Abstract: In order to obtain α-glucan phosphorylase with good heterologous expression level and enzymatic properties and to investigate the impact of structural domains on the enzyme, a novel α-glucan phosphorylase TsGP was screened from the hyperthermophilic archaeon Thermococcus sp. EP1 through database mining and sequence analysis. After confirming that the C-terminal of TsGP was a non-catalytic domain in the three-dimensional structure predicted by AlphaFold2, a C-terminal truncated mutant ΔTsGP was constructed. Heterologous recombinant expression of TsGP and ΔTsGP was performed in E.coli BL21(DE3), and their enzymatic properties were characterized. The results showed that the expression level of ΔTsGP in E.coli was 3.76 times higher than that of TsGP. At the optimal reaction temperature 70 ℃, the specific enzyme activity of ΔTsGP was 23.87 U/mg, which was 1.3 times higher than that of TsGP. Under 50~65 ℃ which was widely applied as industrial catalytic temperature, the thermal stability of ΔTsGP was equivalent to that of TsGP, and the enzyme activity was higher. In addition, ΔTsGP and TsGP had the same substrate specificity, their smallest substrate was maltotriose, and as the substrate chain length increased, their specific enzyme activity gradually increased. Using maltoheptaose as the substrate, the k_cat value of ΔTsGP was 37.09 s⁻¹, which was 1.21 times higher than that of TsGP, but its substrate affinity (K_m=3.30 mmol/L) was reduced by 2.77 times. Through structural analysis and non-catalytic domain truncation strategies, the study successfully improved the expression level of TsGP in E. coli and the specific enzyme activity under industrial enzyme catalytic conditions. The study would provide guidance for the efficient expression and application of αGPase protein and lays the foundation for further optimizing the performance of this enzyme through engineering modification.