Optimization of Ultrahigh-Throughput Screening Assay for Protein Engineering of d-Allulose 3-Epimerase

d-Allulose is the corresponding epimer of d-fructose at the C-3 position, which exhibits a similar taste and sweetness to sucrose. As a low-calorie sweetener, d-allulose has broad application prospects in the fields of medicine, food, and so on. Currently, the production method of d-allulose is mainly the enzymatic conversion of d-fructose by d-allulose 3-epimerase (DAEase). However, the limited specific activity and thermal stability of DAEase restrict its industrial application. Herein, an ultrahigh-throughput screening assay based on the transcription factor PsiR was extensively optimized from the aspects of culture medium components, screening plasmid, and expression host, which enhanced the correction between the fluorescent readout and the enzyme activity. Then, the error-prone PCR (epPCR) library of Clostridium cellulolyticum H10 DAEase (CcDAEase) was screened through the above optimized method, and the variant I228V with improved specific activity and thermal stability was obtained. Moreover, after combining two beneficial substitutions, D281G and C289R, which were previously obtained by this optimized assay, the specific activity of the triple-mutation variant I228V/D281G/C289R reached up to 1.42-fold of the wild type (WT), while its half-life (T_1/2) at 60 °C was prolonged by 62.97-fold. The results confirmed the feasibility of the optimized screening assay as a powerful tool for the directed evolution of DAEase.