A novel, high performance enzyme for starch liquefaction. Discovery and optimization of a low pH, thermostable alpha-amylase.
High throughput screening of microbial DNA libraries was used to identify alpha-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 degrees C without addition of endogenous Ca(2+). 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 Ca(2+) 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 alpha-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.
Richardson
Toby H
TH
Diversa Corporation, San Diego, California 92121, USA. trichardson@diversa.com
Tan
Xuqiu
X
Frey
Gerhard
G
Callen
Walter
W
Cabell
Mark
M
Lam
David
D
Macomber
John
J
Short
Jay M
JM
Robertson
Dan E
DE
Miller
Carl
C
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D005524:Food Technology / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013213:Starch / Q000378:metabolism*; D003313:Zea mays; D000516:alpha-Amylases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D013213:Starch; D000516:alpha-Amylases