Pullulanases are well-known debranching enzymes that hydrolyze α-1,6-glycosidic linkages in starch and oligosaccharides. However, most of the pullulanases exhibit limited activity for practical applications. Here, two sites (787 and 621) lining the catalytic pocket of Bacillus naganoensis pullulanase were identified as being critical for enzymatic activity by triple-code saturation mutagenesis. Subsequently, both sites were subjected to NNK-based saturation mutagenesis to obtain positive variants. Among the variants showing enhanced activity, the enzymatic activity and specific activity of D787C were 1.5-fold higher than those of the wild-type (WT). D787C also showed a 1.8-fold increase in kcat and a 1.7-fold increase in kcat/ Km. In addition, D787C maintained higher activity compared with that of WT at temperatures over 60 °C. All the positive variants showed higher acid resistance, with D787C maintaining 90% residual activity at pH 4.0. Thus, enzymes with improved properties were obtained by saturation mutagenesis at the active site.