Figure 3

PG deacetylase activity of purified Rv1096 protein. A) Acetic acid released by the Rv1096 protein over-expressed in E. coli and M. smegmatis. PG (1 mg/ml) from wild-type M. smegmatis was used as a substrate and mixed with different concentrations of purified Rv1096 (1.22, 2.88, 3.65 or 4.74 μg/ml). After incubation at 37°C for 30 min, acetyl group release was detected using an acetic acid kit. Squares indicate Rv1096 protein over-expressed in E. coli and triangles indicate Rv1096 protein over-expressed in M. smegmatis. Values are means ± SD. B) Time course and concentration curve for Rv1096. Purified Rv1096 protein at 1.22, 2.88 or 3.65 μg/ml was incubated with M. smegmatis PG (1 mg/ml) substrate at 37°C for 5, 10, 15, 30 and 50 min. Plotted values are means ± SD. C) K_m and V_max values for Rv1096 PG deacetylase activity. Kinetic parameters were calculated by a double reciprocal plot. D) Rv1096 protein exhibited a metallo dependent enzymatic activity. Various divalent cations (Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺or Zn²⁺) were added to a final concentration of 0.5 μM. Values are mean ± SD.