基于定向进化的D-阿洛酮糖3-差向异构酶分子改造OA

As the quality of life improves,people's demand for health is also increasing,and sugar substitutes are gradually coming into people's view.D-allulose is a low-calorie,high sweetness,scarce sugar that is an ideal substitute for sucrose.The instrumental en-zyme for industrial preparation of D-allulose is mainly D-allulose 3-epimerase(DAE),however,to meet the demands of industrial produc-tion,the specific activity of DAE still needs to be further increased.In this study,by applying a D-allulose-specific biosensor composed of the transcription factor psiR,combining with fluorescence-activated cell sorting(FACS)fluorescence analysis sorting and 96-well microti-ter plate screening,we finally obtained the Clostridium cellulolyticum H10 DAE(CcDAE)mutant F147L with a 26.21％increase in spe-cific activity,with an optimal temperature of 60 ℃ and an optimal pH of 7.5.This study not only successfully demonstrated that transcrip-tion factor-based biosensors have considerable application value in the directed evolution of proteases,but also provided new ideas for the modification of DAE enzymes.