Discovery and Engineering of Novel Enzymes for Industrial Biocatalysis

Abstract

Enzymes as biocatalysts offer many benefits over traditional catalysis such as working under mild pH and temperature, producing enantiomerically pure products and using less energy than many industrial processes. Enzymes from nature provide us with a huge variety of industrially desirable reactions; however, there is a gap between what biology provides us and what is required for industry. Often the activity, stability or specificity of an enzyme needs to optimised to be industrially viable. An example is carboxylic acid reductase, an enzyme that catalyses the reduction of carboxylic acids to aldehydes. Aldehydes serve as valuable intermediates in many products, yet are costly and difficult to synthesise, therefore, enzymatic conversion is an attractive prospect. In this thesis, we carry out biophysical characterisation of a group of carboxylic acid reductases, determining their structure and multimeric state in solution, inhibition kinetics, and elemental composition using MicroPIXE. Furthermore, we
propose that the enzyme should be reclassified as a nicotino-enzyme, an enzyme family with a tightly bound NADPH co-factor. Lastly, we demonstrate that large domain movements occur when the enzyme binds to its substrate.
The second part of this thesis focuses on enzymes that depolymerise plastics. Plastic pollution is one of the biggest threats facing the environment. The high rate of production of disposable plastic, combined with the recalcitrance of plastic to biodegradation, is resulting in massive accumulation in the environment. In 2016, a bacterium called Ideonella sakaiensis was discovered that could utilise polyethylene terephthalate as a sole carbon source by secretion of an enzyme called PETase. Here we describe PETase in terms of structure and show that it has structural similarities with the cutinase family of enzymes. We use a combination of techniques to show that it is active against aromatic substrates such as polyethylene terephthalate or polyethylene furanoate but not aliphatic substrates such as polybutylene succinate. Furthermore, we used rational design to engineer a variant with improved catalytic activity on polyethylene terephthalate and polyethylene furanoate.

Date of Award	22 Apr 2020
Original language	English
Awarding Institution	University of Portsmouth
Supervisor	John McGeehan (Supervisor)