Submitted to: Protein and Peptide Letters
Publication Type: Peer reviewed journal
Publication Acceptance Date: 8/8/2003
Publication Date: 10/1/2003
Citation: Wong, D., Batt, S.B., Lee, C.C., Robertson, G.H. 2004. Direct screening of libraries of yeast clones for alpha-amylase activity on raw starch. Protein and Peptide Letters. 10:459-468. Interpretive Summary: The production of ethanol in the United States reached a 2.68 billion gallons capacity in 2002. The refinery process involves a key step of cooking starch at high temperatures before adding enzymes for the break down of starch to simple sugars for fermentation. We have been engineering enzymes that can break down raw starch at low temperatures, with the ultimate aim of eliminating the energy intensive and costly cooking step. In the course of this study, we have developed various methods of analyzing the degree of starch breakdown, which would yield a critical assessment of the suitability of the enzymes developed. This report represents the first employment of direct measurement of the extent of breakdown attributed to individual enzymes present in yeast cell cultures, which enables a rapid screening of high activity enzyme mutants. Direct measurement of enzyme action on starch breakdown is important because it determines cost effectiveness of the enzyme in processing applications.
Technical Abstract: High-throughput screening for high-activity barley alpha-amylase mutants expressed in Saccharomyces cerevisiae is hampered by the interference of the glucose used in yeast growth media. In a previous report, it was demonstrated that glycerol could be used as an alternative carbon source, with an unexpected enhancement of synthesis and secretion of the recombinant enzyme (J. Protein Chem. 2002, 21, 419-425). The present investigation employed such a system to enable direct and rapid screening of libraries of yeast clones for alpha-amylase activities on raw starch using a colorimetric method in a microplate format. Specific activities were determined by coupling with quantification of the enzyme concentration in the same medium using chemiluminescent immunodetection.