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 °C in the presence of 1 mM Co2+. The half-life (t1/2) at 50 °C was 13.6 h, and the melting temperature (Tm) was 62.4 °C. It was strictly metal-dependent, and the addition of Co2+ remarkably enhanced its thermostability, with a 5.4-fold increase in t1/2 value at 55 °C and 4.8 °C increase in Tm. 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.
Keywords: D-Allulose; D-Allulose 3-epimerase; Thermoclostridium caenicola; Weakly acidic pH.