Analisis fungsi enzimatik protein katg rekombinan dengan mutasi baru pada mycobacterium tuberculosis yang resisten terhadap isoniazid = Analysis of enzymatic function of recombinant katg proteins harboring new mutations derived from izoniazid resistance mycobacterium tuberculosis
Yuli Setianingsih;
Herawati Aru Sudoyo, supervisor; R. Tedjo Sasmono, supervisor; Asmarinah, supervisor; Andi Yasmono, supervisor; Ahmad Hudoyo, examiner
([Publisher not identified]
, 2013)
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ABSTRAK Tuberkulosis (TB) masih menjadi masalah kesehatan utama di Indonesia. Salah satupenyebab tingginya kasus TB disebabkan adanya resistensi Mycobacteriumtuberculosis (Mtb) terhadap obat anti tuberkulosis. Isoniazid (INH) yang merupakansalah satu obat lini pertama dalam pengobatan TB adalah pro-drug yang akan diubahmenjadi bentuk aktifnya melalui aktivitas protein KatG Mtb. Mutasi pada gen katGyang mengkode protein KatG kemungkinan mempengaruhi aktivitas katalase danperoksidase protein sehingga menyebabkan resistensi Mtb terhadap INH. Dalamstudi ini, dilakukan kontruksi plasmid rekombinan protein KatG tipe liar dan proteinKatG dengan mutasi baru pada residu N330D dan H400Y, serta mutasi yang umumdijumpai pada residu S315T dan S315N. Ekspresi protein KatG rekombinandilakukan menggunakan host E. coli. Over ekspresi kelima protein rekombinan KatGterjadi setelah induksi IPTG. Purifikasi protein KatG rekombinan dilakukanberdasarkan prinsip kromatografi afinitas menggunakan Nikel sepharose. Setelahpurifikasi diperoleh protein KatG yang murni. Aktivitas katalase dan peroksidaseprotein rekombinan KatG diukur pada berbagai konsentrasi substrat yang diperlukandalam pengukuran efisiensi katalitik kedua aktivitas protein KatG. Hasilnyamenunjukkan bahwa mutan protein KatG memiliki efisiensi katalitik yang lebihrendah dari protein KatG tipe liar. Penurunan efisiensi katalitik aktivitas katalasemutan N330D dan H400Y sebesar 31% dan 37% dan untuk aktifitas peroksidasesebesar 39% dan 3% dibandingkan KatG tipe liar. Struktur 3 dimensi protein KatGdari mutan tersebut dibuat menggunakan perangkat Modeller dan divisualisasikanmenggunakan perangkat Pymol. Tidak terdapat adanya perubahan konformasi 3dimensi protein KatG mutan dibandingkan dengan struktur 3 dimensi protein KatGtipe liar. Namun, letak residu N330D dan H400Y yang berada dekat daerah aktifikatan INH pada protein KatG kemungkinan berpengaruh terhadap penurunanaktivitas enzimatik protein KatG. ABSTRACT Tuberculosis (TB) is currently a major health problem in Indonesia. One of the manycauses of the high incident of TB is due to the resistance of the Mycobacteriumtuberculosis (Mtb) to anti-TB drugs. Isoniazid (INH), one of the first line anti-TBdrugs for TB treatment, is a pro-drug that is converted to its active form through theactivity of Mtb KatG protein. Mutations in the katG gene encoding KatG may affectthe catalytic efficiency of the catalase and peroxidase activities of the protein thateventually confers resistance to INH. In this study, recombinant plasmids containingkatG gene that have new mutations on residue N330D dan H400Y, wild type, as wellas mutant proteins with common mutations at residue S315T and S315N wereconstructed. Expression of recombinant KatG was performed using E. coli as anexpression host. Over expression of recombinant KatG was facilitated by IPTGinduction. Purification of recombinant KatG was performed using affinitychromatography employing Nickel sepharose. Pure recombinant proteins wereobtained, and the catalase and peroxidase activities of the recombinant KatG proteinwere measured at various concentration of substrates. Result showed that mutantKatGs have a lower catalytic efficiency for both catalase and peroxidase activitiesthan the wild type protein. Decreasing catalytic efficiency for catalase of mutantsN330D and H400Y were 31% and 37% than that of wild type KatG, while catalyticefficiency for peroxidase of mutants N330D and H400Y were 39% and 3% lowerthan that of wild type. Three dimensional structures of mutant KatGs were generatedusing Modeller and visualized using PyMol softwares. The three dimensionalstructural of mutant KatG showed no conformational change compared with that ofwild type KatG. However, the location of residues N330D and H400Y which are inthe close proximity to the active site of KatG for INH binding is likely to have aneffect on the decreased enzymatic activities of mutant KatG proteins. |
 T-Pdf Tri Yuli Setianingsih.pdf :: Unduh |
| No. Panggil : | T-Pdf |
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| Penerbitan : | [Place of publication not identified]: [Publisher not identified], 2013 |
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| Bahasa : | ind |
| Sumber Pengatalogan : | LibUI ind rda |
| Tipe Konten : | text |
| Tipe Media : | unmediated ; computer |
| Tipe Carrier : | volume ; online resource |
| Deskripsi Fisik : | xviii, 134 pages : illustration ; 28 cm + appendix |
| Naskah Ringkas : | |
| Lembaga Pemilik : | Universitas Indonesia |
| Lokasi : | Perpustakaan UI, Lantai 3 |
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