Path: TopS1-Final ProjectChemistry-FMIPA2017

Optimasi Degradasi Asam Monokloroasetat (MCA) oleh Haloacid Dehalogenase dari Bacillus cereus Strain Lokal dalam Klon Rekombinan pET-bcfd1 dengan Sel Inang E. coli BL21(DE3)

Optimation of Monochloroacetic Acid (MCA) Degradation by Haloacid Dehalogenase from Bacillus cereus Local Strain in pET-bcfd1 Recombinant Clone in E. coli BL21(DE3)

Undergraduate Theses from JBPTITBPP / 2017-09-27 11:42:40
Oleh : Rachmad Ade Pratama , S1 - Department of Chemistry
Dibuat : 2017-09-20, dengan 1 file

Keyword : Kata kunci : MCA, Bacillus cereus, haloacid dehalogenase, klon rekombinan pET- bcfd1.

Asam monokloroasetat (MCA) adalah senyawa organik penting dalam industri. Senyawa ini diperoleh sebagai hasil klorinasi asam asetat yang secara luas digunakan sebagai bahan baku dalam pembuatan selulosa karboksimetil (CMC), herbisida asam 2,4diklorofenoksiasetat (2,4-D) dan asam 2-metil-4-klorofenoksiasetat (MCPA), penghilang cat dan grafiti, plastik, dan senyawa produk lain dalam bentuk ester atau amidanya. Namun, senyawa ini menyebabkan polusi terhadap lingkungan air dan kesehatan manusia karena sifatnya yang toksik, korosif, persisten, dan karsinogen. Senyawa ini banyak ditemukan sebagai polutan dalam air minum dan air limbah. Biodegradasi merupakan salah satu cara untuk mengurangi MCA dengan memanfaatkan organisme penghasil haloacid dehalogenase. Penelitian sebelumnya telah berhasil mengisolasi gen haloacid dehalogenase dari Bacillus cereus strain lokal dalam klon rekombinan pET-bcfd1 dengan sel inang E. coli BL21(DE3). Penelitian ini mempelajari kondisi optimum degradasi MCA oleh sel hidup E. coli BL21(DE3) yang membawa plasmid rekombinan pET-bcfd1 dalam medium LB yang mengandung MCA. Kadar Cl- yang dilepaskan ke medium sebagai hasil reaksi enzimatis haloacid dehalogenase terhadap substrat MCA dipelajari secara spektrofotometri menggunakan metode Bergmann dan Sanik. Pengujian degradasi MCA oleh sel hidup klon rekombinan dilakukan pada rentang konsentrasi MCA 0-1 mM, suhu inkubasi 30 oC dan 37 oC, waktu inkubasi sebelum penambahan IPTG selama 0-12 jam, penambahan konsentrasi IPTG 0-1 mM, waktu induksi selama 1-8 jam, dan suhu induksi 30 oC dan 37 oC pada pH medium 6-10. Hasil uji kuantitatif terhadap ion Cl- yang dilepaskan ke medium menunjukkan bahwa kondisi optimum degradasi ini dicapai pada 0,2 mM MCA, inkubasi 37 oC selama 6 jam, konsentrasi IPTG 0,01 mM, induksi 30 oC selama 2 jam, dan pH medium 7. Pengujian degradasi MCA secara reaksi enzimatis menggunakan ekstrak kasar haloacid dehalogenase dipelajari pada rentang konsentrasi MCA 0-1 mM, suhu reaksi 25 oC-57 oC, dan pH bufer 6-11. Reaksi dilakukan selama 10 menit. Hasil uji menunjukkan bahwa kondisi optimum degradasi ini dicapai pada 0,1 mM MCA, suhu reaksi 37 oC, dan pH bufer 10.

Deskripsi Alternatif :

Monochloroacetic acids (MCAs) are organic compounds industrially important. These compounds are obtained as the chlorination products of acetic acid and have been widely used as building blocks in the production of carboxymethyl cellulose (CMC), 2,4dichlorophenoxyacetic acid (2,4-D) and 2-methyl-4-chlorophenoxyacetic acid (MCPA) herbicide, paint and graffiti removal, plastic, and other forms as its ester or amide. However, these compounds cause water environmental pollution and harm to human health as a result of their toxicity, corrosive, persistence, and carsinogenic effects. These compounds are commonly found as pollutants in drinking water and wastewater. Biodegradation is one way to reduce the pollution caused by these compounds, utilizing an enzyme called haloacid dehalogenase produced by the microorganisms. In the previous research, the haloacid dehalogenase gene from Bacillus cereus local strain has been successfully isolated in E. coli BL21(DE) pET-bcfd1 recombinant clone. This research aims to optimize the MCA degradation by this living E. coli BL21(DE3) carrying pET-bcfd1 recombinant clone in LB medium containing MCA. Liberation of chloride ion in medium as a result of enzimatic reaction catalyzed by haloacid dehalogenase to MCA as the substrate was followed spectrophotometrically as described by Bergmann and Sanik. The MCA degradation perform by the recombinant clone living cells is performed in the ranges of 0-1 mM of MCA concentrations, 30 oC and 37 oC of incubation temperatures, 0-12 hours of incubation periods before adding IPTG, 0-1 mM of IPTG concentrations, 1-8 hours of induction periods, and 30 oC and 37 oC of induction temperatures in medium pHs 6-10. The results indicated that the optimum MCA degradation is achieved in 0,2 mM MCA concentration, incubation at 37 oC for 6 hours, 0,01 mM IPTG, induction at 30 oC for 2 hours, and in medium of pH 7. On the other hand, the enzimatic reaction using haloacid dehalogenase crude enzyme is conducted for 10 minutes in the ranges of MCA concentrations of 0-1 mM, reaction temperatures of 25 oC-57 oC, and bufer pHs of 6-11 suggests that in MCA concentration of 0,1 mM, reaction temperature of 37 oC, and buffer of pH 10 provides optimum condition of MCA degradation.

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  • Enny Ratnaningsih, MS, PhD, Editor: Irwan Sofiyan

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