A d-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for d-psicose production: cloning, expression, purification, and characterization

Abstract

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.