Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion.
The role of N-linked glycosylation on the biological activity of Schwanniomyces occidentalis SWA2 alpha-amylase, as expressed in Saccharomyces cerevisiae, was analysed by site-directed mutagenesis of the two potential N-glycosylation sites, Asn-134 and Asn-229. These residues were replaced by Ala or Gly individually or in various combinations and the effects on the activity, secretion and thermal stability of the enzyme were studied. Any Asn-229 substitution caused a drastic decrease in activity levels of the extracellular enzyme. In contrast, substitutions of Asn-134 had little or no effect. The use of antibodies showed that alpha-amylase was secreted in all the mutants tested, although those containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. alpha-Amylases with substitution at Asn-229 had a 2 kDa lower molecular mass than the wild-type protein, as did the wild-type protein itself after treatment with endoglycosidase F. These findings indicate that Asn-229 is the single glycosylated residue in SWA2. Thermostability analysis of both purified wild-type (T50=50 degrees C, where T50 is the temperature resulting in 50% loss of activity) and mutant enzymes indicated that removal of carbohydrate from the 229 position results in a decrease of approx. 3 degrees C in the T50 of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (Km) but decreases to 1/22 its apparent catalytic efficiency (kcat/Km). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the Sw. occidentalis SWA2 alpha-amylase in both its wild-type and mutant forms.
Yáñez
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E
Centro de Biologia Molecular 'Severo Ochoa' (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Carmona
T A
TA
Tiemblo
M
M
Jiménez
A
A
Fernández-Lobato
M
M
D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D015870:Gene Expression; D006031:Glycosylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics; D013213:Starch / Q000378:metabolism; D013696:Temperature; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D013213:Starch; D000516:alpha-Amylases