Protein Engineering, Vol 11, 783-788, Copyright © 1998 by Oxford University Press
MJ Allen, PM Coutinho and CF Ford
To stabilize Aspergillus awamori glucoamylase (GA), three proline
substitution mutations were constructed. When expressed in Saccharomyces
cerevisiae, 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 S30P
mutation was combined with two previously identified stabilizing mutations:
Gly137-->Ala, and Asn20-->Cys/Ala27-- >Cys (which creates a
disulfide bond between positions 20 and 27). The combined mutants
demonstrated cumulative stabilization as shown by decreased irreversible
thermoinactivation rates between 65 and 80 degrees C. Additionally, two of
the combined mutants outperformed wild- type GA in high-temperature (65
degrees C) saccharifications of DE 10 maltodextrin and were more active
than the wild-type enzyme when assayed using maltose as substrate.
ARTICLES
Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations
Department of Zoology and Genetics, Iowa State University, Ames 50011, USA.
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