Purifikasi dan Karakterisasi Parsial Acetyl-CoA Acetyltransferase (PhaA) Rekombinan

IKE FEBRIYANTI, M. Saifur Rohman, M.Si., M.Eng., Ph.D.; Prof. Ir. Triwibowo Yuwono, Ph.D.

2022 | Skripsi | S1 MIKROBIOLOGI PERTANIAN

Acetyl-CoA acetyltransferase (PhaA) merupakan enzim yang mengkatalisis pembentukan acetoacetyl-CoA dari monomer acetyl-CoA. Enzim ini memiliki peran penting dalam jalur biosintesis polihidroksibutirat (PHB). Sebelumnya, ORF dari gen phaA telah berhasil dikloningkan pada vektor pET28a(+) dan diekspresikan dalam Escherichia coli BL21(DE3), namun kelarutan dari PhaA rekombinan masih belum diketahui. Penelitian ini bertujuan untuk menguji kelarutan dan mempurifikasi PhaA rekombinan yang diekspresikan dalam Escherichia coli. Escherichia coli BL21(DE3) yang membawa pET-phaA dikultivasikan dalam media Luria Bertani dengan 50 µg/mL antibiotik kanamisin. Pada hari berikutnya, kultur sel diinokulasikan ke dalam media Luria Bertani baru dan dikultivasi pada temperatur 37°C hingga OD600 mencapai 0,5. Sel kemudian diinduksi dengan 0,5 mM IPTG dan kultivasi dilanjutkan pada temperatur 15°C selama satu malam. Sel dipanen dengan sentrifugasi. Pelet sel diresuspensi dengan 10 mM larutan penyangga Tris-HCl pH 8,8. Sel kemudian dipecah dengan sonikasi. Hasil sonikasi lalu disentrifugasi untuk memisahkan pelet dan supernatan. Pelet dan supernatan dianalisis untuk menguji kelarutan dari PhaA rekombinan. PhaA rekombinan kemudian dipurifikasi dengan kromatografi penukar anion. Uji kelarutan menunjukkan bahwa sebagian besar PhaA rekombinan berada dalam bentuk yang larut (94,46%). PhaA rekombinan berhasil dipurifikasi dengan kromatografi penukar anion.

Acetyl-CoA acetyltransferase (PhaA) is an enzyme that catalyse the formation of acetoacetyl-CoA from acetyl-CoA monomer. This enzyme plays important role in the pathway of polyhydroxybutirate (PHB) biosynthesis. Previously, the open reading frame of phaA gene has been successfully cloned into pET28a(+) and expressed in Escherichia coli BL21(DE3). The solubility of the recombinant PhaA, however, has not been known yet. Therefore, the objectives of this work were to examine the solubility and purify the recombinant PhaA expressed in E. coli system. The E. coli BL21(DE3) harbouring pET-phaA was cultivated on LB media supplemented with 50 g/ml kanamycin. In the following day the cell culture was transferred to new LB media and cultivated at 37°C until reaching the OD600 of 0,5. The culture was then induced by 0.5 mM IPTG and then cultivation was continued at 15°C for overnight. Cells was harvested by centrifugation followed by resuspension of cell pellets in 10 mM Tris-HCl buffer pH 8,8. The cells were disrupted by sonication and clarified by centrifugation to separate the pellet and supernatant. The separated pellet and supernatant were analysed to determine the recombinant PhaA solubility. The recombinant protein was then purified by anion exchange chromatography. The solubility test indicated that the majority of recombinant PhaA was present in soluble form (94.46%). In addition, the recombinant PhaA was successfully purified by anion exchange chromatography.

Kata Kunci : PhaA, ekspresi, kelarutan, purifikasi