Phenylalanine476 mutation of pullulanase from Bacillus subtilis str. 168 improves the starch substrate utilization by weakening the product beta-cyclodextrin inhibition.

Since beta-cyclodextrin (beta-CD) was the competitive inhibitor of pullulanase, substrate utilization and product inhibition were a conflict in beta-CD production when using pullulanase for debranching to release more convertible substrate. Here, a phenylalanine complexed with cyclodextrin ligand via classic hydrophobic interaction and hydrogen bond in the crystal structure of pullulanase and absolutely conserved in all homologous, was selected to study its contribution in inhibition and to get mutants with lower affinity to beta-CD. Mutants were generated by substituting phenylalanine with tyrosine, valine, histidine, cysteine, aspartic acid or alanine. Enzyme activity assay and ITC data proved that F476 mutants successfully reduced the protein affinity to beta-CD. Although circular dichroism spectra and fluorescence spectroscopy results showed F476 mutation affected the secondary structure and microenvironment of pullulanase, mutants F476C and F476H retained relatively higher enzyme activity and significantly decreased affinity to beta-CD. The conversion rate of starch into beta-CD increased by 6.0% and 6.6%, when adding F476C or F476H in the production process.