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-3 95. 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 ther mostability. 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 a ctivity, but greatly decreased enzyme secretion and thermostability. T he mutant lacking the N-glycan linked to Asn-395 was synthesized very slowly, and was more associated with cell membrane components and susc eptible to proteinase degradation than were wild-type or other mutant glucoamylases. Its secreted form was 30-fold less thermostable than wi ld-type enzyme at pH 4.5. Replacement of Asn-182 by Gln to eliminate d eamidation at this site did not change glucoamylase specific activity or thermostability, while replacement by Asp decreased specific activi ty about 25%, but increased thermostability moderately at pH 4.5 below 70 degrees C. Both mutations of Asn-182 increased glucoamylase produc tion.