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
References
Jan 1, 1986·Methods in Enzymology·Y Shioi, T Sasa
Jan 1, 1983·Journal of Biochemistry·K TanakaT Horio
Dec 1, 1982·Journal of Biochemistry·K TanakaT Horio
Jan 1, 1982·Methods in Enzymology·K J Ellis, J F Morrison
Aug 1, 1994·Journal of Biochemistry·M I Mínguez-MosqueraL Gallardo-Guerrero
Sep 29, 1999·Planta·V ScheumannW Rudiger
Oct 8, 1999·Journal of Biotechnology·R GaffarB Bisakowski
Dec 28, 1999·Proceedings of the National Academy of Sciences of the United States of America·T TsuchiyaK Takamiya
Jan 29, 2000·The Plant Journal : for Cell and Molecular Biology·D Jacob-WilkY Eyal
Oct 25, 2000·Trends in Plant Science·K I TakamiyaH Ohta
Feb 24, 2001·Cellular and Molecular Life Sciences : CMLS·S Hörtensteiner
Oct 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·T TrebitshJ Riov
Feb 12, 2002·Zeitschrift Für Naturforschung. C, a Journal of Biosciences·D Hornero-Méndez, M I Mínguez-Mosquera
Apr 16, 2002·Plant Physiology·Celso Eduardo Benedetti, Paulo Arruda
Dec 1, 1996·Plant Physiology·P. MatileB. Krautler
Jan 29, 2003·Plant & Cell Physiology·Tohru TsuchiyaKen-ichiro Takamiya
Apr 5, 2003·Physiologia Plantarum·María Roca, María Isabel Mínguez-Mosquera
Mar 12, 2004·Annual Review of Plant Physiology and Plant Molecular Biology·Philippe MatileHoward Thomas
Dec 16, 2004·The Plant Cell·Tarja KariolaE Tapio Palva
May 26, 2005·Archives of Biochemistry and Biophysics·Kiani A J ArkusJoseph M Jez
Aug 10, 2005·Journal of Biotechnology·Salwa KarbouneSelim Kermasha
Oct 1, 1987·The New Phytologist·George A F HendryStanley B Brown