Temperature (A) and pH (B) optima and stability (C and D) of the purified GHF43 R_09-02 protein.

<p>The parameters were determined using <i>p</i>NPβX as the substrate. (<b>A</b>) For the optimum temperature determination, the pH was adjusted to 6.0 (sodium acetate 20 mM). (<b>B</b>) The optimum pH was determined in the range of pH 4.0–9.0 at 34°C. The buffers (100 mM) used were as follows: acetate (pH 4.0–6.0), MES (pH 6.0–7.0), HEPES (pH 7.0–8.0) and Tris-HCl (pH 8.0–9.0). In both cases, the <i>k</i>_cat value was determined using an [E] ranging from 0 to 12 nM and a substrate concentration of 70 mM. Activity at 100% refers to 230.3±13.7 s⁻¹ at pH 6.0 and 34°C. (<b>C</b>) The time lost normalised quantification of the R_09-02 activity levels (with <i>p</i>NPβX) at 34°C and pH 6.0 (sodium acetate 20 mM) is shown. Protein (1.5 μg) was incubated, and the activity was determined as described in the <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038134#s2" target="_blank">Methods</a></b>. (<b>D</b>) The effect of chemical reagents and metal ions on the hydrolase activity (<i>p</i>NPβX). The concentrations of the various chemicals ranged from 2 mM (black) and 5 mM (light grey) to 10 mM (dark grey), and the relative activities were defined using the activity ratio without the added chemicals. The optimal pH (6.0) and temperature (34°C) were used in the assays. All of the measurements were analysed in triplicate, and error bars are indicated. The error bars represent the standard deviation of three replicates from a single protein preparation.