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.

Modena, D., Vanoni, M., Englard, S., & Marmur, J. (1986). Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 248(1), 138-150 [10.1016/0003-9861(86)90410-8].

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus

VANONI, MARCO ERCOLE;
1986

Abstract

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.
Articolo in rivista - Articolo scientifico
Scientifica
Glucan 1,4-alpha-Glucosidase; Genes, Fungal; Hydrogen-Ion Concentration; Immune Sera; Molecular Weight; Antibodies, Monoclonal; Antibody Specificity; Hot Temperature; Antigens; Glucosidases; Saccharomyces; Kinetics; Isoenzymes; Substrate Specificity; Species Specificity
English
Modena, D., Vanoni, M., Englard, S., & Marmur, J. (1986). Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 248(1), 138-150 [10.1016/0003-9861(86)90410-8].
Modena, D; Vanoni, M; Englard, S; Marmur, J
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/34292
Citazioni
  • Scopus 38
  • ???jsp.display-item.citation.isi??? 35
Social impact