Hm. Chen et al., EFFECT OF REPLACING HELICAL GLYCINE RESIDUES WITH ALANINES ON REVERSIBLE AND IRREVERSIBLE STABILITY AND PRODUCTION OF ASPERGILLUS-AWAMORI GLUCOAMYLASE, Protein engineering, 9(6), 1996, pp. 499-505
To decrease irreversible thermoinactivation of Aspergillus awamori glu
coamylase, five Gly residues causing helix flexibility were replaced w
ith Ala residues. Mutation of Gly57 did not affect thermostability, Mu
tation of Gly137 doubled it at pHs 3.5 and 4.5 but barely changed it a
t pH 5.5. The Gly139-->Ala mutation did not change thermostability at
pH 3.5, improved it at pH 4.5 and worsened it at pH 5.5, The Gly137/Gl
y139-->Ala/Ala mutation gave 1.5-2-fold increased thermostabilities at
pHs 3.5-5.5, Mutations of Gly251 and Gly383 decreased it at all pHs.
Gly137-->Ala and Gly137/Gly139-->Ala/Ala glucoamylases are the most st
able yet produced by mutation, Guanidine treatment at pH 4.5 decreased
the reversible stabilities of Gly137-->Ala, Gly139-->Ala and Gly137/G
ly139-->Ala/Ala glucoamylases at infinite dilution while not changing
those of Gly251-->Ala and Gly383-->Ala glucoamylases, which is, in gen
eral, opposite to what occurred with thermoinactivation, Mutation of G
ly57 greatly improved the extracellular glucoamylase production by yea
st, that of Gly137 barely affected it and those of Gly139 and of both
Gly137 and Gly139 strongly impeded it. These observations suggest that
alpha-helix rigidity can affect reversible and irreversible glucoamyl
ase stability differently, that the effects of multiple mutations with
in one alpha-helix to improve stability are not always additive and th
at even single mutations can strongly affect extracellular enzyme prod
uction.