Tris inhibits a GH1 β-glucosidase by a linear mixed inhibition mechanism
Fig 1
Effect of Tris on the Sfβgly activity and stability.
(A) Initial rate of 10 mM NPβglc hydrolysis catalyzed by Sfβgly in the absence (○; 13 μM.min − 1) and presence of 40 mM Tris (●; 7.9 μM.min − 1). (B) Initial rate of 10 mM NPβglc hydrolysis catalyzed by Sfβgly previously incubated with 300 mM Tris at 30 °C for 18 h (●; 15 μM.min − 1) and without Tris (○; 13 μMmin − 1). Both experiments, shown in A and B, were performed with 0.09 μM Sfβgly. Data are mean and standard deviation of three determinations of the product formed in each incubation time using three separate assays with the same enzyme sample. The substrate was prepared in 100 mM phosphate buffer pH 6.0. Activity assays were performed at 30 °C. (C) Tests aiming at the detection of the transglycosylation reaction catalyzed by Sfβgly (0.009 μM). Production of p-nitrophenolate (○) and glucose (*) from 20 mM NPβglc in the presence of 30 mM Tris. The substrate was prepared in 100 mM phosphate buffer pH 6.0. Activity assays were done at 30 °C. Data are mean and standard deviation of three determinations of the product formed in each incubation time using the same enzyme sample. (D) Standard curve of p-nitrophenolate with (○) and without (○) 120 mM Tris. Slopes are 4420 and 4721 Abs415nm. μM-1, respectively. (E) Standard curve of glucose with (○) and without (○) 120 mM Tris. Slopes are 1007 and 935 Abs415nm. μM-1, respectively. R2 are higher than 0.99.