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 appreciable sucrase activity. The fungus produced sucrase and cellobiase constitutively in different media but with different activity ratios. The kinetics of secretion of the two enzymes was similar under in vivo and in vitro conditions. HPGPLC analysis of the culture filtrates indicated the presence of both sucrase and cellobiase in the same protein fractions 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)2SO4 precipitation was different with different amounts of associated sucrase activity present in the culture filtrate. The (NH4)2SO4-precipitated cellobiase fraction also contained cellobiases in proteins of widely varied molar mass ranges. However, none of the low-molar mass proteins other than the 450-kDa enzyme could be purified, as all low-molar-mass fractions spontaneously aggregated to the 450-kDa enzyme. Hydrophobic chromatography of the (NH4)2SO4-precipitated fractions followed by HPGPLC of the eluted active fraction yielded both cellobiase-free sucrase and a very low sucrase-containing cellobiase fraction. The cellobiase fraction, homogeneous in PAGE, was also a high-molar-mass protein complex dissociating into a number of protein bands on SDS-PAGE.(ABSTRACT TRUNCATED AT 250 WORDS)
Roy
S B
SB
Department of Applied Biochemistry, Indian Institute of Chemical Biology, Calcutta, India.
Ghosh
A K
AK
Sengupta
S
S
Sengupta
S
S
D001487:Basidiomycota / Q000201:enzymology*; D002241:Carbohydrates; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D013393:Sucrase / Q000378:metabolism; D001617:beta-Glucosidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D002241:Carbohydrates; D009097:Multienzyme Complexes; D001617:beta-Glucosidase; D013393:Sucrase