Development of a high-throughput purification method and a continuous assay system for chlorophyllase.
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 inhibitory effects of metal ions and esterase inhibitors, and the effect of functional group-modifying reagents validated the utility of the plate-based system. The combined purification and assay system provides a convenient and rapid method for the assessment of chlorophyllase activity.
Arkus
Kiani A J
KA
Donald Danforth Plant Science Center, St. Louis, MO 63132, USA.
Jez
Joseph M
JM
D001681:Biological Assay / Q000379:methods; D002265:Carboxylic Ester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism*; D002734:Chlorophyll / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli; D017343:Genes, Plant / Q000502:physiology; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009596:Nitrophenols / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry; D014908:Triticum / Q000378:metabolism
C471213:His-His-His-His-His-His; D009596:Nitrophenols; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; D006639:Histidine; C008642:4-nitrophenyl acetate; D002265:Carboxylic Ester Hydrolases; C019875:chlorophyllase