AMPLIFIKASI DAN KARAKTERISASI GEN EXOPOLYPHOSPHATASE (ppx) DARI BAKTERI TOLERAN URANIUM

MUSAADAH, Prof. Ir. Triwibowo Yuwono, Ph.D.;Prof. Dr. rer. nat. Agus Taftazani, M.Sc.

2015 | Tesis | S2 Bioteknologi

Penggunaan uranium (U) sebagai bahan bakar alternatif penghasil energi menyebabkan kontaminasi U yang bersifat toksik dan radioaktif. Biomineralisasi U menggunakan fosfat inorganik (Pi) merupakan salah satu metode remediasi kontaminan U yang efektif. Biomineralisasi melibatkan metabolisme polyP yang dikontrol oleh gen ppk dan ppx. Gen ppx mengode enzim exopolyphosphatase yang berperan dalam hidrolisis polyP menjadi Pi bebas. Penelitian ini bertujuan untuk mengisolasi dan mengetahui karakter gen ppx pada mikrobia toleran uranium di Indonesia. Penelitian ini dimulai dengan kegiatan skrining isolat bakteri toleran 0,4mM Uranium yang diisolasi dari limbah radioaktif cair aktivitas rendah Pusat Sains dan Teknologi Akselerator (PSTA) BATAN Yogyakarta secara kualitatif berdasarkan kemampuan tumbuh pada medium bebas-fosfat dan kuantitatif berdasarkan kemampuan akumulasi polyP. Isolat terpilih selanjutnya diidentifikasi secara molekuler dengan metode amplifikasi gen 16S rRNA; diuji kemampuan degradasi polyP, biomineralisasi logam berat Cu2+ dan Cd2+; dan diamplifikasi dan dikarakterisasi gen ppx-nya. Hasil skrining didapatkan isolat potensial polyP yaitu isolat A67I. Berdasarkan hasil analisa gen 16S rRNA isolat A67 I termasuk dalam genus Acinetobacter. Isolat A67I diketahui mampu melepas Pi sebesar 50,70% pada jam ke-10 setelah inkubasi. Isolat A67I terbukti mampu mengurangi konsentrasi logam berat Cu2+ dan Cd2+ sekitar 3,73% untuk Cu2+ dan 2,34% untuk logam Cd2+ setelah 24 jam inkubasi. Gen ppx dari isolat A67I berhasil diisolasi dengan metode PCR menggunakan primer F1-R1 dan F2-R2 berupa single band DNA dengan ukuran ±1500 bp. Penelitian ini berhasil membuktikan bahwa mikroba toleran uranium di Indonesia memiliki gen ppx yang menyandi enzim exopolyphosphatase yang penting dalam proses mendegradasi polyP menjadi Pi bebas.

The use of uranium as an alternative fuel causes radioactive waste. Biomineralisation is one of the bioremediation methods used for removing u contaminant from environment. Biominerallisation is a process of chelating metal using microbial metabolites such as phosphate. polyP metabolism is an essential process for this case. It is known that polyP metabolism is controlled by ppk and ppx genes. The ppx gene encodes an exopolyphosphatase enzyme which plays a key role in polyP hiydrolysis to excess Pi, while ppk gene encodes a polyphosphate kinase enzyme which controlled a polyP synthesis. The aims of this study was to charachterise ppx gene present in from uranium tolerant microbes in Indonesia. The study was conducted by qualitative and quantitative screening of the 0,4 mM uranium-tolerant bacteria isolated from low-level liquid radioactive waste in Pusat Sains dan Teknologi Akselerator (PSTA) BATAN, Yogyakarta. Qualitative screening was based on the isolate ability to grow in the phosphate-free media, while quantitative screening relied on the isolates ability to uptake and accumulate polyP. The polyP metabolism was also analysed by assaying the polyP degradation and metal removal. Finally, the ppx gene was amplified and sequenced. A potential isolate designated as A67I, was identified belongs to Acinetobacter genus. A67 I isolate released about 50.70% Pi after 10 hour of incubation and it removed Cu2+ and Cd2+ ions from the media after 24 hour of incubation. Cu2+ and Cd2+ ions decreased 3.73% and 2.34%, respectively, following the incubation of the isolate in the media. The amplification resulted a single band of DNA of approximately 1.500 bp in length. The amplified ppx gene was found to contain only a partial sequence of exopolyphosphatase gene sequence. However, the blastN analysis showed that it showed similarities with ppx gene of Acinetobacter radioresitens NBRC strain and other species. The result of this study trus suggest that the uranium-tolerant bacterial isolate rarbons ppx gene encoding exopolyphosphatase enzyme.

Kata Kunci : Uranium, polyP, Amplifikasi, Karakterisasi, gen ppx