Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus.
A major extracellular endoglucanase purified to homogeneity from Thermoascus aurantiacus had a M(r) of 34 kDa and a pI of 3.7 and was optimally active at 70-80 degrees C and pH 4.0-4.4. It was stable at pH 2.8-6.8 at 50 degrees C for 48 h and maintained its secondary structure and folded conformation up to 70 degrees C at pH 5.0 and 2.8, respectively. A 33-amino acid sequence at the N terminus showed considerable homology with 14 microbial endoglucanases having highly conserved 8 amino acids (positions 10-17) and Gly, Pro, Gly, and Pro at positions 8, 22, 23, and 32, respectively. The enzyme is rich in Asp (15%) and Glu (10%) with a carbohydrate content of 2.7%. Polyclonal antibodies of endoglucanase cross-reacted with their own antigen and with other purified cellulases from T. aurantiacus. The endoglucanase was specific for polymeric substrates with highest activity toward carboxymethyl cellulose followed by barley beta-glucan and lichenan. It preferentially cleaved the internal glycosidic bonds of Glc(n) and MeUmbGlc(n) and possessed an extended substrate-binding site with five subsites. The data indicate that the endoglucanase from T. aurantiacus is a member of glycoside hydrolase family 5.
Parry
N J
NJ
Food Materials Science Division, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK.
Beever
D E
DE
Owen
E
E
Nerinckx
W
W
Claeyssens
M
M
Van Beeumen
J
J
Bhat
M K
MK
D001203:Ascomycota / Q000201:enzymology*; D002152:Calorimetry, Differential Scanning; D002266:Carboxymethylcellulose Sodium / Q000737:chemistry; D002480:Cellulase / Q000737:chemistry* / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D005110:Extracellular Space / Q000201:enzymology; D005936:Glucans / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000502:physiology; D013696:Temperature; D013816:Thermodynamics
D005936:Glucans; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium; C015968:lichenin