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Title: Towards increasing the efficiency of Rubisco, through the use of carbonic anhydrase mimetics
Author: O'Donnelly, Kerry
ISNI:       0000 0004 5917 5745
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Increasing photosynthetic efficiency remains one of the few routes left for substantial increases in crop yields. Rubisco, the enzyme responsible for fixating carbon dioxide from the atmosphere, is one of the major bottlenecks of photosynthesis. Some organisms have evolved carbon concentrating mechanisms, utilising carbonic anhydrase, to increase photosynthetic efficiency, by increasing local CO2 concentrations around Rubisco. This project aims to chemically mimic the role of carbonic anhydrase, through the use of small metal-ligand complexes, and to test their ability to perturb Rubisco's activity. In order to achieve this goal, Rubisco had to be first isolated, and a robust Rubisco assay developed, to screen the efficacy of the CA mimetics. A hydrophobic interaction chromatography protocol for the isolation of Rubisco was developed, which eliminated the requirement of salt removal before column chromatography, reducing the number of purification steps in comparison to existing methods. The rapid protocol was employed to obtain highly pure and highly active Rubisco from Spinacia oleracea and Brassica oleracea. Furthermore, the protocol revealed, for the first time, the presence of hydrophobically distinct populations of Rubisco. A fast and sensitive 384-well plate Rubisco enzymatic assay was also developed, providing a robust platform to screen and test the efficacy of the carbonic anhydrase mimetics on Rubisco's activity. A suite of existing and novel carbonic anhydrase mimetics were synthesised. A comparison of the pH-dependant CO2/HCO3- catalytic rates revealed a number of insights into the parameters that promote high interconversion rates of HCO3- to CO2: i) high electron-donating ligands, and hydrophobic ligands for 'substrate channelling' increased HCO3- dehydration rates, ii) higher pKa values for Zn-OH2, favoured the interconversion of HCO3- to CO2, iii) Zn(II) was shown to have the highest catalytic rate for HCO3- dehydration, which was attributed to the weaker binding of HCO3- to Zn(II) than to Cu(II), Co(II) and Ni(II). Additionally, this thesis provides the first proof of concept data for the use of chemical tools that could help towards mitigating the loss of Rubisco's inefficiencies. Zn(II) mimetics were shown to alter Rubisco's activity, which was attributed to a change in local CO2 concentrations, following the addition of the mimetics.
Supervisor: Barter, Laura ; Long, Nicholas ; Woscholski, Rudiger Sponsor: Engineering and Physical Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available