Functional characterization of d-allulose 3-epimerase and high-throughput screening of its highly active mutants

Abstract:

[Background] The rare sugar d-allulose is an ideal low-calorie sweetener substitute with broad application prospects in food processing and medical health. d-allulose 3-epimerase (DAEase) is the key enzyme that is responsible for the isomerization of d-fructose to d-allulose.[Objective] To mine new enzyme genes from the enzyme database to broaden the library resources of DAEase and obtain new enzyme molecules with excellent catalytic performance by rational design and screening.[Methods] A novel DAEase from Microbacterium oxydans (MoDAEase) was expressed in Escherichia coli, and the enzymatic properties of MoDAEase were characterized. The molecular structure modeling and rational design were performed, and the potential mutant amino acids were screened by a high-throughput screening platform.[Results] The recombinant target protein was expressed in a soluble form, with the optimal reaction conditions of 60 ℃ and pH 8.0, and it remained stable at 40–60 ℃ and pH 6.5–8.5. The mutant M110S/P249G with 5.35-fold enhanced catalytic efficiency was identified and it exhibited excellent catalytic performance when being used to convert 500 g/L and 750 g/L d-fructose.[Conclusion] We mined a novel DAEase from M. oxydans and obtained its mutant with significantly improved catalytic activity, which provides both theoretical and practical bases for the enzymatic synthesis of d-allulose.