No abstract

Chemical trapping studies combined with optical and electron paramagnetic resonance measurements were employed to examine the mechanisms of the aqueous photochemistry of methyl-benzoquinone (mBQ) at both low and high quinone concentrations. At low [mBQ], dimethylsulfoxide (DMSO) reacted with a photogenerated intermediate to form a methyl radical, but methane did not, thereby unequivocally excluding the hydroxyl radical. DMSO at concentrations between 50 mM and 2 M completely suppressed the formation of the hydroxylated quinone, while only slowing the formation of the hydroquinone, suggesting reaction with either the triplet state or an intermediate arising from the triplet. Addition of Cl-, a putative physical quencher of the triplet, inhibited the DMSO reaction both noncompetitively and competitively in a fashion similar to that observed previously with nitrite, formate, and salicylic acid, thus providing further evidence for a reactive intermediate distinct from the triplet. This intermediate is attributed to a water-quinone exciplex. The relative yield of the methyl radical from the DMSO reaction decreased with increasing [mBQ], suggesting that at high concentrations, a bimolecular reaction of the triplet with the ground-state quinone outcompetes the formation of the quinone-water exciplex.

D-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for D-psicose production from D-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a D-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using D-psicose as the substrate, the apparent K(m), k(cat), and catalytic efficiency (k(cat)/K(m)) were 27.4 mM, 49 s⁻¹, and 1.78 s⁻¹ mM⁻¹, respectively. Under the optimal conditions, the equilibrium ratio of D-fructose to D-psicose was 69:31. For high production of D-psicose, 216 g/L D-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate D-fructose, indicating that the enzyme was a potential D-psicose producer for industrial production.