrophilic on surface. In pharmaceutical industry, CD is used to improve the solubility or to cover the unpleasant taste and smell of hydrophobic substances which is fit to CD cavity. CD is a product from enzymatic reaction between cyclodextrin glycosyltransferase (CGTase) and starch as substrate. In general, there are three types of CD produced by enzymatic reaction, i.e. a, [3 and y-CD. 13-CD is used more common in pharmaceuticals than other CDs because of its low cost production and simple preparation. The purpose of this study was to isolate and to purify 13-CD which is produced by recombinant CGTase (rCGTase) from Bacillus sp. A2-5a and to characterize the level of purity and yield of 13-CD. rCGTase was produced from Escherichia coli BL21(DE3) carrying piExpress 401 plasmid which was inserted by cgtase synthetic gene from Bacillus sp. A2-5a. An amount of 0.15 UA rCGTase enzyme was added to 0.5 % cooked sagoo starch at pH 6, 55 °C, for 8 hours to produce 13-CD. The purification of f3-CD was done by two methods. The first method was one-step purification with addition of complexing agent, cyclohexane. The second method was two-step purification, where the first step was 13-CD isolation by crystallization in water and the second step was 13-CD purification with cvclohexane. Identity, purity level and yield of (3-CD were determined by UV-V is spectrophotometry, High Performance Liquid Chromatography (HPLC), and Powder X-ray Diffraction (PXRD). [3-CD had been successfully isolated and purified by both methods. The final yield obtained by the first method was 46.81±17.48 % with level of purity 82.23±1.54 % and the ratio of a-:13-:y-CD was 3:94:3. The yield obtained by the second method was 54.21±3.75 % with level of purity 80.24±4.55 % and the ratio of a-:(3¬:y-CD was 4:90:6 (13-CD isolation step), whereas the final yield was 6.49±1.57 % with level of purity 95.23±5.05 % and the ratio of a-:13-.7-CD was 2:96:2. Characterization by PXRD gave specific peaks for [3-CD at 20= 12.62°; 22.5° and 34.70° and indicated that the p-CD obtained by the both methods was still in the form of uncomplete crystal.