Abstract
Oxidative cleavage of alkenes is a widely employed process allowing oxyfunctionalization to corresponding carbonyl compounds. Recently, a novel biocatalytic oxidative alkene cleavage activity on styrene derivatives was identified in TM1459 from Thermotoga maritima. In this work we engineered the enzyme by site-saturation mutagenesis of active site amino acids to increase its activity and to broaden its substrate scope. A high-throughput assay for the detection of the ketone products was successfully developed. Several variants with up to twofold improved conversion level of styrene derivatives were successfully identified. Especially, changes in or removal of the C-terminus of TM1459 increased the activity most significantly. These best variants also displayed a slightly enlarged substrate scope.
Highlights
The carbonyl functionality is widespread in nature and can be found in a broad range of compounds, including metabolites or hormones, in form of aldehyde and ketone
All chemicals were purchased from Sigma-Aldrich or Carl Roth GmbH, if not stated otherwise. p-Chloro-acetophenone (97%) and p-Chloro-α-methylstyrene (95%) were obtained from Lancaster (Alfa Aesar, Ward Hill, MA, USA)
TM1459 from the thermophilic bacterium Thermotoga maritima consists of 114 amino acids and was crystallized as a dimer (Jaroszewski et al, 2004)
Summary
The carbonyl functionality is widespread in nature and can be found in a broad range of compounds, including metabolites or hormones, in form of aldehyde and ketone. Several enzymes are known to catalyze oxidative alkene cleavage reactions to different extent – in many cases as minor (undesired) side reaction, such as hemedependent enzymes with oxygen (Bugg, 2011; Efimov et al, 2011) or hydrogen peroxide as oxidant (Ortiz de Montellano et al, 1987; Tuynman et al, 2000; Bougioukou and Smonou, 2002) or iron and non-iron metal-dependent enzymes (Leuenberger et al, 2001; Marasco and Schmidt-Dannert, 2008) They belong to different enzyme classes including peroxidases, mono- and dioxygenases or laccases and display different protein structures, and the reaction mechanisms (if known) differ (Rajagopalan et al, 2013a). One of the most efficient examples, where alkene cleavage is the main reaction, is a Mn(III)-dependent proteinase A like enzyme from Trametes hirsuta
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