Development of a high-throughput purification method and a continuous assay system for chlorophyllase

Analytical Biochemistry
Kiani A J Arkus, Joseph M Jez

Abstract

In the degradation of chlorophyll, chlorophyllase catalyzes the initial hydrolysis of the phytol moiety from the pigment. Since chlorophyll degradation is a defining feature of plant senescence, compounds inhibiting chlorophyllase activity may delay senescence, thereby improving shelf life and appearance of plant products. Here we describe the development of a 96-well plate-based purification and assay system for measuring chlorophyllase activity. Integrated lysis and immobilized metal affinity chromatography plates were used for purifying recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase from Escherichia coli. Chlorophyllase assays using chlorophyll as a substrate showed that the immobilized fusion protein displayed kinetic parameters similar to those of recombinant enzyme purified by affinity chromatography; however, the need to extract reaction products from a multiwell plate limits the value of this assay for high-throughput screening applications. Replacing chlorophyll with p-nitrophenyl-ester substrates eliminates the extraction step and allows for continuous measurement of chlorophyllase activity in a multiwell plate format. Determination of steady state kinetic constants, pH rate profile, the inh...Continue Reading

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Citations

Apr 11, 2013·Chemical Reviews·Chad P SatoriEdgar A Arriaga
Mar 1, 2008·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Kiani A J Arkus, Joseph M Jez

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