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 awamoriglucoamylase, five Gly residues causing helix flexibility werereplaced with Ala residues. Mutation of Gly57 did not affectthermostability. Mutation of Gly137 doubled it at pHs 3.5 and4.5 but barely changed it at pH 5.5. The Gly139Ala mutationdid not change thermostability at pH 3.5, improved it at pH4.5 and worsened it at pH 5.5. The Gly137/Gly139Ala/Ala mutationgave 1.5–2-fold increased thermostabilities at pHs 3.5–5.5.Mutations of Gly251 and Gly383 decreased it at all pHs. Gly137Alaand Gly137/Gly139Ala/Ala glucoamylases are the most stable yetproduced by mutation. Guanidine treatment at pH 4.5 decreasedthe reversible stabilities of Gly137Ala, Gly139Ala and Gly137/Gly139Ala/Alaglucoamylases at infinite dilution while not changing thoseof Gly251Ala and Gly 383Ala glucoamylases, which is, in general,opposite to what occurred with thermoinactivation. Mutationof Gly57 greatly improved the extracellular glucoamylase productionby yeast, that of Gly137 barely affected it and those of Gly139and of both Gly137 and Gly139 strongly impeded it. These observationssuggest that -helix rigidity can affect reversible and irreversibleglucoamylase stability differently, that the effects of multiplemutations within one -helix to improve stability are not alwaysadditive and that even single mutations can strongly affectextracellular enzyme production.