Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold

A disulfide bridge-constrained cellulose binding domain (CBD,) derived from the cellobiohydrolase Ce17A from Trichoderma reesei has been investigated for use in scaffold engineering to obtain novel binding proteins. The gene encoding the wild-type 36 aa CBDWT domain was first inserted into a phagemi d vector and shown to be functionally displayed on M13 filamentous phage as a protein III fusion protein with retained cellulose binding activity. A c ombinatorial library comprising 46 million variants of the CBD domain was c onstructed through randomization of 11 positions located at the domain surf ace and distributed over three separate beta -sheets of the domain. Using t he enzyme porcine alpha-amylase (PPA) as target in biopannings, two CBD var iants showing selective binding to the enzyme were characterized. Reduction and iodoacetamide blocking of cysteine residues in selected CBD variants r esulted in a loss of binding activity, indicating a conformation dependent binding. Interestingly, further studies showed that the selected CBD varian ts were capable of competing with the binding of the amylase inhibitor acar bose to the enzyme. In addition, the enzyme activity could be partially inh ibited by addition of soluble protein, suggesting that the selected CBD var iants bind to the active site of the enzyme. (C) 2000 Wiley-Liss, Inc.