Nick closing activity of <i>w</i>Bm-LigA and biochemical characterization.

<p>(A) Effect of enzyme substrate ratio on nick closing activity. Incubation of an equimolar ratio of enzyme and substrate caused rapid product formation and quick saturation (close squares). Decreasing the enzyme/substrate ratio to 0.04 or 0.02 produced slower product formation (open squares and triangles res.). (B) Effect of varying concentration of NAD⁺ cofactor. Increasing concentrations of NAD⁺ showed a rise in <i>w</i>Bm-LigA activity which plateaued at about 10 µM. (C) Effect of temperature on enzymatic activity ranging from 0°C –50°C. Enzyme showed maximum activity at 25°C. (D) Nick closing activity at pH ranging between 6.5 and 9.0 with highest activity at 8.0. (E) Effect of divalent cations on enzyme activity. Enzyme was active in presence of 10 mM MgCl₂, MnCl₂ and CaCl₂. However, MnCl₂ and CaCl₂ showed less product formation as compared to the MgCl₂. Enzyme showed no activity in presence of NiCl₂ and CoCl_2. (F) Effect of different concentrations of monovalent cations including NaCl, KCl and NH₄Cl. Maximal activity achieved at 5 mM NaCl which was completely inhibited at 100 mM. In presence of KCl, maximum activity was obtained at 25 mM showing a sharp decline thereafter. Activity increases with increasing concentration of NH₄Cl up to 10 mM, beyond this the enzymatic activity declined. (G) Effect of varying concentrations of EDTA on the ligation activity of <i>w</i>Bm-LigA. Error bars represent standard errors calculated from two independent experiments.