Characterization of trehalase activities from the thermophilic fungus Scytalidium thermophilum.
The thermophilic fungus Scytalidium thermophilum produced large amounts of intracellular and extracellular trehalase activity when grown on starch as the sole carbon source. The specific activity of the purified proteins: 1700 U (mg protein)-1 (extracellular) and 3700 U (mg protein)-1 (intracellular), was many times higher than the values reported for other microbial sources. The apparent molecular mass of the native enzymes was estimated to be 370 kDa (extracellular trehalase) and 398 kDa (intracellular trehalase) by gel-filtration chromatography. Analysis by SDS-PAGE showed unique polypeptide bands of approx. 82 kDa (extracellular trehalase) and 85 kDa (intracellular trehalase), suggesting that the native enzymes were composed of five subunits. The carbohydrate content of extracellular and intracellular trehalases was estimated to be 81% and 51%, respectively. Electrofocusing indicated a pI of 3.7 and 3.4, respectively, for the extracellular and intracellular enzymes. Both trehalases were highly specific for trehalose and were stimulated by calcium and manganese. Calcium and manganese also protected both trehalases from thermoinactivation. Inhibition was observed in the presence of aluminium, mercurium, copper, zinc, EDTA, ADP, and ATP. Apparent Km values, for the extracellular and intracellular trehalases, were 3.58 mM and 2.24 mM, respectively. The optimum of pH for the extracellular and the intracellular trehalase was 6.0, and the optimum of temperature 60 degrees C and 65 degrees C, respectively.
Kadowaki
M K
MK
Departamento de Biologia, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Brazil.
Polizeli
M L
ML
Terenzi
H F
HF
Jorge
J A
JA
D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D007700:Kinetics; D003904:Mitosporic Fungi / Q000201:enzymology*; D014198:Trehalase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*
D014198:Trehalase