A　new　pullulanase-encoding　gene,　pulSL3,　was　cloned　from　Alkalibacterium　sp.　SL3　and　expressed　in　Escherichia　coli.　Deduced　PulSL3　contains　2026　amino　acid　residues　and　consists　of　a　putative　signal　peptide,　four　carbohydrate-binding　modules,　an　E_set　pullulanase　domain,　an　α-amylase　domain,　and　a　pullulanase　domain.　The　purified　rPulSL3　exhibited　pullulanase　activity　only.　To　identify　the　functions　of　the　α-amylase　and　pullulanase　domains,　the　N-　and　C-terminus　truncated　mutants　(pulSL3N　and　pulSL3C)　were　also　expressed　in　E.　coli.　The　purified　rPulSL3N　had　pullulanase　activity,　while　rPulSL3C　had　no　either　α-amylase　or　pullulanase　activity.　Both　rPulSL3　and　rPulSL3N　exhibited　maximum　activities　at　pH　9.0　and　50　°C,　and　were　highly　active　and　stable　at　the　concentration　of　NaCl　up　to　4　M　and　resistant　to　a　few　surfactants　and　detergents.　In　comparison　to　the　wild　type,　rPulSL3N　had　better　thermostability　(retaining　62.9　vs.　33.6%　activity　at　52　°C　for　30　min),　higher　substrate　affinity　(with　Km　of　0.10　vs.　0.15　mg　mL−1)　and　higher　catalytic　efficiency　(836.6　vs.　520.3　mL　mg−1　s−1).　These　excellent　properties　make　rPulSL3N　potential　for　the　application　in　the　industries　of　detergent,　food,　and　environmental　remediation.　©　2020　Elsevier　Ltd