Date of Award
Doctor of Philosophy
Chemical and Biological Engineering
Peter J. Reilly
The Aspergillus awamori glucoamylase gene was modified by cassette mutagenesis, and eleven mutated enzymes were expressed by Saccharomyces cerevisiae;Four mutations were constructed to change the essential Trp 120 residue in subsite 4 to Tyr, Phe, His, and Leu. All the mutations bound maltose (G2) and isomaltose (iG2) more strongly than wild-type glucoamylase. A subsite map of the Tyr 120 mutation showed significantly higher affinities for D-glucosyl residues in subsites 1 and 2, suggesting an interaction of Trp 120 with residues located there;Three carboxylic acid residues in the active site, Asp 176, Glu 179, and Glu 180, were mutated to discover their role in catalysis. The Glu-Gln 179 mutation resulted in almost complete loss of activity with little change in binding, suggesting that Gln 179 is catalytically active. The Glu-Gln 180 mutation suggests that Glu 180 provides a strong bond with [alpha]-(1 → 4)-linked D-glucosyl residues in subsite 2, but is not involved in catalysis. The Asp-Asn 176 mutation resulted in a large decrease in catalytic activity and a moderate increase in binding of G2, iG2, and maltoheptaose (G7). A subsite map of this mutation suggests that Asp 176 may interact with Trp 120 and also be catalytically active;Four residues likely to be in the active site, based on homology with related enzymes, Tyr 116, Leu 177, Trp 178, and Asn 182, were changed to Ala, His, Arg, and Ala, respectively. The Tyr-Ala 116 mutation yielded similar though less dramatic results than the Trp 120 mutations, suggesting it has a similar role in catalysis. The Leu 177, Trp 178, and Asn 182 residues were suspected of affecting the selectivity of [alpha]-(1 → 4)- against [alpha]-(1 → 6)-D-glucosidic bond hydrolysis. The Leu-His 177 mutation showed moderate decreases in binding and catalytic rates. The Trp-Arg 178 mutation showed a substantial decrease in catalytic rate and a two-fold increase in selectivity of iG2 over G2 hydrolysis. The Asn-Ala 182 mutation gave slightly lower catalytic rates with an increased binding of G2 and decreased binging of iG2. This more than doubled the selectivity of G2 over iG2 hydrolysis with little effect on the catalytic rate.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Michael R. Sierks
Sierks, Michael R., "Mutagenesis of the active site of glucoamylase from Aspergillus awamori " (1988). Retrospective Theses and Dissertations. 11184.