OPTIMATION ON MONOCHLORO ACETATE (MCA) DEGRADATION BY HALOACID DEHALOGENASE FROM PSEUDOMONAS AERUGINOSA LOCAL STRAIN IN PET-PAEDD RECOMBINANT CLONE IN E.COLI BL21 (DE3)

• Main Author: DHIA ANDARI NIM: 10513089, AYUNDA
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/21353
• Institution: Institut Teknologi Bandung

Haloacid is an organic compound widely used as a raw materials for pesticides, insectisides, herbisides, and as starting materials for the synthesis of various chemical compounds such as plastic and PVC. However, haloacid is a harmful polutant for the environment because it is persistent in the environment and it is toxic to living things. One way to deal with haloacid polutant is by bioremediation using microorganism capable of producing haloacid dehalogenase, an enzyme that catalyzes the chemical bond disconnection between C atom and halogen. Previous research has succeded in constructing a <br /> <br /> recombinant clone of pET-paed-d in E.coli BL21 (DE3). This recombinant clone contains a gene encoding for haloacid dehalogenase from a local strain of Pseudomonas aeruginosa. This research aims to find the optimum condition to degrade monochloro acetate (MCA) by the recombinant clone pETpaed- <br /> <br /> d in E.coli BL21 (DE3). The activity test is carried out quantitatively using Bergman-Sanik colorimetry method, by measuring the amount of Cl- produced from the degradation of MCA in LB medium. Optimization in vivo was done with the variation of MCA concentration from 0 – 0,5 mM as a substrate, pH of the medium from 3 – 10, incubation temperature at 30°C and 37°C, duration of incubation between 0 – 8 hours, IPTG concentration from 0 – 0,01 mM, induction temperature at 30°C and 37°C, and duration of induction between 0 – 16 hours. On the other hand, optimization in vitro was performed with the variation of MCA concentration from 0 – 0,6 mM as a substrate, pH of the enzymatic reaction from 5 – 11, and the temperature of enzymatic reaction from 0 - 70°C. The optimal degradation <br /> <br /> condition is the condition that produce the highest percentile of MCA degradation. The result of the in <br /> <br /> vivo activity test showed that the optimal condition is reached when the MCA concentration is 0,1 mM, pH of the medium is 7, incubation is performed at 37°C for 4 hours, the IPTG concentration is 0,007 mM, and induction is performed at 30°C for 12 hours. The result of the in vitro activity test showed that the optimal condition is reached when the MCA concentration is 0,55 mM, pH of the enzymatic reaction is 9, and the temperature of the enzymatic reaction is 55°C.