TY - JOUR
T1 - Development of a high-throughput purification method and a continuous assay system for chlorophyllase
AU - Arkus, Kiani A.J.
AU - Jez, Joseph M.
N1 - Funding Information:
We thank the Beachy lab for access to their plate reader and Edgar Cahoon for helpful discussions. This work was supported by funds from the Donald Danforth Plant Science Center and by a Collaborative Research and Education Grant from DuPont. K.A.J.A. was supported by an NSF-Research Experiences for Undergraduates Grant (NSF-DBI-0244155).
PY - 2006/6/1
Y1 - 2006/6/1
N2 - 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.
AB - 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.
KW - Chlorophyll
KW - Chlorophyll degradation
KW - Chlorophyllase
KW - Hydrolase
UR - http://www.scopus.com/inward/record.url?scp=33646358200&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2006.03.034
DO - 10.1016/j.ab.2006.03.034
M3 - Article
C2 - 16643837
AN - SCOPUS:33646358200
SN - 0003-2697
VL - 353
SP - 93
EP - 98
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -