Category
Poster - Basic
Description
This project sought the development of a systematic purification method for peroxidase enzymes from crude plant sources. Peroxidases represent valuable biotechnological tools for a wide range of applications; however, the majority of these applications have been developed utilizing a single member of the peroxidase family, that being the enzyme isolated from horseradish root. Development of a universal purification strategy that is effective for the isolation of peroxidases from a vast number of plant sources may open the door for the development of novel applications, or identify more effective peroxidase enzymes for many of the current applications. This prepared method is a simple step-by-step process, starting from the freshly harvested plant, and produces a partially purified sample of selected plant peroxidases that are higher in specific activity. The method involves grinding crude plant material in 20mM buffer, followed by the addition of 40% ammonium sulfate, which causes precipitation of high molecular weight contaminating protein. The high salt soluble fraction is subjected to liquid chromatography involving a phenyl-Sepharose resin and produces a highly enriched peroxidase enzyme. Two assays were used in this process, a guaiacol assay to determine enzyme activity and a Bradford assay to determine the amount of protein in the samples. These tests were conducted throughout each step of the purification process to confirm that the highest specific activity was found in the partially purified fraction of each sample.
Systematic Purification Scheme for Peroxidase Library Samples
Poster - Basic
This project sought the development of a systematic purification method for peroxidase enzymes from crude plant sources. Peroxidases represent valuable biotechnological tools for a wide range of applications; however, the majority of these applications have been developed utilizing a single member of the peroxidase family, that being the enzyme isolated from horseradish root. Development of a universal purification strategy that is effective for the isolation of peroxidases from a vast number of plant sources may open the door for the development of novel applications, or identify more effective peroxidase enzymes for many of the current applications. This prepared method is a simple step-by-step process, starting from the freshly harvested plant, and produces a partially purified sample of selected plant peroxidases that are higher in specific activity. The method involves grinding crude plant material in 20mM buffer, followed by the addition of 40% ammonium sulfate, which causes precipitation of high molecular weight contaminating protein. The high salt soluble fraction is subjected to liquid chromatography involving a phenyl-Sepharose resin and produces a highly enriched peroxidase enzyme. Two assays were used in this process, a guaiacol assay to determine enzyme activity and a Bradford assay to determine the amount of protein in the samples. These tests were conducted throughout each step of the purification process to confirm that the highest specific activity was found in the partially purified fraction of each sample.
Comments
Undergraduate