STABILIZATION OF ASPERGILLUS-AWAMORI GLUCOAMYLASE BY PROLINE SUBSTITUTION AND COMBINING STABILIZING MUTATIONS

To stabilize Aspergillus awamori glucoamylase (GA), three proline subs titution mutations were constructed. When expressed in Saccharomyces c erevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability. The S3 0P mutation was combined with two previously identified stabilizing mu tations: Gly137-->Ala, and Asn20-->Cys/Ala27-->Cys (which creates a di sulfide bond between positions 20 and 27). The combined mutants demons trated cumulative stabilization as shown by decreased irreversible the rmoinactivation rates between 65 and 80 degrees C. Additionally, two o f the combined mutants outperformed wild-type GA in high-temperature ( 65 degrees C) saccharifications of DE 10 maltodextrin and were more ac tive than the wild-type enzyme when assayed using maltose as substrate .