Probing the mechanism of CH-activation by lytic polysaccharide monooxygenases

Abstract

The recently discovered lytic polysaccharide monooxygenases are of increasing interest for biocatalytic applications as their oxidative mode of action could be potentially exploited for C-H-activation chemistries in industrial or pharmaceutical relevant building blocks. Although the oxidative mode of action opens up new opportunities in theory, the mechanism still remains poorly understood, as well as the reaction stoichiometry for these carbohydrate degrading enzymes as it is still unclear whether these enzymes use molecular oxygen and a reductant for their catalytic activity or can rely on hydrogen peroxide as an electron donor. Furthermore, mutagenesis studies and directed evolution approaches are difficult to carry out so far due to the limitation in strong and reliable assays available today. In this project the objective is to tackle the problems involved in LPMO evolution for biocatalysis applications. First of all, the enzymes were expressed in E. coli as an expression host, delivering quick and easy production of high yields of protein. The isolated proteins were used for initial activity studies exploiting uncoupling reactions and peroxidase substrates. These initial studies allowed determination of enzyme activity although not towards the natural substrates. Additionally, the degradation of a mixture of labelled cello-oligosaccharides was analysed via MALDI (matrix assisted laser desorption ionisation) mass spectrometry and it was shown that one enzyme candidate that has been described as active towards oligomeric substrates possessed activity towards i-tag labelled cello-oligosaccharides. This now allows development of high-throughput assays required for in depth mutational studies for the structure-function relationships and directed evolution attempts on LPMOs.

Date of Award	1 Aug 2020
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Anthony Green (Supervisor) & Sam Hay (Supervisor)