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 were unaffected except for a decrease in Ser460-->Pro glucoamylase. Thermostability was increased in Ser436-->Pro glucoamylase, unchanged in Ala435-->Pro glucoamylase and decreased in Ala27-->Pro, Ala393-->Pro glucoamylases. As measured by circular dichroism, mutant glucoamylases Ala435-->Pro and Ser436-->Pro resisted unfolding caused by guanidine hydrochloride at pH 4.5 and 25 degrees C better than wild-type glucoamylase, whereas mutant glucoamylases Ala27-->Pro, Ala393-->Pro and Ser460-->Pro were more susceptible to unfolding than wild-type glucoamylase, reaching a level of 50% unfolded enzyme at guanidine hydrochloride concentrations 0.50-0.75 M lower than that of the wild-type enzyme. Mutations Ala435-->Pro and Ser436-->Pro are located in a non-regular structure, which is assumed to be stabilized by these mutations. The Ala27-->Pro residue is partially buried, which may result in unfavorable steric 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 eliminates an O-glycosylation site, which could explain how these mutations destabilized glucoamylase.
Li
Y
Y
Department of Zoology and Genetics, Iowa State University, Ames 50011, USA.
Reilly
P J
PJ
Ford
C
C
D001230:Aspergillus / Q000201:enzymology*; D001483:Base Sequence; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019791:Guanidine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009994:Osmolar Concentration; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013696:Temperature
D011392:Proline; D005087:Glucan 1,4-alpha-Glucosidase; D019791:Guanidine