ARS | Publication request: In Vitro Evolution of High-Activity Barley Alpha-Amylase for Raw Starch Digestion

Submitted to: American Association Of Cereal Chemists Meetings
Publication Acceptance Date: July 22, 2004
Publication Date: September 19, 2004
Citation: Wong, D., Robertson, G.H., Batt, S.B., Lee, C.C. 2004. In Vitro Evolution Of High-Activity Barley Alpha-Amylase For Raw Starch Digestion. American Association Of Cereal Chemists Meetings, September 19-22, 2004, San Diego, California. Abstract.

Technical Abstract: Direct enzymatic digestion or disassembly of starch is a desirable alternative to high-temperature enzymatic digestion. The principle advantage in fuel ethanol processes is to reduce overall energy use. Conventional cooking may consume an energy equivalent of up to 20% of the fuel value of the ethanol produced. This low temperature alternative has been elusive because there are only a few raw-starch digesting enzymes and these are from a limited number of sources. This report describes research directed to evolve enzymes in vitro with improved ability to digest raw starch. The parent enzymes, barley a-amylase low and high pI isozymes were expressed in Saccharomyces cerevisiae, and subjected to in vitro evolution employing screens based on soluble dye-labeled starch. Enhanced total activity (>100X) and protein specific activity (>30X) for the high pI form relative to the parent enzyme have been observed. All five mutations identified are located on the surface wall of the (a/b)8 barrel and are distant from the essential residues in the active site.