CONSTRUCTION OF DNA ENCODING CYCLODEXTRIN GLUCANOTRANSFERASE FROM LOCAL BACILLUS ISOLATE INTO EXPRESSION VECTOR FOR EXPRESSION IN Escherichia coli AND CHARACTERIZATION OF ITS EXPRESSION PRODUCT
Cyclodextrin glucanitransferase (CGTase, EC. 2.4.1.19) is an enzyme catalyzes cyclodextrin (CD) formation from starch. Due to its unique form, CD is widely used in pharmaceutical formulation. Bacillus sp. LT1B and PT2B are local CGTase-producing bacteria isolated and characterized in Pharmaceutic...
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| Format: | Theses |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/44456 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Cyclodextrin glucanitransferase (CGTase, EC. 2.4.1.19) is an enzyme catalyzes
cyclodextrin (CD) formation from starch. Due to its unique form, CD is widely
used in pharmaceutical formulation. Bacillus sp. LT1B and PT2B are local
CGTase-producing bacteria isolated and characterized in Pharmaceutical
Biotechnology Laboratory ITB. Enzimatic characteristics of CGTase from both
isolates are highy potential for production of CD at industrial scale. The objective
of this research was to clone DNA encoding CGTase of both isolates into
expression vector pET-16b_rec and to characterize its expression product.
Cloning method used in this research was QuickStep Cloning. Correction of
coding sequence error was done by SDM-SPRINP PCR. The result was
confirmed by sequencing analysis of recombinant plamid. Optimization of
overproduction condition includes amount of IPTG, time and temperature of
induction. Produced rCGTase was analyzed by SDS-PAGE for determining its
molecule weight and zymograph for detecting its activity. In cloning step, singlestranded megaprimers (MP) was produced using For_QS and MR (MP I) also
Rev_QS and MF (MP II) primer pairs. Optimized consentration ratio of forward
and reverse primer to produce MP I and MP II were 25:1 (50 °C) and 20:1 (53 °C)
respectively. Annealing temperature of megaprimer recombination into vector was
52 °C. Optimized condition of induction temperature and time for rCGTase
overproduction was 37 °C for 3 hours using 0.1 mM final concentration of IPTG.
rCGTase was expressed as inclusion body (IB) and solubilized using 8M urea 1.5
mL/110 mg IB. rCGTase was refolded and purified simultaneously in affinity
nickel column. Experimental size of rCGTase LT1B and PT2B was 78.2 kDa and
77.5 kDa respectively. Zymograph result showed rCGTase positively possessing
starch hydrolysis and ?-cyclization activity after refolding process.
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