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 K-m and V-max values of this enzyme (for polygalacturonic acid) were 3.23 mg/mL and 0.15 mu mol/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 t(1/2) 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 (Delta E#) for heat inactivation was 5.341 kJ/mol, and the thermodynamic activation parameters Delta G#, Delta H#, and Delta S# were also calculated, revealing a potential application for the industry.