Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation.
Aspergillus awamori glucoamylase is a secreted glycoprotein containing N-linked carbohydrate recognition sites at Asn-171, Asn-182 and Asn-395. Site-directed mutagenesis was performed at Asn-182 and Asn-395 to determine whether these residues were N-glycosylated by Saccharomyces cerevisiae, to investigate the function of any glycans linked to them, and to determine the effect of their deamidation on glucoamylase thermostability. Asn-171 and Asn-395, but not Asn-182, were N-glycosylated. Deletion of the glycan N-linked to Asn-395 did not affect specific activity, but greatly decreased enzyme secretion and thermostability. The mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susceptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wild-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate deamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activity about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase production.
Department of Chemical Engineering, Iowa State University, Ames 50011.
H M
HM
Chen
C
C
Ford
P J
PJ
Reilly
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001230:Aspergillus / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics
D001216:Asparagine; D004247:DNA; D005087:Glucan 1,4-alpha-Glucosidase