This　study　aimed　to　clone　the　novel　α-glucosidase　gene　Aga432　from　Paenibacillus　sp.　and　enhance　its　catalytic　activity　through　site-directed　mutagenesis.　A　gene　fragment　encoding　α-glucosidase　was　successfully　amplified　from　the　genomic　DNA　of　Paenibacillus　sp.,　comprehensive　sequence　analysis　was　performed,　and　homology　modeling　and　molecular　docking　were　employed　to　construct　gene-engineered　strains.　Eight　positive　mutant　strains　were　identified,　among　which　the　enzymatic　properties　of　recombinant　Aga432　and　the　highest　relative　activity　mutant　AT-2　were　characterized.　Additionally,　the　dispersing　effects　of　recombinant　α-glucosidases　Aga432　and　AT-2　on　biofilms　were　explored,　and　their　toxicity　to　mouse　embryo　fibroblasts　was　evaluated.　The　results　revealed　that　the　specific　activity　of　Aga432　was　45.05　U/mg,　while　the　mutant　AT-2　exhibited　a　significantly　enhanced　specific　activity　of　84.09　U/mg.　Although　the　optimal　reaction　temperature　and　pH　for　AT-2　were　essentially　unaltered　relative　to　Aga432,　its　thermal　stability　was　significantly　enhanced,　and　it　exhibited　heightened　stability　under　acidic　conditions.　The　Km　of　mutant　AT-2　was　2.18　times　that　of　Aga432,　the　Vmax　was　3.19　times,　the　Kcat　was　2.33　times,　and　the　Kcat/Km　was　1.07　times　that　of　Aga432.　In　vitro　cellular　assays　indicated　that　Aga432　and　AT-2　at　concentrations　of　15.0~30.0　μg/mL　were　non-toxic　and　exhibited　good　cell　compatibility.　Biofilm　dispersal　assays　demonstrated　that　both　recombinant　α-glucosidases　at　concentrations　ranging　from　10.0　to　50.0　μg/mL　significantly　dispersed　bacterial　biofilms　(P＜0.0001).　The　thermostability　of　α-glucosidase　Aga432　was　successfully　enhanced　through　molecular　modification　in　this　study,　laying　a　foundation　for　the　development　of　novel　α-glucosidases　and　providing　a　reference　for　future　targeted　modification　research.　©　The　Author(s)　2025.