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Asp238→Asn Creates a Novel Consensus N ‐Glycosylation Site in Aspergillus awamori Glucoamylase
Author(s) -
Allen Martin J.,
Fuchs Erica B.,
Ford Clark F.
Publication year - 2002
Publication title -
starch ‐ stärke
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.62
H-Index - 82
eISSN - 1521-379X
pISSN - 0038-9056
DOI - 10.1002/1521-379x(200209)54:9<385::aid-star385>3.0.co;2-s
Subject(s) - thermostability , aspergillus awamori , mutant , glycosylation , wild type , extracellular , chemistry , biochemistry , enzyme , glycan , microbiology and biotechnology , saccharomyces cerevisiae , biology , glycoprotein , yeast , gene
A single mutation, Asp238→Asn (D238N), of Aspergillus awamori glucoamylase (GA) was identified that increases extracellular production of the enzyme in Saccharomyces cerevisiae at 37 °C. The mutant was isolated as a suppressor of Gly396→Ser (G396S), a previously isolated temperature‐sensitive mutation that decreases the thermostability and extracellular production of GA expressed in S. cerevisiae . Culture supernatants of the double mutant G396S/D238N contained much more GA than supernatants of G396S at 33.5 and 37 °C but not at 30 °C. Additionally, culture supernatants of the D238N contained 1.5 to 2‐fold more GA than supernatants of wild‐type when grown at 37 °C but not at 30 or 33.5 °C. The D238N mutation creates a consensus N‐glycosylation site in GA. Mass spectrometry showed that the molecular weight of D238N was 2319 Da greater than that of the wild‐type GA and that of D238N/G396S was 3094 Da greater than that of G396S, suggesting the presence of an additional N ‐linked glycan at residue 238. No difference in thermostability or activity was observed between the G396S and G396S/D238N mutants or between wild‐type and D238N GAs, and D238N did not affect intracellular GA levels at 30 or 37 °C.