SUBSTITUTION OF ASPARAGINE RESIDUES IN ASPERGILLUS-AWAMORI GLUCOAMYLASE BY SITE-DIRECTED MUTAGENESIS TO ELIMINATE N-GLYCOSYLATION AND INACTIVATION BY DEAMIDATION
Hm. Chen et al., SUBSTITUTION OF ASPARAGINE RESIDUES IN ASPERGILLUS-AWAMORI GLUCOAMYLASE BY SITE-DIRECTED MUTAGENESIS TO ELIMINATE N-GLYCOSYLATION AND INACTIVATION BY DEAMIDATION, Biochemical journal, 301, 1994, pp. 275-281
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.