AbstractBACKGROUNDRare sugars have become promising ‘sugar alternatives’ because of their low calories and unique physiological functions. Among the family of rare sugars, d‐allulose is one of the sugars attracting interest. Ketose 3‐epimerases (KEase), including d‐tagatose 3‐epimerase (DTEase) and d‐allulose 3‐epimerase (DAEase), are mainly used for d‐allulose production.RESULTSIn this study, a putative xylose isomerase from Caballeronia insecticola was characterized and identified as a novel DAEase. Caballeronia insecticola DAEase displayed prominent enzymatic properties, and 150 g L−1 d‐allulose was produced from 500 g L−1 d‐fructose in 45 min with a conversion rate of 30% and high productivity of 200 g L−1 h−1. Furthermore, DAEase was employed in a phosphorylation–dephosphorylation cascade reaction, which significantly increased the conversion rate of d‐allulose. Under optimized conditions, the conversion rate of d‐allulose was approximately 100% when the concentration of d‐fructose was 50 mmol L−1.CONCLUSIONThis research described a very beneficial and facile approach for d‐allulose production based on C. insecticola DAEase. © 2022 Society of Chemical Industry.