The characterisation of acetophenone monooxygenase
Date
2011-04-04
Authors
Mutondo, Moola Siseho
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Abstract
Catechols are important precursors in the production of pharmaceuticals but they are
associated with many problems such as their instability and susceptibility to oxidation
and polymerisation. In order to avoid these problems, acylcatechols are used.
However, disadvantages exist in the industrial production of acylcatechols. The
production of acylcatechols industrially involves chemical syntheses that require large amounts of hazardous peracids, and accumulation of large amounts of undesirable
byproducts. The processes used also have low yields. A solution to these problems
associated with the production of acylcatechols lies in the use of biocatalytic means of synthesising acylcatechols.
Baeyer-Villiger monooxygenases (BVMOs) have been found to have the ability to
yield key chiral products of value in various chemoenzymatic syntheses used in
industry. This study investigates the potential of the BVMOs to synthesise
acylcatechols thereby avoiding the use of complex and hazardous procedures.
Preliminary screening of the ability of various bacteria to perform Baeyer-Villiger
oxidations to produce acylcatechols showed that Pseudomonas fluorescens ACB and
Arthrobacter sp. M5 have notable potential to catalyse such reactions.
In this study, the acetophenone monooxygenase produced by Arthrobacter sp. M5
was selected for further investigation. The acetophenone monooxygenase gene was
cloned in Escherichia coli HB101 in order to enable sequence analysis studies of the
gene. This also facilitated greater manipulation of the gene in a host (E. coli) that is
well studied and easy to apply in the overexpression of the acetophenone
monooxygenase gene. Overexpression of the acetophenone monooxygenase gene is
necessary because it allows for greater amounts of the acetophenone monooxygenase
to be produced for optimisation studies that enable the enzyme to be tailored to the
needs of industry. After optimisation, the acetophenone monooxygenase can be used
to biocatalytically produce valuable acylcatechols thus avoiding the drawbacks
associated with chemical syntheses.
A library of Arthrobacter sp. M5 total DNA was made in E. coli HB101 and was
screened for Baeyer-Villiger monooxygenase activity using two colorimetric
methods. The library was also screened using degenerate probes by Southern blotting.
Description
Keywords
catechol , Monooxygenase , Phenacetin