Carbonic anhydrase from Nicotiana tabacum leaves

In this study, carbonic anhydrase (CA: carbonate hydrolyase; E.C.4.2.1.1) was purified from adult Nicotiana tabacum leaves and studied biochemically. The enzyme was purified twice times by using $(NH_4)_2SO_4$ precipation and DEAE-cellulose column chromatography, and its activity was determined for two different substrates ($CO_2$ and p-nitrophenyl acetate). The enzyme obtained from the ion exchange column was purified 40.7 fold and the purity was controlled by 3%-10% discontinuous SDS-PAGE. The pH of the purified enzyme varied between 6.0 and 7.2, the optimum being 6.9. $V_{max}$ and $K_m$ values were calculated with p-nitrophenyl acetate (0.1524 mM, 0.5446 mM, respectively) as substrate. The optimum temperature for the enzyme was $40^circ C$. The molecular weight of the enzyme and of the subunits were found to be approximately $approx$ 137.000 daltons and $approx$ 22.800 daltons, respectively. The results indicate that 6 subunits are present. Changes in enzyme activity were determined in the presence of caffeine, nicotine, metal ions and some chemicals.

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