Purification, characterization, kinetic properties, and thermal behavior of extracellular polygalacturonase produced by filamentous fungus Tetracoccosporium sp.
For the first time, a polygalacturonase from the culture broth of Tetracoccosporium sp. was isolated and incubated at 30 degrees C in an orbital shaker at 160 rpm for 48 h. The enzyme was purified by ammonium sulfate precipitation and two-step ion-exchange chromatography and had an apparent molecular mass of 36 kDa, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Its optimum activity was at pH 4.3 and 40 degrees C, and the Km and Vmax values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 micromol/min, respectively. Ag+, Co2+, EDTA, Tween-20, Tween-80, and Triton X-100 stimulated polygalacturonase activity whereas Al3+, Ba2+, Ca2+, Fe2+, Fe3+, Ni2+, Mg2+, Mn2+, and SDS inhibited it. In addition, iodoacetamide and iodoacetic acid did not inhibit enzyme activity at a concentration of 1 mM, indicating that cysteine residues are not part of the catalytic site of polygalacturonase. We studied the kinetic properties and thermal inactivation of polygalacturonase. This enzyme exhibited a t1/2 of 63 min at 60 degrees C and its specific activity, turnover number, and catalytic efficiency were 6.17 U/mg, 113.64 min-1, and 35.18 mL/(min.mg), respectively. The activation energy (DeltaE#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters DeltaG#, DeltaH#, and DeltaS# were also calculated, revealing a potential application for the industry.
Aminzadeh
Saeed
S
Department of Biochemistry and Biophysics, Faculty of Science, Tarbiat Modares University, Tehran, Iran.
Naderi-Manesh
Hossein
H
Khajeh
Khosro
K
Naderi-Manesh
Mehdi
M
D004789:Enzyme Activation; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D007700:Kinetics; D011096:Polygalacturonase / Q000737:chemistry* / Q000302:isolation & purification*; D013696:Temperature
D005656:Fungal Proteins; D011096:Polygalacturonase