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

Area of Science:

• Biochemistry
• Plant Science
• Enzymology

Background:

• Chlorophyll degradation, catalyzed by chlorophyllase, is a key process in plant senescence.
• Inhibiting chlorophyllase could extend the shelf life and improve the appearance of plant products.

Purpose of the Study:

• To develop a 96-well plate-based purification and assay system for measuring chlorophyllase activity.
• To enable rapid assessment of chlorophyllase activity for screening potential senescence-delaying compounds.

Main Methods:

• Recombinant hexahistidine-tagged Triticum aestivum (wheat) chlorophyllase was purified using immobilized metal affinity chromatography in 96-well plates.
• Chlorophyllase activity was assayed using both chlorophyll and p-nitrophenyl-ester substrates.
• Kinetic parameters, pH profiles, and inhibitor effects were determined using the plate-based system.

Main Results:

• The immobilized enzyme displayed similar kinetic parameters to the purified enzyme.
• Using p-nitrophenyl-ester substrates allowed for continuous measurement, overcoming limitations of chlorophyll substrate assays.
• The plate-based system was validated for assessing enzyme kinetics and inhibitor effects.

Conclusions:

• A convenient and rapid 96-well plate system for chlorophyllase purification and assay was successfully developed.
• This system facilitates the assessment of chlorophyllase activity and the screening of potential senescence inhibitors.
• The developed assay system has significant implications for improving the shelf life of plant products.