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Aspergillus awamori glucoamylase (GA) is an enzyme involved in industrial com starch processing. Genetic and biochemical approaches were used to smdy the mechanisms governing GA thermostability. Three proline substimtion (Xaa-Pro) mutations were constructed that were predicted to increase the enzyme's stability by decreasing its conformational entropy of unfolding. When expressed ui Saccharomyces cerevisiae Ser30-Pro increased, Asp345-Pro did not alter and Glu408-Pro greatly decreased GA stability as measured by resistance to irreversible thermoinactivation relative to the wildtype enzyme. The Ser30-Pro mutation was combined with other previously identified stabilizing mutations to examine whether combining such mutations could cumulatively stabilize the GA. The SerSO-Pro mutation cumulatively stabilized the enzyme when combined with the Asn20-Cys/Ala27-Cys mutations, which create a disulfide bond between positions 20 and 27. Similarly, when SerSO-Pro was combined with a Glyl37-Ala mutation the enzyme was cumulatively stabilized. The combined mutant Asn20-Cys/Ala27-Cys/Ser30-Pro/Glyl37-Ala was the most stable variant constructed and increased the enzyme's activation energy for thermoinactivation by 4.4 kJ/moI at 65°C and its melting temperature (the temperature at which the enzyme was 50% inactivated after 10 minutes) by 3.9°C relative to wild-type GA. None of the combined mutants decreased the enzyme's activity. The Asn20-Cys/Ala27-Cys/Ser30-Pro/Glyl37-Ala and Ser30-Pro/Glyl37-Ala mutants increased resistance to irreversible thermoinactivation in the presence of 1.71 M glucose and outperformed wild-type GA in a high-temperamre ix (65°C) saccharification of DE 10 maltodextrin. Using random mutagenesis, an AspZSS-Asn mutation was identified that increased the expression of recombinant GA by S. cerevisiae when grown at elevated temperatures in both wild-type and Gly396-Ser genetic backgroimds. This may be due to increased resistance to intraand/or extra-cellular proteolysis conferred by the Asp238-Asn amino acid substimtion. The screening method used to identify the Asp238-'Asn mutation may be useful to identify other mutations which could increase the expression of other productiondeficient GA mutants. |
