Purification and characterization of the cell-wall-associated and extracellular alpha-glucosidases from Saccharomycopsis fibuligera.
Cell-wall-associated and extracellular alpha-glucosidases were purified to homogeneity from Saccharomycopsis fibuligera KZ growing on a medium containing cellobiose as the sole source of carbon; this substrate has the greatest inducing effect on the production of both forms of the enzyme. Depending on the source of carbon, 75-90% of the enzyme is associated with cell wall, from which it can be completely released by 1% Triton X-100 at 25 degrees C in 2 h. Both enzymes are glycoproteins in monomeric form with an apparent molecular mass of 132 kDa estimated by SDS/PAGE and 135 kDa estimated by gel filtration. N-linked carbohydrate accounts for 12% of the total mass. Both forms exhibited optimum activity at pH 5.5 and seem to be stable in the pH range 4.0-8.0 on incubation at 4 degrees C for 24 h. The cell-wall-associated form had an optimum activity at 42.5 degrees C and was stable in the absence of substrate up to 30 degrees C, while the extracellular form had optimal activity at 52.5 degrees C and was stable up to 40 degrees C. Both forms are unable to renature after thermal inactivation. The cell-wall-associated and extracellular alpha-glucosidases cleaved the same kind of substrates, from maltose to maltoheptaose, isomaltase and panose, although showing different rates of hydrolysis, and had little or no activity with polysaccharides. The extracellular form cross-reacts with antibody raised against the cell-wall-associated form, and both forms show the same peptide pattern after cleavage with chymotrypsin. The amino acid sequences of six peptides from both forms show marked similarity to those of Schwanniomyces occidentalis glucoamylase.
Reiser
V
V
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Gasperík
J
J
D000595:Amino Acid Sequence; D002473:Cell Wall / Q000737:chemistry / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005110:Extracellular Space / Q000737:chemistry / Q000201:enzymology; D006596:Hexosaminidases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017830:Octoxynol / Q000494:pharmacology; D010449:Peptide Mapping; D012442:Saccharomycopsis / Q000201:enzymology*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature; D000520:alpha-Glucosidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D007527:Isoenzymes; D017830:Octoxynol; D006596:Hexosaminidases; D000520:alpha-Glucosidases