CONTROLLING SUBSTRATE PREFERENCE AND TRANSGLYCOSYLATION ACTIVITY OF NEOPULLULANASE BY MANIPULATING STERIC CONSTRAINT AND HYDROPHOBICITY IN ACTIVE-CENTER

The substrate specificity and the transglycosylation activity of neopu llulanase was altered by site-directed mutagenesis on the basis of inf ormation from a three-dimensional structure predicted by computer-aide d molecular modeling. According to the predicted three dimensional str ucture of the enzyme-substrate complex, it was most likely that Ile-35 8 affected the substrate preference of the enzyme, Replacing Ile-358 w ith Trp, which has a bulky side chain, reduced the acceptability of al pha-(1-->6)-branched oligo- and polysaccharides as substrates. The cha racteristics of the I358W-mutated enzyme were quite different from tho se of wild type neopullulanase and rather similar to those of typical starch-saccharifying alpha-amylase, In contrast, replacing Ile-358 wit h Val, which has a smaller side chain, increased the preference for al pha-(1-->6)-branched oligosaccharides and pullulan as substrates. The transglycosylation activity of neopullulanase appeared to be controlle d by manipulating the hydrophobicity around the attacking water molecu le, which is most likely used to cleave the glucosidic linkage in the hydrolysis reaction. We predicted three residues, Tyr-377, Met-375, an d Ser-422, which were located on the entrance path of the water molecu le might be involved, The transglycosylation activity of neopullulnase was increased by replacing one of the three residues with more hydrop hobic amino acid residues; Y377F, M375L, and S422V. In contrast, the t ransglycosylation activity of the enzyme was decreased by replacing Ty r-377 with hydrophilic amino acid residues, Asp or Ser.