| International Journal of Current Microbiology and Applied Sciences |
| ISSN: 2319-7706 Volume 2 Number 6 (2013) pp. 70-79 |
| Optimization of Mycobacterium tuberculosis DHFR production from recombinant Saccharomyces cerevisiae |
| Archana Raju, Manisha A. Khedkar and Mariam S. Degani* |
| Department of Pharmaceutical Sciences and Technology, Institute of Chemical Technology, N. P. Marg, Matunga, Mumbai 400019, India *Corresponding author e-mail: msms.degani@ictmumbai.edu.in |
| A B S T R A C T |
| Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate in a NADPH dependent manner. The production of Mycobacterium tuberculosis (Mtb) DHFR by a recombinant strain of Saccharomyces cerevisiae (Y182) shake-flask culture was optimized by identifying the most significant medium components which affect DHFR production (dextrose, yeast extract, peptone) by Response surface methodology (RSM) to determine the optimal concentrations of these components using Design Expert Version 6.0.10. The Mtb DHFR enzyme activity increased from 32.03 ± 0.042 U/l in basal medium to 67.01 ± 0.23 U/l in the optimized medium containing 1% yeast extract, 2.36% peptone and 2.68% dextrose. An enzyme assay was developed to assess the purity of the isolated enzyme by testing for IC50 values against known inhibitors. |
| Keywords |
| MtbDHFR, Recombinant yeast; Response Surface Methodology; Sonication; Enzyme Assay. |