Production of nitrile hydrolysing biocatalysts from Rhodococcus rhodochrous

Soobben, Marushka
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Biocatalysis plays an important role in initiating environmentally-friendly processes during chemical synthesis. This involves the utilisation of enzymes from various biological systems during the synthesis of chemicals. The use of biocatalysts provides a sustainable alternative to metal catalysts when manufacturing pharmaceuticals and chemicals. Members of the genus Rhodococcus have been found to host many enzymes that can be utilised in biocatalysis due to their highly diverse physiological and metabolic functions. Rhodococcus rhodochrousis able to code for both a nitrilase and a nitrile hydratase which are necessary for the bioconversion of nitriles to carboxylic acids. Here, dimethylformamide successfully induced nitrilases from R. rhodochrous A29, A99 and ATCC BAA-870. These strains were cultured using a benchtop bioreactor. Benzonitrile was used as a substrate for an activity assay where benzoic acid was successfully produced. This was shown by using TLC, FT-IR and NMR spectroscopy. Next, Escherichia coliBL21 (DE3) containing two plasmids encoding a cobalt nitrile hydratase from R. rhodochrous ATCC BAA-870 was cultured in a bioreactor and induced with IPTG. Protein quantification showed an increase in total cellular proteins after induction. Benzamide was successfully produced by using benzonitrile as the substrate during an activity assay indicating successful heterologous expression of a nitrile hydratase. In addition, 100% conversion rate was observed with a minimum of 32 U of enzyme activity for both enzymes. In future, the process whereby these enzymes are produced should be optimised and various substrates should be tested to determine the substrate specificity of nitrile hydrolysing enzymes from these strains
A dissertation Submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, 2020