Efficient expression and characterization of D-allulose 3-epimerase from Novibacillus thermophilus

Abstract:

[Background] d-allulose is an excellent sugar substitute with high sweetness and low calories. d-allulose 3-epimerase (DPEase) catalyzes the epimerization of d-fructose to produce d-allulose, being an essential enzyme in the enzymatic production of d-allulose. [Objective] To improve the potential of DPEase for industrial application, we realized heterologous expression of this enzyme and characterized the enzymatic properties. [Methods] A DPEase (NtDPEase) from Novibacillus thermophilus was expressed in Komagataella phaffii under the regulation of the glyceraldehyde-3-phosphatedehydrogenase (GAP) constitutive promoter. The enzymatic properties of the recombinant protein were then characterized. [Results] The transformant was incubated in a 5 L fermenter for high cell density fermentation (108 h), with the highest enzyme activity of 201.3 U/mL. The recombinant enzyme was purified to reach the electrophoretic purity, with a molecular weight of 35 kDa. This enzyme showed the best performance at pH 7.0 and 60 ℃ and good stability within the ranges of pH 6.0-8.0 and temperatures below 45 ℃. Furthermore, the enzyme was used to convert d-fructose with different concentrations (100-500 g/L) to produce d-allulose, which scored the highest conversion rate of 29.0%. [Conclusion] This study achieves the efficient expression of NtDPEase in K. phaffii for the first time, providing a theoretical and practical basis for the enzymatic production of d-allulose.