AUTHOR=Fink Matthias , Trunk Sarah , Hall Mélanie , Schwab Helmut , Steiner Kerstin 

TITLE=Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.01511

DOI=10.3389/fmicb.2016.01511

ISSN=1664-302X

ABSTRACT=<p>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 <italic>Thermotoga maritima</italic>. 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.</p>