0 or above 60

When the refolding experiments were carri

0 or above 6.0.

When the refolding experiments were carried out under acidic conditions (pH range between 2.0 and 6.0), the recombinant Af-Tth showed 4THase activity. The maximum activity was obtained when the refolding was carried out at pH 4.0 (Table 1a). When nitric acid was used instead of sulfuric acid for pH adjustment and 0.4 M ammonium nitrate instead of 0.4 M ammonium sulfate was also used, the activity could be detected after refolding at pH 4.0. Therefore, it was the acidity and not the sulfate from acidification with sulfuric acid that conferred activity on 4THase. Because considerable refolding has been successfully performed in the presence of glycerol, the effects of glycerol find more concentrations were evaluated. Refolding to provide an active protein was performed in the presence of 0–50% glycerol, with the maximum 4THase activity observed with 30% (Table 1b). The effect of 14–60-h incubation periods was also evaluated, but longer dialysis and incubation periods did not have a significant effect on the refolding yield. The effects IDH inhibitor review of the initial protein concentration were also evaluated because

a high initial protein concentration has been reported to lead to aggregation and poor recovery of refolded protein (Singh & Panda, 2005). When inclusion bodies were solubilized in a 6 M guanidine hydrochloride solution containing 10 mM dithiothreitol at a concentration of 0.01 mg mL−1, 95% of the recombinant protein was recovered in the soluble fraction. However,

very low specific activity (2.8 U mg−1) was detected at that concentration. About 90% of the recombinant protein in the soluble fraction may not be successfully refolded Sorafenib price in spite of its being in soluble form. On the other hand, when inclusion bodies were solubilized in the buffer at concentrations of 0.05–0.5 mg mL−1, 25–45% of the recombinant protein was recovered in the soluble fraction. At a concentration of >1.0 mg mL−1, almost all proteins aggregated and the yield of the refolded protein was <10%. The highest yield of soluble 4THase, with a specific activity of 19.8 U mg−1, was obtained when the refolding was performed at the high initial protein concentration of 0.5 mg mL−1. The primary structure of Af-Tth showed a similarity to PQQ-dependent enzymes such as PQQ-dependent dehydrogenases. Recently, the 4THase (Ac-TetH) from Acidithiobacillus caldus, which is an acidophile and obtains energy for growth from the oxidation of reduced inorganic sulfur compounds, has been suggested to contain quinoid compounds as a cofactor (Rzhepishevska et al., 2007). Refolding experiments in the presence and absence of 70 μM PQQ revealed no significant effect on the activation of the enzyme activity. We further attempted to detect quinoid compounds in the refolded enzyme (the specific activity was 20 U mg−1) by NBT-glycinate staining.

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