Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus.
In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000941:Antigens / Q000276:immunology; D005800:Genes, Fungal; D005087:Glucan 1,4-alpha-Glucosidase / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D005959:Glucosidases / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera / Q000276:immunology; D007527:Isoenzymes / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D012440:Saccharomyces / Q000201:enzymology*; D013045:Species Specificity; D013379:Substrate Specificity
D000911:Antibodies, Monoclonal; D000941:Antigens; D007106:Immune Sera; D007527:Isoenzymes; D005959:Glucosidases; D005087:Glucan 1,4-alpha-Glucosidase