d-Allulose is an attractive
noncaloric sugar substitute
with numerous health benefits, which can be biosynthesized by d-allulose 3-epimerases (DAEases). However, enzyme instability
under harsh industrial reaction conditions hampered its practical
applications. Here, we developed a continuous spectrophotometric assay
(CSA) for the efficient analysis of d-allulose in a mixture.
Furthermore, a high-throughput screening strategy for DAEases was
developed using CSA by coupling DAEase with a NADH-dependent ribitol
dehydrogenase, enabling high-throughput screening of DAEase variants
with desired properties. The variant M15S/P40N/S209N exhibited a half-life
of 22 h at 60 °C and an 8.7 °C increase of the T5060 value, with a 1.2-fold increase of activity.
Structural modeling and molecular dynamics simulations indicated that
the improvement of thermostability and activity was due to some new
hydrogen bonds between chains at the dimer interface and between the
residue and the substrate d-fructose. This work offers a
robust tool and theoretical basis for the improvement of DAEases,
which will benefit the enzymatic biosynthesis of d-allulose
and promote its industrial application.