Submitted to: Journal of Protein Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/28/2004
Publication Date: 10/1/2004
Citation: Wong, D., Batt, S.B., Lee, C.C., Robertson, G.H. 2004. High-activity barley alpha-amylase by directed evolution. Journal of Protein Chemistry, 23:53-460. Interpretive Summary: Alpha-amylases are the major enzyme used in the conversion of agricultural crops to value-added products in the high volume biofuels and sweetener industries. The enzymes are used to convert starch to dextrins prior to fermentation (biofuels) or isomerization (sweeteners). We have been engineering novel alpha-amylases that could effectively digest starch without the need for high temperature cooking. Libraries of enzyme mutants were created by random mutation and shuffling of the gene sequences, and screened for active variants using high-throughput methods previously developed by our group. This report represents the first employment of directed evolution techniques to enabling a substantial increase in the enzyme activity of barley alpha-amylase. It describes a practical and proven route to further improve the performance of alpha-amylases that could be tailored to meet the various demands of industrial processes.
Technical Abstract: Barley alpha-amylase isozyme 2 was cloned into and constitutively secreted by Saccharomyces cervisiae. The gene coding for the wild-type enzyme was subjected to error-prone PCR and gene shuffling. Libraries of mutants were screened by halo formation on starch agar plates, followed by high-throughput liquid assay using dye-labeled starch as the substrate. The concentration of recombinant enzyme in the culture supernatant was determined by immunodetection, and used for the calculation of specific activity. After three rounds of mutation/shuffling, one mutant (Mu322) showed 1000 times the total activity and 30 times the specific activity of the wild-type enzyme. Comparison of the amino acid sequence of this mutant with the wild type revealed 5 substitutions: Q44H, R303K and F325Y in domain A, and T94A and R128Q in domain B. Two of these mutations (Q44H and R303K) result in amino acids highly conserved in cereal alpha-amylases. R303K and F325Y are located in the raw starch-binding fragment of the enzyme molecule.