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 enzyme for industrial preparation of D-allulose is mainly D-allulose 3-epimerase (DAE), however, to meet the demands of industrial production, 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 microtiter plate screening, we finally obtained the Clostridium cellulolyticum H10 DAE (CcDAE) mutant F147L with a 26.21% increase in specific activity, with an optimal temperature of 60 ℃ and an optimal pH of 7.5.This study not only successfully demonstrated that transcription factor-based biosensors have considerable application value in the directed evolution of proteases, but also provided new ideas for the modification of DAE enzymes.
SUN Mingjing
,
LIU Zhanzhi
,
LIU Shuhan
,
WANG Liuxin
,
WANG Sheng
,
WU Jing
. Directed evolution-based molecular modification of D-allulose 3-epimerase[J]. Food and Fermentation Industries, 2026
, 52(1)
: 25
-30
.
DOI: 10.13995/j.cnki.11-1802/ts.042005
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