Expression of Haloacid Dehalogenase Gene from Pseudomonas aeruginosa in pET-paed-d Recombinant Clone with Escherichia coli ArcticExpress Host Cells (DE3)

• Main Author: Hairuddin, Muhammad
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/41973
• Institution: Institut Teknologi Bandung

Organohalogen is one of organic compounds that widely used in various industrial fields as active ingedients in pesticides, plastics, precursors, and organic reaction solvents. However, organohalogen compounds are xenobiotic that are toxic, difficult to degrade, and can be transformed into harmful metabolites that pollute the environment. One way to degrade these compounds, is bioremediation using microorganisms with dehalogenase activity. One of microorganisms that have dehalogenase activity is Pseudomonas aeruginosa. Pseudomonas aeruginosa has haloacid dehalogenase gene that have been cloned, named paed-d , and have been expressed using pET-30a vector in E. coli BL21 (DE3). Previous research stated that the activity of the enzyme produced in E. coli BL21 (DE3) was still not optimum due to improper protein folding. This study aims to study the expression of paed-d genes and the activity of enzymes produced by recombinant pET-paed-d clones in E. coli ArcticExpress (DE3). E. coli ArcticExpress (DE3) has 2 chaperons, namely Cpn 10 and Cpn 60 which are expected to help the process of folding protein so that it can increase the activity of the haloacid dehalogenase enzyme produced. One unit of dehalogenase activity is expressed as the number of enzymes needed to release I f.U110l halide ion per minute. The haloacid dehalogenase activity was determined using the colorimetric method developed by Bergmann and Sanik. The specific activity of the enzyme was determined by measuring the protein content in the sample using the Bradford method. Expression of paed-d gene in E. coli BL21 (DE3) host cell was carried out under optimum conditions with MCA concentration of 0.1 mM, medium pH 9, incubation 37 o C for 4 hours, IPTG concentration 0.007 mM, induction at 30 ° C for 6 hours, whereas expression in E. coli ArcticExpress (DE3) host cell was carried out under the same conditions, only the induction temperature was carried out at 15 ° C. The results obtained indicate that the specific acti v ity of haloacid dehalogenase from E. coli ArcticExpress (DE3) for addition of 0.1 mM MC A and I mM is 1.5 times hi gher than haloacid dehalogenase from E. coli BL21 ( 0£3 ). Th e s pec ific acti v i ty of h a l oa cid deh a l ogenase f ro m E. coli A rctic Ex press ( D E3) for th e additi o n of 0.1 mM and I mM MCA were 0.471 U I m g protein and 0.478 U I mg protein respectively, while the specific activity of haloacid dehalognase from E. coli BL21 (DE3) for addi t ion of 0.1 mM and I mM MCA, each is 0.334 U I m g protein and 0.344 U I mg protein.