Expression of the Schwanniomyces occidentalis SWA2 amylase in Saccharomyces cerevisiae: role of N-glycosylation on activity, stability and secretion

摘要

pThe role of N-linked glycosylation on the biological activity of iSchwanniomyces occidentalis/i SWA2 α-amylase, as expressed in iSaccharomyces cereiv/iisiae/i, 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 α-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. α-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 (iT/isub50/sub = 50 °C, where iT/isub50/sub 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 °C in the iT/isub50/sub of the enzyme. The Gly-229 mutation does not change the apparent affinity of the enzyme for starch (iK/isubi/im/sub) but decreases to 1/22 its apparent catalytic efficiency (ik/isubcat/sub/iK/isubi/im/sub). These results therefore indicate that glycosylation at the 229 position has an important role in the extracellular activity levels, kinetics and stability of the iSw. occidentalis/i SWA2 α-amylase in both its wild-type and mutant forms./p