Purification and partial characterization of a recombinant Chlorophyllide a oxygenase (CAO)

The plant Arabidopsis thaliana converts Chlorophyll a to Chlorophyll b via the enzyme Chlorophyllide a Oxygenase (CAO). Chlorophyll a and b differ only at position 3, i.e., a methyl and aldehyde group, respectively. Work described in this report focuses on in vitro conversion of Chlorophyll a to b. The cao gene was cloned into a pMAL expression vector containing a N-terminal maltose binding tag which was used to transform E. coli expression host Rosetta facilitating isolation and expression of recombinant CAO protein. Cells were grown overnight, harvested by centrifugation, and the cell pellet resuspended in 20 ml low-salt buffer (LSB: 10 mM phosphate buffer pH 7.0 containing 30 mM NaCl, 10 mM beta-ME and 1 mM EGTA). Cells were lysed using a French press (1000 psi) and cell debris pelleted by centrifugation (4000 rpm for 20 minutes). Supernatant material was loaded onto an amylose column and the maltose binding protein purified using a Biologic Duoflow Chromatography system (Bio-Rad). Protein was eluted with LSB containing 50 mM maltose at a flow rate of 0.25 ml/min. Purified recombinant CAO protein was added to Synechocystis wild type lysate and conversion of Chlorophyll a to b monitored using thin layer chromatography and mass spectrometry.