Discovery of minimalistic biocatalysts via biocatalytic self-assembly

  • Krystyna Duncan

Student thesis: Doctoral Thesis


Efficient and selective catalysis of chemical reactions and biological pathways by enzymes has long inspired bio- and supramolecular chemists. Many have tried to mimic this enzymatic activity through the design of de novo catalysts however the proficient rate of an enzyme has not yet been matched by a synthetic mimic. Short peptide sequences have emerged as potentially useful molecules in the design and discovery of synthetic catalysts. Their side chains provide the same chemical functionality as found in proteins and can also manipulate the local environment to favor catalysis. They may also form self-assembled nanostructures which can be utilized to present ordered functionality as well as demonstrating the ability to display catalytic and binding functionalities in a multivalent fashion. This has been shown to improve catalytic rates although they still do not match that of natural enzymes. In contrast to designing catalysts, there are several approaches to screen libraries of peptide sequences for transition state binding with the advantage being that sequence can be directly linked to functionality. Phage display in particular is a useful tool for the discovery of new peptide catalysts based on specific enzymatic activity.By utilizing enzymatically triggered self-assembly of an aromatic peptide amphiphile, Fmoc-Thr-Leu-OMe, formed in situ by condensation of the amino acid precursors, in combination with a phage display peptide library; a new methodology has been developed to identify peptide catalysts for specific enzymatic function based on a direct link between sequence and activity. The self-assembly of the precursors in the presence of the catalytic peptide results in a gel aggregate at the tip of the phage allowing this extra weight to be used to separate the phage from the bulk sample by centrifugation before being amplified and sequenced. It was possible to characterize the amidase and esterase activity of the phage identified in this manner by several techniques including TEM, HPLC, fluorescence as well as UV-Vis spectroscopy, demonstrating by several means that the sequences identified do possess (modest) amidase and esterase functionality. In addition to the full characterization of the catalytic phage which demonstrated the desired activity, the peptide sequences alone were also characterized for amidase and esterase enzymatic functionality using ester hydrolysis assays based on fluorescence and UV-Vis spectroscopy.Of the sequences identified by phage display, one cysteine containing peptide sequence demonstrated significant rate enhancement in comparison to the remaining histidine containing samples therefore it was studied further to gain an insight into the mechanism of catalysis utilizing CD and UV-Vis spectroscopy. Cysteine is a known catalytic amino acid and is the main source of nucleophilic attack in the presence of a basic amino acid. Terminal amino groups have also been found to enhance the rate at which ester bonds are broken and this is demonstrated by the tripeptide KYF. CD, TEM, fluorescence and UV-Vis spectroscopy were utilized to demonstrate the self-assembling propensity as well as catalytic activity of KYF and again show an understanding of the catalytic mechanism. This system self-assembles at low concentrations to form short fibrils, although in this case, the formation of nanostructures in the sample does not enhance the catalysis as seen in literature examples.;Next, as combinatorial methodology was successfully developed for the identification of amidase and esterase active peptides, the same technology was taken advantage of to screen for phosphatase activity. Fmoc-Tyr(PO₄)³⁻-Ala-OH and Fmoc-Phe-Tyr(PO₄)³⁻-OH form self-assembled hydrogel materials on the addition of alkaline phosphatase. Fibrous networks develop trapping
Date of Award13 Jun 2016
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsEPSRC (Engineering and Physical Sciences Research Council)
SupervisorRein Ulijn (Supervisor) & Peter Halling (Supervisor)

Cite this