The invention discloses a method for synthesizing UDP-glucose through cooperation of UDP-glucose pyrophosphorylase and
cellobiose phosphorylase by a one-pot method, and belongs to the technical field of
enzyme engineering. The method comprises the following steps: selecting UDP-glucose pyrophosphorylase (UGPase) from mycelia thermophilus and
cellobiose phosphorylase (CBP) from
clostridium thermocellum, and carrying out catalytic reaction by taking
cellobiose and UTP as raw materials, so as to obtain UDP-glucose. Experimental results show that heat-resistant UGPase is coupled with CBP, substrates cellobiose and UTP can be efficiently and highly selectively converted into UDP-glucose under mild conditions, and accumulation of an
intermediate product glucose-1-
phosphoric acid is extremely low. According to the invention, a synergistic
reaction system with matched conditions is constructed, and a novel and efficient method for synthesizing UDP-glucose by a one-pot method is provided.