A Novel, High Performance Enzyme for Starch Liquefaction

By T. Richardson, X. Tan, G. Frey, W. Callen, M. Cabell, D. Lam, John L. Macomber, J. Short, D. Robertson, Carl W. Miller. Journal of Biological Chemistry, 2002

167 citations recorded by the source corpus.

High throughput screening of microbial DNA libraries was used to identify α-amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95 °C without addition of endogenous Ca2+. After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca2+ and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one α-amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.

Subfields: Biomedical Engineering, Biotechnology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/16724f21b27d863f73c79aa468540f339c37be6c

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Citations in the corpus

Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes (2009, methodology): “In fact, two DNA shuffling strategies have earlier been developed based on the use of type IIB or type IIs restriction enzymes [17,18,19,20].”