Abstract
Based on a series of experimental results using neopullulanase and the structural similarities, we previously reported a general idea for an enzyme super family, α-amylase family, including α-amylase, pullulanase/isoamylase, cyclomaltodextrin glucanotransferase, 1, 4-α-D-glucan branching enzyme, neopullulanase, and related enzymes. We also demonstrated a hypothesis that the enzymes belonging to α-amylase family can be altered each other by manipulating substratebinding specificity or by changing the environment of the active center. A unique enzyme, neopullulanase, was used to explore further into the hypothesis. The three-dimensional structure of neopullulanase was predicted by a homology modeling procedure. A computer simulated docking-study in three-dimension was also employed to investigate the enzyme-substrate structures. Based on the simulated docking-study, we introduced some replacement of the amino acid residues which are most likely to affect the substrate preference of neopullulanase by site-directed mutagenesis. By manipulating the amino acid residues, the enzyme specificities toward α- (1→6) -branched oligosaccharides and pullulan were reduced or increased. The characteristics of the mutated neopullulanase which was reduced in its specificity toward α- (1→6) -branched oligosaccharides and pullulan was quite different from those of wild-type enzyme and rather similar to those of α-amylase. Some experimental keys to explore our hypotheses are also discussed.
