PURIFICATION AND CHARACTERIZATION OF THE CELL-WALL-ASSOCIATED AND EXTRACELLULAR ALPHA-GLUCOSIDASES FROM SACCHAROMYCOPSIS-FIBULIGERA

Cell-wall-associated and extracellular alpha-glucosidases were purifie d to homogeneity from Saccharomycopsis fibuligera KZ growing on a medi um containing cellobiose as the sole source of carbon; this substrate has the greatest inducing effect on the production of both forms of th e enzyme. Depending on the source of carbon, 75-90% of the enzyme is a ssociated 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 estimate d by SDS/PAGE and 135 kDa estimated by gel filtration. N-linked carboh ydrate accounts for 12% of the total mass. Both forms exhibited optimu m 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 optima l activity at 52.5 degrees C and was stable up to 40 degrees C. Both f orms 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, a lthough showing different rates of hydrolysis, and had little or no ac tivity 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 am ino acid sequences of six peptides from both forms show marked similar ity to those of Schwanniomyces occidentalis glucoamylase [Dohmen, Stra sser, Dahlems and Hollenberg (1990) Gene 95, 111-121] and some other r elated hydrolases.