The characterisation of acetophenone monooxygenase

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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.
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Keywords
catechol , Monooxygenase , Phenacetin
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