Practical oxyfunctionalisation biocatalysts by engineering monooxygenases into peroxyzymes.

Introduction

Chemistry is far away from being a mature science: many desirable transformations are still out of scope. One important example is the selective (oxy)functionalisation of non-activated C-H bonds, which still represents a dream reaction of organic chemistry. This is because balancing high reactivity (needed for the activation of inert C-H bonds) with selectivity is difficult to achieve. Enzymes, specifically monooxygenases, are catalysts that principally solve this challenge.

Challenges with Monooxygenases

Monooxygenases, however, are not practical catalysts for organic chemistry. This is because they have evolved to enable the survival of their host organisms and not to suit the needs of organic chemists.

Complex Molecular Architecture

In particular, the complex molecular architecture of monooxygenases necessitates:

• O2
• Stoichiometric reductants
• Additional catalytic components

These factors, together with mechanistic challenges arising from their complex molecular architecture, impede their chemistry-wide application.

Project Goals

PeroxyZyme aims at solving these issues and establishing evolved monooxygenases (peroxyzymes) as practical catalysts for organic chemistry.

Key Innovations

Peroxyzymes will be able to function with simple hydrogen peroxide rather than via the natural, albeit complex and vulnerable electron transport chains. This fundamental change in the monooxygenases catalytic mechanisms will be achieved by a mechanism-driven and experimentally validated semi-rational engineering approach.

Characterization and Improvement

Evolved peroxyzymes will be characterised using up-to-date (ultra)fast spectroscopy to identify:

1. Catalytic bottlenecks
2. Possible inactivation mechanisms

This molecular understanding will provide the basis for further improvement of first-generation peroxyzymes.

Practical Applications

The practical usefulness of evolved peroxyzymes will be demonstrated on a preparative scale by using them in non-aqueous reaction media, enabling high product concentrations and space-time yields.