Abstract: The  maltase-glucoamylase (MGAM) from the large fungus Ganoderma lucidum  as the research object,  and a mutant D246A with significantly reduced enzyme activity was successfully constructed by using methods such as homologous sequence alignment,  homologous modeling,  substrate docking,  and site-specific mutation. The characterization results of enzymatic properties show that  the optimal reaction temperature decreases from 65 ℃ for wild type (WT) to 60 ℃， and the heat tolerance of the mutant decreases. The optimal pH value increases from 6.0 for WT to 7.0,  which is beneficial for the growth of engineering bacteria.  The half-life decreases from 2.0 h for WT to 1.5 h,  the stability of enzyme decreases. The enzyme kinetics results show that  the enzyme kinetics curve of mutant D246A conforms to the Michaelis equation, and   compared with the WT, the K_m value increases,  indicating a decrease in affinity of enzyme and substrate.  V_max decreases to 1/4 of its original value.

Key words: site-specific mutation, heterologous expression, property characterization,  , maltase-glucoamylase