SUBCLONING OF CGTASE ALFA THERMOCOCCUS SP. B1001 SYNTHETIC GENE TO PETL6B AND CHARACTERIZATION OF ITS EXPRESSION PRODUCT IN ESCHERICHIA COLI BL21(DE3)

• Main Author: Azizah Kharisnaeni, Siti
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/66664
• Institution: Institut Teknologi Bandung

CGTase is enzyme that catalyzes cyclodextrins (CDs) from starch. CDs are widely used in pharmacy as solubilizer, stability and bioavailability enhancer, also to reduce unpleasant smells or tastes. With the increasing need for CDs, production of CGTases with better characteristics is needed. In industry, CDs are produced in high temperature, hence thermostable enzymes are needed. Laboratory of Pharmaceutical Biotechnology ITB has successfully generated recombinant alpha CGTase (rCGTase a) which is encoded by alpha cztase synthetic geneof Thermococcus sp. B1001 for production in periplasm of E. coli BL21(DE3). Expression level of that gene in pJexpres 414 expression vector (pJx_cgtaseglia) was very low. The aim of this research is to subclone alpha cgtase synthetic gene into pET16b, hence the expression of the gene will be better. Subcloning was performed for two difference location of expression, periplasm and cytoplasm. The synthetic gene in pJxcgtase_alfa was amplified by PCR separately with each specific primers resulting in 2264 by (periplasm) and 2218 by (cytoplasm) of PCR product. The PCR product was ligated into pGEMT cloning vector and was transformed into E. colt TOP 10. Recombinant plasmids (pGEMTcgt alfasito and pGEMT cgtalla_peri) have been confirmed by analysis of migration, restriction, and PCR. Synthetic genes in recombinant pGEMT were digested with restriction enzymes BamHE and NcoI, and ligated ittto the linear pETl6b expression vector, which has been digested with the same enzyme. Recombinant pETl6b (pET16b cgt alfa sito and pET16b_cgt_alfa_peri) were --_-ansformed into E. colt TOP10 and confirmed by the same method before. Cytoplasmic and periplasmic rCGTase proteins produced in E. coil B1-21 (DE3) with 0.1 mM IPTG induction at 25 °C for 6 hours. The 80 kDa proteins were obtained and have starch hydrolysis activity. Periplasmic rCGTase protein was isolated from 5 triL culture using PeriPreps Peoplasting Kit, then was analyzed by SDS PAGE and was observed its starch hydrolysis activity. Cytoplasmic rCGTase recombinant protein was produced at a scale of 100 mL.The protein mainly was obtained as insoluble inclusion body and the rest is soluble protein. Both inclusion body and soluble rCGTase have starch hydrolysis activity. Expression level of alpha cgtase gen that has subcloned into pETl6b increased 1,8 times (cytoplasm) and 1,1 times (periplasm) compared to its expression level in pJexpress 414.