OPTIMATION OF HALOACID DEHALOGENASE PRODUCTION FROM KLEBSIELLA PNEUMONIAE ITB1 BY RECOMBINANT CLONE OF PET- HAKP1 IN ESCHERICHIA COLI BL21 (DE3) HOST CELL USING RESPONSE SURFACE METHOD (RSM)

• Main Author: Irish Sukandar, Sarah
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/47662
• Institution: Institut Teknologi Bandung

Organohalogens are naturally existing compounds which also synthesized by people for various needs. One example of organohalogens is monochloroacetic acid (MCA). MCA is widely used in chemical industries as intermediate compounds, such as in the production of carboxy methyl cellulose (CMC) as emulsion agent, as a raw material for the production herbicides, plastics, surfactants, shampoos, liquid soaps, and many more. However, monochloroacetic acid is a pollutant because this compound is toxic, persistent, corrosive, and carcinogen. One effort to decrease the effect of MCA pollutant is by performing bioremediation that taking advantage of microorganisms. Klebsiella pneumoniae is one of bacteria that able to degrade MCA. However, this bacterium is pathogen. Previous research has been successfully cloned the haloacid dehalogenase gene from K. pneumoniae into pGEM-T vector and subcloned it into pET-30a(+) expression vector to produce pET-hakp1 recombinant clone in Escherichia coli BL21 (DE) host cell. This research’s aim to find an optimum condition in the production of haloacid dehalogenase from this recombinant clone using Response Surface Methodology (RSM). Independent variables chosen are incubation temperature after induction, incubation time after induction, and inducer isopropyl ?-d-1- thiogalactopyranoside (IPTG) concentration. Haloacid dehalogenase activity is determined by measuring the amount of chloride ions released as the product of MCA degradation by colorimetry of Bergmann-Sanik method. The amount of produced protein in the mixture is determined using Bradford method. The optimum condition for haloacid dehalogenase production in E. coli BL21 (DE3) pET-hakp1 is observed at 37°C induction temperature, 4 hours induction time, and 1,8 M IPTG inducer with maximum enzyme specific activity of 57,04 U/mg. One unit of dehalogenase activity is defined as enzymes amounts needed to produce 1 ?M free chloride ion per minute