Parker, B.M.;
(2009)
Directed evolution of an L-aminoacylase biocatalyst.
Doctoral thesis , UCL (University College London).
Abstract
Enzymes from extreme environments possess highly desirable traits of activity and stability under process conditions. One such example is L-aminoacylase (E.C. Number 3.5.1.14) from the thermophilic archaeon Thermococcus litoralis (TliACY), which catalyzes the hydrolysis of the amide bond between the nitrogen and the carbonyl group of an N-protected L-amino acid. As this reaction is enantiomerspecific, L-aminoacylase is often used to resolve racemic mixtures in the preparation of chiral intermediates. Using protein engineering techniques, the capabilities of such biocatalysts can be extended. This thesis seeks to compare the ability of libraries created by error-prone PCR and structure-guided mutagenesis methods to identify residues governing substrate specificity. Libraries were constructed to screen for variants which showed a shift in substrate specificity towards aliphatic amino acids, whilst maintaining the preferred benzoyl protecting group. An existing fluorescent screen for proteases was adapted for the high-throughput screening of mutant L-aminoacylase libraries, and was demonstrated to be capable of quantitatively detecting millimolar quantities of product. From an error-prone PCR library over the dimerization region of the enzyme, 10000 variants were screened against a variety of N-benzoylated amino acid substrates. Sequence alignment and homology model construction allowed a 3-D model of TliACY to be built from its closest neighbours in the PDB. Phylogenetic comparison methods were used to identify regions and residues of significance, which were then examined using sitedirected saturation mutagenesis. Purification and characterisation of selected variants of TliACY examined two mutants in detail: S4C (S100T / M106K) which exhibited a 300% improvement in catalytic efficiency over wild-type on the Nbenzoyl valine substrate, and S3B (F251K) which showed a shift in substrate preference against aliphatic amino acid substrates.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | Directed evolution of an L-aminoacylase biocatalyst |
| Language: | English |
| Additional information: | Authorisation for digitisation not received |
| UCL classification: | UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Engineering Science > Dept of Biochemical Engineering |
| URI: | https://discovery.ucl.ac.uk/id/eprint/18724 |
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