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.

Bioconversion of d-glucose to d-psicose with immobilized d-xylose isomerase and d-psicose 3-epimerase on Saccharomyces cerevisiae spores

“…ST-24, Agrobacterium tumefaciens, Rhodobacter sphaeroides SK011, Clostridium cellulolyticum H10, Clostridium scindens 35704, Clostridium bolteae, Treponema primitia ZAS-1, Ruminococcus sp., Mesorhizobium loti, Desmospora sp, and Clostridium sp. have been characterized and employed for d-psicose synthesis [9,11,12,20,21,32,33,[36][37][38][39][40]. DTEases from A. tumefaciens, C. cellulolyticum H10, C. scindens 35704, C. bolteae, T. primitia, Ruminococcus sp., Desmospora sp, and Clostridium sp.…”

Production of d-psicose from d-fructose by whole recombinant cells with high-level expression of d-psicose 3-epimerase from Agrobacterium tumefaciens

“…The maximum activity of double-site variant DPEase from A. tumefaciens has previously been reported at pH 8.0 and 50 C (23). The reaction of whole recombinant cells expressing DPEase from C. bolteae (16) or C. cellulolyticum (17) has been performed at pH 6.5 and 55 C or at pH 8.0 and 55 C, respectively. The halflives of whole recombinant cells expressing the double-site variant DPEase from A. tumefaciens at 45, 50, 55, 60, and 65 C using the same concentration of cells were 4,812, 2,310, 468, 150, and 42 min, respectively (Fig.…”

“…D-Psicose has mainly been produced from D-fructose via the reactions of enzymes, including D-tagatose 3-epimerases (DTEases) from Pseudomonas cichorii (12) and Rhodobacter sphaeroides (13) and D-psicose 3-epimerases (DPEases) from Agrobacterium tumefaciens (14), Ruminococcus sp. (15), Clostridium bolteae (16), Clostridium cellulolyticum (17), Clostridium scindens (18), Clostridium sp. (19), and Desmospora sp.…”

“…D-Psicose production by whole cell reactions has been reported for only two recombinant Escherichia coli cells. DPEases from C. bolteae (16) and C. cellulolyticum (17) are expressed in these cells. However, the productivity of D-psicose by these cells is low for industrial applications.…”

Advances in the enzymatic production of l-hexoses

“…8437 (Zhang et al 2013c), Clostridium sp. BNL1100 (Mu et al 2013), Clostridium bolteae (Jia et al 2014), Dorea sp. CAG317 , and Treponema primitia (Zhang et al 2016).…”