EFFECT OF REPLACING HELICAL GLYCINE RESIDUES WITH ALANINES ON REVERSIBLE AND IRREVERSIBLE STABILITY AND PRODUCTION OF ASPERGILLUS-AWAMORI GLUCOAMYLASE

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.