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چکیده
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Pectinases are one of the most important industrial enzymes and hydrolyze pectin to galacturonic acid. Various methods are used for pectinase purification from microbial sources including precipitation and chromatography. Nowadays many studies focused on purification and immobilization of enzymes by magnetic beads. In this investigation purification and immobilization of Piriformospora indica pectinase by kappa carrageenan-chitosan was investigated. P. indica was cultured on YPG medium supplemented with apple pectin for 6 days. The slurry was filtered by centrifugation at 10,000 rpm at 4 ºC for 15 min. Total protein content and pectinase activity determined by Bradford assay and DNS method, respectively. One unit of pectinase activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol galacturonic acid per min under the assay condition. Synthesized magnetic nanoparticle characterized by XRD, FTIR and TEM analysis. Pectinase purification was initiated by salting out of crude proteins by ammonium sulfate, continued by dialysis and terminated by magnetic precipitation. According to the SDS-PAGE results, purification degree by using nanomagnetics was increased and showed higher purification. The highest protein content and pectinase activity of purified samples were 0.187mg/ml and 0.668U/ml, respectively. The immobilization was confirmed by SEM analysis. Activity of pectinase which immobilized on kappa carrageenan-chitosan nanomagnetics was 3.084 U/ml. In conclusion, kappa carrageenan-chitosan nanomagnetics could open new insights for purification and immobilization of pectinase. Further studies on pectinase immobilization are ongoing.
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