Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 7/1/1999
Publication Date: N/A
Citation: Interpretive Summary: Many industrial fermentation and enzymatic conversion processes rely on the breakdown of grain starch to glucose by enzymes. The production of fuel ethanol and sweeteners are two important examples. The conversion to glucose employs cooking gelatinization, enzymatic liquefication, and saccharification. The conversion has high chemical, capital, and energy demands that could be significantly reduced if we could tailor enzymes for this conversion to operate at low temperature and against solid starch. This research program is applyiing methods of combinatorial chemistry or directed molecular evolution. In these methods existing enzymes are modified randomly to create large libraries of variants that are tested and selected to achieve the desired activities. This report describes a facet of this research in which basic information about the properties of starch degradiing enzymes was sought. This task applied a new tool in which a random library of peptide molecules were bound to the enzymes and then analyzed for the binding sequence. This basic information is an important step in the design of strategies for altering the structure of the target enzyme molecules and expands our fundamental knowledge about properties of agriculturally important enzymes.
Technical Abstract: Peptide ligands that bind to pancreatic a-amylase were isolated from bacteriophage libraries displaying random 15-mer peptides by iterative affinity selection and amplication. The DNA sequences of tight-binding clones selected were determined. The phage display ligands with the highest binding activity contained high content of Arg, Tyr and Trp residues, and shared a short consensus sequence of Arg-X-Tyr-Trp. These clones were shown to exhibit considerable affinity towards barley a-amylase by TU titering. The dissociation constants for the phage displayed peptide binding to pancreatic and barley a-amylases suggest cross-reactivity with comparable interactions.