Mj. Allen et al., STABILIZATION OF ASPERGILLUS-AWAMORI GLUCOAMYLASE BY PROLINE SUBSTITUTION AND COMBINING STABILIZING MUTATIONS, Protein engineering (Print), 11(9), 1998, pp. 783-788
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
.