Characterization of a Recombinant d-Allulose 3-epimerase from Thermoclostridium caenicola with Potential Application in d-Allulose Production

In recent years, with the increasing public health awareness, low-calorie rare sugars have received more attention on a global scale. d-Allulose, the C-3 epimer of d-fructose, is a representative rare sugar. It displays high sweetness and excellent physiological functions, but only provides a caloric value of 0.4 kcal/g. d-Allulose 3-epimerase (DAEase) is indispensable in d-allulose production. In this study, a putative DAEase from Thermoclostridium caenicola was identified and characterized. The novel T. caenicola DAEase displayed maximum activity at pH 7.5 and 65 degrees C in the presence of 1 mM Co2+. The half-life (t(1/2)) at 50 degrees C was 13.6 h, and the melting temperature (T-m) was 62.4 degrees C. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t(1/2) value at 55 degrees C and 4.8 degrees C increase in T-m. Furthermore, DAEase displayed high relative activity (89.0%) at a weakly acidic pH 6.5 and produced 139.8 g/L d-allulose from 500 g/L d-fructose, achieving a conversion ratio of 28.0%. These findings suggest that T. caenicola DAEase is a promising biocatalyst for the production of d-allulose.