Regulation (alteration) of activity and conformation of sucrase by coaggregation with cellobiase in culture medium of Termitomyces clypeatus.
Extracellular sucrase (S) of Termitomyces clypeatus was aggregated with cellobiase (C) in culture filtrate and coaggregates of sucrase to cellobiase with different activity ratios (S/C) were obtained during purification. Specific activity of the enzyme decreased significantly, after purification of sucrase free from cellobiase. Purified sucrase was characterized as a glycoprotein of molar mass around 55kDa as indicated by SDS-PAGE and HPGPLC. K(m) and V(max) of the purified enzyme were determined as 34.48 mM and 13.3 U/mg, respectively, at optimum temperature (45 degrees C) and pH (5.0). Substrate affinity and reaction velocity of the purified enzyme, free from cellobiase, was lowered by approximately 3.5 and 55 times, respectively, than that of the enzyme obtained from culture filtrate. The instant regain of sucrase activity up to the extent of 41% was obtained on in vitro addition of cellobiase (free from sucrase) to the enzyme in incubation mixture. Conformation of the enzyme free from cellobiase appeared to be significantly different from that of the coaggregate, as analyzed by circular dichroic and light scattering spectroscopy. It was concluded that activity and conformation of sucrase is regulated (altered) by heteroaggregation with cellobiase in the fungus.
Mukherjee
Sumana
S
Department of Applied Biochemistry, Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Calcutta 700032, India. sumankhowala@iicb.res.in
Basak
Soumen
S
Khowala
Suman
S
D000363:Agaricales / Q000737:chemistry; D001487:Basidiomycota / Q000201:enzymology; D002942:Circular Dichroism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008970:Molecular Weight; D011487:Protein Conformation; D012680:Sensitivity and Specificity; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet; D013393:Sucrase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013395:Sucrose; D013696:Temperature; D001617:beta-Glucosidase / Q000737:chemistry* / Q000378:metabolism
D013395:Sucrose; D001617:beta-Glucosidase; D013393:Sucrase