Thermostability Improvement of the D-Allulose 3-Epimerase from Dorea sp CAG317 by Site-Directed Mutagenesis at the Interface Regions

D-Allulose is a low-calorie sweetener and has broad applications in the food, cosmetics, and pharmaceutical industries. Recently, most studies focus on D-allulose production from D-fructose by D-allulose 3-epimerase (DAEase). However, the major blocker of industrial production of D-allulose is the poor thermostability. In this study, site-directed mutagenesis at the interface regions of Dorea sp. DAEase was carried out, and the F154Y/E191D/I193F mutation was obtained. The mutant protein displayed much higher thermostability, with a t(1/2) value of 20.47 h (50 degrees C) and a T-m value of 74.18 degrees C. Compared with the wild-type DAEase, the t(1/2) value at 50 degrees C increased by 5.4-fold, and the T-m value increased by 17.54 degrees C. In the D-allulose production from 500 g/L D-fructose, 148.2 g/L D-allulose could be obtained by F154Y/E191D/I193F mutant protein. The results suggest that site-directed mutagenesis at the interface regions is an efficient approach for improving the thermostability of DAEase.