Abstract: Based on genomic annotation information, this study took the lead in exploring the β-1,3-/α-1,3-glucan synthetic pathway of Rhizobium pusense. It focused on the action mechanism and potential catalytic efficiency of β-D-fructofuranosidase, the key enzyme in the first step of this synthetic pathway. High-throughput bioinformatics statistical and comparative analysis was conducted by targeting and mining data of β-D-fructofuranosidase from other microbial sources. The results indicated that the sequence and structure of β-D-fructofuranosidase from Rhizobium pusense showed a high degree of similarity and conservation. It was clarified that the key sites Tyr196 and Asp257 in its active region played a crucial role in catalyzing the substrate. Through high - throughput targeted mining of the database, it was found that among the microorganisms with potential β-D-fructofuranosidase expression, there were 6007 bacterial sources and 746 fungal sources. Among them, the β-D-fructofuranosidase of the representative bacterial strain Francisella tularensis OHARA and the fungal strain Fusarium fujikuroi IMI 58289 had relatively high potential catalytic efficiencies, which were 10.73 and 27.40 L·mol⁻¹·s⁻¹ respectively. Further analysis suggested that differences in the size of the active cavity space and the bottleneck radius of β-D-fructofuranosidase from different sources were one of the reasons for the differences in their catalytic efficiencies. This study provides an important basis for a profound understanding of the synthesis mechanism of β-1,3-/α-1,3-glucan in Rhizobium pusense. It also lays the foundation for the development and utilization of other microbial resources for the efficient fermentation production of β-1,3-/α-1,3-glucan, and is of great significance for promoting the development of related fields.