title: Engineering of TM1459 fromThermotoga maritimafor Increased Oxidative Alkene Cleavage Activity.
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 fromThermotoga 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.
authors.affiliation: Austrian Centre of Industrial Biotechnology Graz, Austria.
authors.forename: Matthias
authors.initials: M
authors.lastname: Fink
authors.affiliation: Austrian Centre of Industrial Biotechnology Graz, Austria.
authors.forename: Sarah
authors.initials: S
authors.lastname: Trunk
authors.affiliation: Department of Chemistry, University of Graz Graz, Austria.
authors.forename: Mélanie
authors.initials: M
authors.lastname: Hall
authors.affiliation: Austrian Centre of Industrial BiotechnologyGraz, Austria; Institute of Molecular Biotechnology, Graz University of TechnologyGraz, Austria.
authors.forename: Helmut
authors.initials: H
authors.lastname: Schwab
authors.affiliation: Austrian Centre of Industrial Biotechnology Graz, Austria.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Steiner
keywords: TM1459; alkene cleavage; cupin; enzyme engineering; styrene