AbstractBackgroundD‐Allulose is one of the most well‐known rare sugars widely used in food, cosmetics, and pharmaceutical industries. The most popular method for D‐allulose production is the conversion from D‐fructose catalyzed by D‐allulose 3‐epimerase (DAEase). To address the general problem of low catalytic efficiency and poor thermostability of wild‐type DAEase, D‐allulose biosensor was adopted in this study to develop a convenient and efficient method for high‐throughput screening of DAEase variants.ResultsThe catalytic activity and thermostability of DAEase from Caballeronia insecticola were simultaneously improved by semi‐rational molecular modification. Compared with the wild‐type enzyme, DAEaseS37N/F157Y variant exhibited 14.7% improvement in the catalytic activity and the half‐time value (t1/2) at 65°C increased from 1.60 to 27.56 h by 17.23‐fold. To our delight, the conversion rate of D‐allulose was 33.6% from 500‐g L−1 D‐fructose in 1 h by Bacillus subtilis WB800 whole cells expressing this DAEase variant. Furthermore, the practicability of cell immobilization was evaluated and more than 80% relative activity of the immobilized cells was maintained from the second to seventh cycle.ConclusionAll these results indicated that the DAEaseS37N/F157Y variant would be a potential candidate for the industrial production of D‐allulose.