EFFECT OF INTRODUCING PROLINE RESIDUES ON THE STABILITY OF ASPERGILLUS-AWAMORI

In Aspergillus awamori glucoamylase, Ala27, Ala393, Ala435, Ser436 and Ser460 were replaced with proline residues, in order to stabilize the enzyme by forming more rigid peptide backbones, Specific activities w ere unaffected except for a decrease in Ser460-->Pro glucoamylase, The rmostability was increased in Ser436-->Pro glucoamylase, unchanged in Aal435-->Pro glucoamylase and decreased in Ala27-->Pro, Ala393-->Pro g lucoamylases. As measured by circular dichroism, mutant glucoamylases Ala435-->Pro and Ser436-->Pro resisted unfolding caused by guanidine h ydrochloride at pH 4.5 and 25 degrees C better than wild-type glucoamy lase, whereas mutant glucoamylases Ala27-->Pro, Ala393-->Pro and Ser46 0-->Pro were more susceptible to unfolding than wild-type glucoamylase , reaching a level of 50% unfolded enzyme at guanidine hydrochloride c oncentrations 0.50-0.75 M lower than that of the wild-type enzyme, Mut ations Ala435-->Pro and Ser436-->Pro are located in a non-regular stru cture, which is assumed to be stabilized by these mutations, The Ala27 -->Pro residue is partially buried, which may result in unfavorable st eric contact and/or regional strains; mutation Ala393-->Pro results in loss of a hydrogen bond, since the N of the proline residue does not have an extra hydrogen to act as donor; and mutation Ser480-->Pro elim inates an O-glycosylation site, which could explain how these mutation s destabilized glucoamylase.