Towards the Industrial Application of the Baeyer-Villiger Monooxygenase MO14 from Rhodococcus jostii

The Baeyer-Villiger reaction is a key reaction in organic synthesis, due to the utility of the addition
of an oxygen atom adjacent to a carbonyl group. This reaction is also useful in an industrial
setting and Baeyer-Villiger monooxygenases are often capable of performing this reaction in an
exceptionally regio- and enantio-selective manner. This remarkable selectivity means that they are
excellent biocatalyst targets for a number of industrially relevant syntheses, including the stereoselective
synthesis of lactones and sulfoxides and also the resolution of racemic species, including
�-hydroxyketones.
This PhD project focussed on the enzyme MO14, encoded by the gene ro03437 from the bacterium
Rhodococcus jostii sp. RHA1. MO14 has previously demonstrated particularly high regioand
enantioselectivity in the conversion of the model BVMO substrate, bicyclo[3.2.0]hept-2-en-6-
one. This enzyme, along with several others from the same organism, was selected for study of
the activity and MO14 in particular has been singled out due to its remarkable breadth of substrate
scope and S-selective character.
All of the selected enzymes were tested against a selection of industrially relevant targets, then
focus concentrated on MO14, as it demonstrated the most interesting biocatalytic activities. A variety
of purification strategies were examined for the purification of MO14, with several potential
lines of enquiry identified for the full purification of this enzyme. A study of the transformation of
bicyclo[3.2.0]hept-2-en-6-one was conducted, with several variables of the reaction assessed, followed
by investigation of the ability of this enzyme to transform a range of prochiral sulfides. As a
precursor to industrial applications, a series of scale-up reactions were conducted using MO14 to
examine the potential for use on a scale much larger than standard laboratory investigation. Finally,
a series of mutants were generated to examine the origin of the exceptional selectivity exhibited by
this enzyme.