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. 1994 Jul 1;301(Pt 1):275–281. doi: 10.1042/bj3010275

Substitution of asparagine residues in Aspergillus awamori glucoamylase by site-directed mutagenesis to eliminate N-glycosylation and inactivation by deamidation.

H M Chen 1, C Ford 1, P J Reilly 1
PMCID: PMC1137172  PMID: 8037681

Abstract

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.

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