Modulation of biorecognition of glucoamylases with Concanavalin A by glycosylation via recombinant expression.
Various types of glucoamylases were prepared to modulate their biospecific interaction with Concanavalin A. Glucoamylase Glm was isolated from the native yeast strain Saccharomycopsis fibuligera IFO 0111. Two glycosylated recombinant glucoamylases Glu's of S. fibuligera HUT 7212 were expressed and isolated from the strains Saccharomyces cerevisiae and one, nonglycosylated, from Escherichia coli. The biospecific affinity of those preparations to Concanavalin A was investigated and compared with the commercially available fungal glucoamylase GA from Aspergillus niger. All glycosylated enzymes showed affinity to Concanavalin A characterized by their precipitation courses and by the equilibration dissociation constants within the range from 1.43 to 4.17 x 10(-6) M (determined by SPR method). The results suggested some differences in the interaction of Con A with the individual glucoamylases. The highest affinity to Con A showed GA. The recombinant glucoamylase Glu with the higher content of the saccharides was comprised by two binding sites with the different affinity. The glucoamylases with the lowest affinity (Glm and Glu with a lower content of saccharides) also demonstrated a nonspecific interaction with Con A in the precipitation experiments. The minimal differences between the individual glucoamylases were determined by the inhibition experiments with methyl-alpha-d-mannopyranoside.
Mislovicová
D
D
Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia. chemmisl@savba.sk
Masárová
J
J
Hostinová
E
E
Gasperík
J
J
Gemeiner
P
P
D001234:Aspergillus niger / Q000201:enzymology; D001665:Binding Sites; D011232:Chemical Precipitation; D003208:Concanavalin A / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005087:Glucan 1,4-alpha-Glucosidase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008766:Methylmannosides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012442:Saccharomycopsis / Q000201:enzymology; D020349:Surface Plasmon Resonance
D004791:Enzyme Inhibitors; D008766:Methylmannosides; D011994:Recombinant Proteins; D003208:Concanavalin A; C008466:methylmannoside; D005087:Glucan 1,4-alpha-Glucosidase