ABSTRACT
In the present work the radish (Raphanus sativus L.) was used as the low-cost alternative source of peroxidase. The enzyme was immobilized in different supports: coconut fiber (CF), calcium alginate microspheres (CAMs) and silica SBA-15/albumin hybrid (HB). Physical adsorption (PA) and covalent binding (CB) as immobilization techniques were evaluated. Immobilized biocatalysts (IBs) obtained were physicochemical and morphologically characterized by SEM, FTIR and TGA. Also, optimum pH/temperature and operational stability were determined. For all supports, the immobilization by covalent binding provided the higher immobilization efficiencies-immobilization yield (IY%) of 89.99 ± 0.38% and 77.74 ± 0.42% for HB and CF, respectively. For CAMs the activity recovery (AR) was of 11.83 ± 0.68%. All IBs showed optimum pH at 6.0. Regarding optimum temperature of the biocatalysts, HB-CB and CAM-CB maintained the original optimum temperature of the free enzyme (40 °C). HB-CB showed higher operational stability, maintaining around 65% of the initial activity after four consecutive cycles. SEM, FTIR and TGA results suggest the enzyme presence on the IBs. Radish peroxidase immobilized on HB support by covalent binding is promising in future biotechnological applications.
Subject(s)
Enzymes, Immobilized/chemistry , Peroxidase/chemistry , Plant Proteins/chemistry , Raphanus/enzymology , Enzyme StabilityABSTRACT
It is possible to analyze peroxidase (POD) from different vegetable sources by electrophoresis. Zymography, i.e., a SDS-PAGE method to detect enzyme activity, is used to specifically detect POD activity and to visualize the total protein profile. For this purpose, we describe how a radish homogenate is prepared and submitted first to electrophoresis, and then, the POD activity present in the gel is reactivated and selectively stained using guaiacol as substrate. After scanning the gel, the same gel is further stained with Coomassie blue to determine the whole protein profile of the sample.