Nitrile hydratase enantioselectivity as a function of genetic diversity and directed evolution

Schmid, Kate
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The South African soil isolate Rhodococcus rhodochrous ATCC BAA-870 encodes a low-molecular mass nitrile hydratase (L-NHase), similar to that of from R. rhodochrous J1, which may be used to produce enantiopure β-substituted amides for use in peptidomimetics and other pharmaceuticals. This study aimed to compare the protein sequence of this enzyme with other known and novel NHase, heterologously express enzyme, identify residues involved in enantioselectivity of the L-NHase for β-substituted nitrile substrates and finally to carry out directed evolution on the recombinantly expressed L-NHase to improve its enantioselectivity. Multiple South African soil isolates were genetically screened to identify any NHase genes. The NHase genes from R. rhodochrous ATCC BAA-870, R. rhodochrous J1, five South African Rhodococcal soil isolates and other reported NHase genes were compared to determine if patterns of gene sequence and substrate usage occurred. Homology modelling and previous studies were used to visualize residues within the substrate channel that may interact with substrates. Directed evolution using iterative saturation mutagenesis (ISM) and the combinatorial active site saturation test (CASTing) was done to introduce mutations to the selected residues. Chiral high performance liquid chromatography was attempted to test for enantioselectivity of the recombinant L-NHase. All of the Rhodococcus soil isolates were found to possess a gene for L-NHase but not H-NHase. With the L-NHases from R. rhodochrousJ1 and BAA-870, they formed three separate homologous genetic groups, however these not appear to have associated substrate usage patterns. Database mining revealed a possible origin for the R. rhodochrous J1 H-NHase. ISM-PCR was successful in introducing mutations within the beta subunit of the recombinant L-NHase but unfortunately not in the target region of the gene. The L-NHase from R. rhodochrous ATCC BAA-870 was successfully recombinantly expressed and was able to fully hydrolyse the substrates 4-methoxybenzonitrile, 3-amino-3-p-tolyl propanenitrile and 3-amino-3-(4-methoxyphenyl) propanenitrile, although enantioselectivity could not be quantified due to HPLC column degradation. Although some methods used in this study require optimisation, this is a promising approach to modify and possibly improve the enantioselectivity of the L-NHase from R. rhodochrous ATCC BAA-870 for β-substituted amides.
A thesis submitted to the Faculty of Science, University of the Witwatersrand Johannesburg in fulfilment for the requirements of the degree of Master of Science, 2020