Submitted to: American Chemical Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 10/29/2004
Publication Date: 10/29/2004
Citation: Batt, S.B., Wong, D., Lee, C.C., Wagschal, K.C., Robertson, G.H. 2004. Directed evolution of barley alpha-amylase for increased activity. American Chemical Society Western Regional Meeting, Sacramento, CA. Paper No. 144 (Abstracts).
Technical Abstract: The alpha-amylase enzyme is used in the industrial degradation of starch to sugars for the production of ethanol and other value-added chemicals. By improving the enzymes used in the biorefining of starch, the cost of producing fuel ethanol and other bioproducts could be decreased. We are engineering alpha-amylase enzymes for increased activity. The barley alpha-amylase isozyme 2 gene was cloned into an expression vector in Saccharomyces cerevisiae. Libraries of mutant alpha-amylase genes were created by PCR random mutagenesis and gene shuffling. High-throughput enzyme activity screening was conducted using soluble starch or dye-labeled starch substrates. Immunodetection was used to measure the concentration of recombinant enzymes in liquid culture samples. Enzyme mutants with increased activity were selected and subjected to additional cycles of mutation and gene shuffling, screening, and selection. We used directed evolution to develop barley alpha-amylase mutants with higher activity than the native barley alpha-amylase.