Mutagenesis of the active site of glucoamylase from Aspergillus awamori

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1988
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Sierks, Michael
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Peter J. Reilly
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Chemical and Biological Engineering

The function of the Department of Chemical and Biological Engineering has been to prepare students for the study and application of chemistry in industry. This focus has included preparation for employment in various industries as well as the development, design, and operation of equipment and processes within industry.Through the CBE Department, Iowa State University is nationally recognized for its initiatives in bioinformatics, biomaterials, bioproducts, metabolic/tissue engineering, multiphase computational fluid dynamics, advanced polymeric materials and nanostructured materials.

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The Department of Chemical Engineering was founded in 1913 under the Department of Physics and Illuminating Engineering. From 1915 to 1931 it was jointly administered by the Divisions of Industrial Science and Engineering, and from 1931 onward it has been under the Division/College of Engineering. In 1928 it merged with Mining Engineering, and from 1973–1979 it merged with Nuclear Engineering. It became Chemical and Biological Engineering in 2005.

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1913 - present

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  • Department of Chemical Engineering (1913–1928)
  • Department of Chemical and Mining Engineering (1928–1957)
  • Department of Chemical Engineering (1957–1973, 1979–2005)
    • Department of Chemical and Biological Engineering (2005–present)

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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.

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Fri Jan 01 00:00:00 UTC 1988