Characterisation and modification of a series of esterases for cold temperature applications

Togneri, Peter Daniel (2015) Characterisation and modification of a series of esterases for cold temperature applications. PhD thesis, University of Glasgow.

Abstract

The purpose of this project was to generate low temperature active esterases for a constituent of laundry detergent, to permit the hydrolysis of fat and oil stains in cold water washing. To determine how well the esterase could work against substrate bound to a surface at low temperatures, it was decided to utilise the esterases as ligands in atomic force microscopy to measure affinity of said esterases against non-emulsified substrates.

Early work involved the identification and characterisation of a novel family of esterases from extremophilic origins as possible ligands for use in the method described. Additional work was performed on testing the theory behind atomic force microscopy by developing ligands from thoroughly understood lipolytic enzymes and measuring any changes in activity. Finally, work was performed on making a psychrophilic variant through mutagenesis from an esterase that had not been previously studied in this project.

Results indicated that there was one candidate from the novel family which showed great activity against long-chain substrates at the desired temperature, but experiments indicated that no member of the novel family were monomers, and were thus deemed unsuitable for use with atomic force spectroscopy.

For this reason, other psychrophilic, monomeric esterase were explored. One such candidate was found, variants were made through directed evolution which were found to have increased activity over the wild-type form.

Results from collaborators working with atomic force microscopy using ligands designed in this project indicated that it was valid as a method for analysing esterase affinity to substrate.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Molecular biology, protein engineering, enzymology, directed evolution.
• Subjects: Q Science > QR Microbiology
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Rosser, Prof. Susan
• Date of Award: 2015
• Depositing User: Mr Peter Togneri
• Unique ID: glathesis:2015-6242
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 21 Apr 2015 11:03
• Last Modified: 02 May 2018 12:20
• URI: https://theses.gla.ac.uk/id/eprint/6242