Engineering D-allulose 3-epimerase from Clostridium cellulolyticum for improved thermostability using directed evolution facilitated by a nonenzymatic colorimetric screening assay

D-Allulose 3-epimerase (DAEase) is a biocatalyst of concern for D-allulose enzymatic synthesis; however, it exhibits comparatively low thermal stability. To facilitate the directed evolution of DAEase, a nonenzymatic colorimetric assay was developed for high-throughput screening of enhanced DAEase mutants. One desirable mutant, MT4 (S38F/F42N/A70P/T119P), was screened based on purifying selection of the randomly mutagenesis library and positive screening of the randomly shuffled library. Compared to the wild-type, the combinational mutant MT4 had higher catalytic activity (1.23-fold) and showed 5, 6.2, and 6.92 °C increases in Topt, T50, and Tm values, respectively. Furthermore, at 60 °C, the t1/2 value of mutant MT4 was 7.30nullh, exhibiting an 11.4-fold increase in comparison to the wild-type. When the mutant MT4 was used in D-allulose production, the yield reached 28.3%, higher than the 25.1% achieved by the wild-type. Dynamic property and structural change analysis demonstrated that the enhanced properties of mutant MT4 were largely attributed to the β4-α4 loop rigidification and active-site tunnel entrance constriction. This work expands the industrial application value of DAEase and provides a robust method as the basis for further protein engineering to achieve better performance of this enzyme.