Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: April 13, 2004
Publication Date: June 13, 2004
Citation: Wong, D., Batt, S.B., Lee, C.C., Robertson, G.H. Engineering apha-amylase for enhanced activity by molecular evolution. American Society of Biochemistry and Molecular Biology Annual Meeting, June 12-16, 2004, Boston, Massachusetts. Abstract No. B38. Technical Abstract: Alpha-Amylase is the major enzyme in starch hydrolysis for the production of sweeteners and bioethanol. We attempted to engineer barley a-amylase to achieve high activity using directed evolution. The gene coding the wild-type enzyme was cloned into Saccharomyces cervisiae, and subjected to repeated cycles of error-prone PCR and DNA shuffling. Enzyme variants were screened for activity using starch plates and liquid assays with soluble starch and dye-labeled starch as the substrate, respectively. Enzyme concentration was determined by chemiluminescent detection. An enzyme mutant was isolated showing 1000 times the total activity and 30 times the specific activity of the wild type. Three of the five amino acid substitutions Q44H, R303K, and F325Y were located in domain A, with the latter two mutations occurred within the raw starch/substrate binding region. The remaining two mutations, T94A and R128Q, were found in domain B. Q44H and R303K resulted in substitutions of amino acids highly conserved in cereal alpha-amylases.