Kinetic Analysis of Catalase on Recombinant Catalase-Peroxidase from the Clinical Isolate of Mycobacterium tuberculosis L26
Tuberculosis (TB) is a deadly infection disease caused by a bacterium called Mycobacterium tuberculosis (M. tuberculosis). Recently, TB control has become more difficult due to the emergence of multidrug-resistant tuberculosis (MDR-TB). The MDR-TB is defined as a strain which is resistant to at leas...
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| Format: | Final Project |
| Language: | Indonesia |
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| Online Access: | https://digilib.itb.ac.id/gdl/view/32250 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Tuberculosis (TB) is a deadly infection disease caused by a bacterium called Mycobacterium tuberculosis (M. tuberculosis). Recently, TB control has become more difficult due to the emergence of multidrug-resistant tuberculosis (MDR-TB). The MDR-TB is defined as a strain which is resistant to at least two of the first-line TB drugs, isoniazid (INH) and rifampicin (RIF). Resistant M. tuberculosis to INH is generally caused by point mutations in the katG gene encoding catalase-peroxidase (KatG). However, which amino-acid residues of KatG affects the resistant M. tuberculosis, needs further investigations. This research was aimed to determined kinetic parameters of catalase on four types of KatG, KatG wildtype (KatGwt), KatG from clinical isolate L26 (KatGL26) and two of mutants KatG (Trp397Arg and Leu415Pro). All variants of KatG were expressed in Escherichia coli BL21(DE3) and were purified by an affinity chromatography using a Nickel-Nitrilotriacetic Acid (Ni-NTA) matrix. The catalase activity and its kinetic parameters of KatG was determined by degradation of H2O2 at a wavelength, ? 240 nm. Expression and purification of four KatGs had successfully performed with a result of a predominant band at ~80 kDa on SDS-PAGE gel, corresponding to the size of KatG. Compared to KatGwt, KatGL26 and two of KatG mutants (Trp397Arg and Leu415Pro) lost its catalase activity by 92.48, 69.88 and 80.29%, respectively. The kcat/Km value of catalase of KatGwt, KatGL26 and mutant KatG Trp397Arg were 2.53 ? 104, 3.07 ? 105 and 1.55 ? 104 M–1 s–1, respectively. Structural analysis on modelled KatG mutants using PyMol showed that conformation of the cross-link structure in the active site of KatGL26 and two of mutants KatG (Trp397Arg and Leu415Pro) had disrupted. Taken together, the kinetic and modelling structure analysis suggest that Trp397 and Leu415 are important residues for the catalase activity. |
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