EXPRESSION OF HALOACID DEHALOGENASE GENE FROM BACILLUS CEREUS IN PET-BCFD1 RECOMBINANT CLONE IN ESCHERICHIA COLI ARCTICEXPRESS (DE3) HOST

• Main Author: PUTERI PUSPASERUNI (NIM:10513055), NADYA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29502
• Institution: Institut Teknologi Bandung

Organohalogen is one of the xenobiotic compounds that has toxic, hard to degrade, could transformed into harmful metabolite, and became pollutant to our environtment. Bioremediation of organohalogen could be done using microorganism that produce haloacid dehalogenase, e.g Bacillus cereus. Bacillus cereus has haloacid dehalogenase gene that have been cloned, named bcfd1, and had been expressed in pET-30a using E.coli BL21 (DE3). Previous research showed that expression of haloacid dehalogenase in pET-bcfd1 using E.coli BL21 (DE3) produced enzym that quite optimum because of disfolded protein. This research was delivered to study the expression of halaocid dehalogenase from Bacillus cereus in pET-bcfd1 recombinant clone in Escherichia coli ArcticExpress (DE3). E.coli ArcticExpress known has chaperons that could help making a good protein folding so it could have better activities of haloacid dehalogenase. Recombinant plamsid of pET-bcfd1 was isolated using alkaline lysis from E.coli TOP10, confirmed by restriction gene analysis, and inserted into E.coli ArcticExpress (DE3) using heat shock methode. Transformant was being selected using gentamicin and kanamicin on LB médium and confirmed by size analysis between pET-30a and pET-bcfd1 and also Re-PCR. Activties of haloacid dehalogenase was measured using Bergmann-Sanik methode, which was calculating amounts of Cl- that has been released from catalytic reactions. Specified activities of haloacid dehalogenase was determined using Bradford methode, which was calculating amounts of protein on sample. Expression in E.coli ArcticExpress (DE3) was induced with 0.01 mM IPTG at 15°C for 2 and 24 hours, also expression of halaocid dehalogenase in E.coli BL21 (DE3) was induced with 0.01 mM IPTG at 30°C for 2 and 24 hours. Specified activities of haloacid dehalogenase from E.coli ArcticExpress (DE3) after being induced for 2 and 24 hours each was 0.162 U/μg proteins and 0.039 U/μg proteins. On the other hand, specified activities of haloacid dehalogenase from E.coli BL21 (DE3) after being induced for 2 and 24 hours each was 0.052 U/μg proteins and 0.047 U/μg proteins. So, it could be concluded that activities of haloacid dehalogenase that was produced from E.coli ArcticExpress (DE3) after being induced for 2 and 24 hours were 3 times higher than expressed in E.coli BL21 (DE3). Induction throughout 24 hours gave the same specified activites on both expression in E.coli BL21 (DE3) and E.coli ArcticExpress (DE3).