EXPRESSION OF THE SCHWANNIOMYCES-OCCIDENTALIS SWA2 AMYLASE IN SACCHAROMYCES-CEREVISIAE - ROLE OF N-GLYCOSYLATION ON ACTIVITY, STABILITY ANDSECRETION

The role of N-linked glycosylation on the biological activity of Schwa nniomyces occidentalis SWA2 alpha-amylase, as expressed in Saccharomyc es cerevisiae, was analysed by site-directed mutagenesis of the two po tential N-glycosylation sites, Asn-134 and Asn-229. These residues wer e replaced by Ala or Gly individually or in various combinations and t he effects on the activity, secretion and thermal stability of the enz yme were studied. Any Asn-229 substitution caused a drastic decrease i n activity levels of the extracellular enzyme. In contrast, substituti ons of Asn-134 had little or no effect. The use of antibodies showed t hat alpha-amylase was secreted in all the mutants tested, although tho se containing substitutions at Asn-229 seemed to have a lower rate of synthesis and/or higher degradation than the wild-type strain. alpha-A mylases 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 (T-50 = 50 degrees C, where T-50 is the tem perature resulting in 50% loss of activity) and mutant enzymes indicat ed that removal of carbohydrate from the 229 position results in a dec rease of approx. 3 degrees C in the T-50 of the enzyme. The Gly-229 mu tation does not change the apparent affinity of the enzyme for starch (K-m) but decreases to 1/22 its apparent catalytic efficiency (k(cat)/ K-m). These results therefore indicate that glycosylation at the 229 p osition has an important role in the extracellular activity levels, ki netics and stability of the Sw. occidentalis SWA2 alpha-amylase in bot h its wild-type and mutant forms.