The kinetic characterization of mutated cyanobacterial dual-functioning fructose/sedoheptulose bisphosphate using a malachite green colorimetric assay

Abstract: The increased human dependence on fossil-based fuels, industrial chemicals, and structural materials has caused a drastic increase in atmospheric CO2 levels during the past 50 years. Consequences of this have been observed in the disruption of terrestrial and aquatic ecosystems. Bioengineering of photosynthetic organisms capable of converting CO2 into biomass and value-added organic molecules provide a promising solution to the problem. However, faster CO2 fixation rates are needed in order to meet industrial scale production capacities. Previously published studies have mainly focused on the overexpression of Calvin cycle enzymes or engineering of ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) whereas fewer have looked into the engineering of enzymes downstream of RuBisCO. In this thesis, a malachite green-based colorimetric assay was optimized and used in characterizing the kinetic properties of wild type bifunctional cyanobacterial fructose/sedoheptulose bisphosphatase (cy-F/SBPase) and variants of the enzyme with respect to the hydrolysis of one of its substrates (fructose 1,6-bisphosphate). The assay was validated by successfully assaying a set of previously kinetically characterized cy-F/SBPase variants as well as the wild type. Subsequently, a group of rationally de novo designed mutants of the enzyme was kinetically characterized. This way, a crude understanding of new aspects of the protein structure-function relationship was established, which could potentially guide future protein engineering efforts on cy-F/SBPase. The developed malachite green assay could also complement such research efforts as a result of its multiplexed format which is desirable when assaying larger protein libraries.