DEVELOPMENT OF HIGH-MOLAR-MASS CELLOBIASE COMPLEX BY SPONTANEOUS PROTEIN-PROTEIN INTERACTION IN THE CULTURE FILTRATE OF TERMITOMYCES-CLYPEATUS

The 450 kDa cellobiase from Termitomyces clypeatus which migrates as a single band on IEF, PAGE and SDS-PAGE, was found to possess appreciab le sucrase activity. The fungus produced sucrase and cellobiase consti tutively in different media but with different activity ratios. The ki netics of secretion of the two enzymes was similar under in vivo and i n vitro conditions. HPGPLC analysis of the culture filtrates indicated the presence of both sucrase and cellobiase in the same protein fract ions of different molar mass, even in the 30-kDa protein fraction. No free sucrase or cellobiase could be detected in the culture filtrates. It was also observed that fractionation of cellobiase by (NH4)(2)SO4 precipitation was different with different amounts of associated sucra se activity present in the culture filtrate. The (NH4)(2)SO4-precipita ted cellobiase fraction also contained cellobiases in proteins of wide ly varied molar mass ranges. However, none of the low-molar mass prote ins other than the 450-kDa enzyme could be purified, as all low-molar- mass fractions spontaneously aggregated to the 450-kDa enzyme. Hydroph obic chromatography of the (NH4)(2)SO4-precipitated fractions followed by HPGPLC of the eluted active fraction yielded both cellobiase-free sucrase and a very low sucrase-containing cellobiase fraction. The cel lobiase fraction, homogeneous in PAGE, was also a high-molar-mass prot ein complex dissociating into a number of protein bands on SDS-PAGE. I t was suggested that the 450-kDa cellobiase was not liberated by the f ungus as a preformed enzyme complex but that the complex developed thr ough interaction of cellobiase with sucrase under in vine conditions a nd the possibility of the involvement of other proteins in the aggrega tion cannot be excluded.