CONSTRUCTION AND ENZYMATIC CHARACTERIZATION OF RECOMBINANT CYCLODEXTRIN GLYCOSYLTRANSFERASE A2-5a MUTEIN

• Main Author: Kurniawan, Siendy
• Format: Theses
• Language: Indonesia
• Subjects: Farmakologi dan terapeutik
• Online Access: https://digilib.itb.ac.id/gdl/view/45737
• Institution: Institut Teknologi Bandung

Cyclodextrin glycosyltransferase (CGTase; EC 2.4.1.19) is an enzyme that produces ?-,?- and ?- cyclodextrins (CDs) from starch. Ratio of each CD is varied, depending on the type of CGTase. Several studies on CGTase showed that residues of the 145-152 loop, that formed the -7 subsite in CGTase, are essential in the formation of large CD. In this study, the recombinant CGTase A2-5a was mutated at sequence encoding residues 144-152 to produce rCGTase mutein (A144V/L145D/ ?(146-151)/A152I) which then was characterized for its enzymatics profile. The study was started by construction of the mutant cgtase using PCR based site-directed mutagenesis. Changing of the codons was performed by using mutant primers that was designed in this study. Confirmation of the mutant of cgtase was done by using migration and sequencing analyses. Mutant cgtase was transformed into Escherichia coli BL21(DE3) for overproduction of rCGTase mutein by induction of 0.1 mM IPTG for 6 hours at 26 C. The mutein was purified by Ni-NTA resin. Enzyme characterization such as product specificity, starch hydrolysis, ?-cyclization activity, optimum temperature, optimum pH, and enzyme kinetics were compared with those of native enzyme. Mutant cgtase has been successfully constructed using PCR based site-directed mutagenesis and was confirmed using migration and sequencing analyses. Native and mutein rCGTases have been oveproduced in LB medium and were purified by using affinity chromatography with Ni-NTA column as a 76.39 kDa protein in size. Product specificity, starch hydrolysis and ?-cyclization activity of rCGTase mutein showed no difference when compared to native. Optimum temperature and pH to produce ?-CD were also not changed. The kinetics parameters of rCGTase mutein slightly changed compared to native in producing CD-?using preheated soluble starch. In this research, mutant cgtase has been successfully constructed and rCGTase mutein has been characterized where the mutation does not affect all parameters except for kinetics of the enzyme.