Abstract
The specificity of the neopullulanase from Bacillus stearothermophilus was altered by protein engineering. The amino acid residues constituting the active center of the neopullulanase were tentatively identified according to a molecular model of Taka-amylase A and homology analysis of the amino acid sequences of neopullulanase, Taka-amylase A, and other amrlolytic enzymes. When one of the putative catalytic sites (Glu-357, Asp-424, and Asp-328) were replaced by the opposite charged (His) or non-charged (Gin or Asn) amino acid residue, neopullulanase activities toward α-(1→4)- and α-(1→6)-glucosidic linkages disappeared . When the putative substrate-binding sites were replaced, the specificities of the mutated neopuliulanases toward α-(1→4)- and α-(1→6)-glucosidic linkages were obviously different from that of the wild-type enzyme. This finding proves that one active center of the neopullulanase participated in the dual activity toward α-(1→4)- and α-(1→6)-glucosidic linkages. Some mutated neopullulanases exhibited higher specificities toward the α-(1→4) linkages relative to the wild-type enzyme. The mutated enzymes exhibiting higher specificities toward theα-(1→6) linkages were also obtained. These mutated neopullulanases were tested for the production of panose, a branched oligosaccharide which might be used as an anticariogenic sweetener . The production ratio of panose from puliulan was significantly increased by using the mutated neopullulanase which exhibited higher specificity toward the α-(1→4)-glucosidic linkage. In contrast, the production ratio of panose was obviously decreased by using the mutated neopullulanase which exhibited higher specificity toward the α-(1→6)-glucosidic linkage.
