ABSTRACT
Oleate hydratases (Ohys, EC 4.2.1.53) are a class of enzymes capable of selective water addition reactions to a broad range of unsaturated fatty acids leading to the respective chiral alcohols. Much research was dedicated to improving the applications of existing Ohys as well as to the identification of undescribed Ohys with potentially novel properties. This study focuses on the latter by exploring the genus Rhodococcus for its plenitude of oleate hydratases. Three different Rhodococcus clades showed the presence of oleate hydratases whereby each clade was represented by a specific oleate hydratase family (HFam). Phylogenetic and sequence analyses revealed HFam-specific patterns amongst conserved amino acids. Oleate hydratases from two Rhodococcus strains (HFam 2 and 3) were heterologously expressed in Escherichia coli and their substrate scope investigated. Here, both enzymes showed a complementary behaviour towards sterically demanding and multiple unsaturated fatty acids. Furthermore, this study includes the characterisation of the newly discovered Rhodococcus pyridinivorans Ohy. The steady-state kinetics of R. pyridinivorans Ohy was measured using a novel coupled assay based on the alcohol dehydrogenase and NAD+-dependent oxidation of 10-hydroxystearic acid.
Subject(s)
Bacterial Proteins/metabolism , Hydro-Lyases/metabolism , Oleic Acid/metabolism , Rhodococcus/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Biocatalysis , Fatty Acids, Unsaturated/chemistry , Fatty Acids, Unsaturated/metabolism , Genome, Bacterial/genetics , Hydro-Lyases/chemistry , Hydro-Lyases/genetics , Hydrogen-Ion Concentration , Kinetics , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Rhodococcus/classification , Rhodococcus/genetics , Substrate Specificity , TemperatureABSTRACT
The application of purified enzymes as well as whole-cell biocatalysts in synthetic organic chemistry is becoming more and more popular, and both academia and industry are keen on finding and developing novel enzymes capable of performing otherwise impossible or challenging reactions. The diverse genus Rhodococcus offers a multitude of promising enzymes, which therefore makes it one of the key bacterial hosts in many areas of research. This review focused on the broad utilization potential of the genus Rhodococcus in organic chemistry, thereby particularly highlighting the specific enzyme classes exploited and the reactions they catalyze. Additionally, close attention was paid to the substrate scope that each enzyme class covers. Overall, a comprehensive overview of the applicability of the genus Rhodococcus is provided, which puts this versatile microorganism in the spotlight of further research.
Subject(s)
Chemistry Techniques, Synthetic , Rhodococcus/metabolism , Biocatalysis , Catalysis , Hydrolases/metabolism , Metabolic Networks and Pathways , Nitriles/metabolism , Oxidation-Reduction , Oximes/metabolism , Sulfur CompoundsABSTRACT
The first enantioselective synthesis was the selective addition of cyanide to benzaldehyde catalysed by a hydroxynitrile lyase (HNL). Since then these enzymes have been developed into a reliable tool in organic synthesis. HNLs to prepare either the (R)- or the (S)-enantiomer of the desired cyanohydrin are available and a wide variety of reaction conditions can be applied. As a result of this, numerous applications of these enzymes in organic synthesis have been described. Here the examples of the last decade are summarised, the enzyme catalysed step is discussed and the follow-up chemistry is shown. This proves HNLs to be part of main stream organic synthesis. Additionally the newest approaches via immobilisation and reaction engineering are introduced.
Subject(s)
Aldehyde-Lyases/metabolism , Nitriles/chemistry , Nitriles/chemical synthesis , Aldehyde-Lyases/chemistry , Chemistry Techniques, Synthetic , Enzymes, Immobilized/chemistry , Enzymes, Immobilized/metabolism , Stereoisomerism , Substrate SpecificityABSTRACT
Enantiomerically pure chiral amines are ubiquitous chemical building blocks in bioactive pharmaceutical products and their synthesis from simple starting materials is of great interest. One of the most attractive strategies is the stereoselective installation of a chiral amine through C-H amination, which is a challenging chemical transformation. Herein we report the application of a multienzyme cascade, generated in a single bacterial whole-cell system, which is able to catalyze stereoselective benzylic aminations with ee values of 97.5%. The cascade uses four heterologously expressed recombinant enzymes with cofactors provided by the host cell and isopropyl amine added as the amine donor. The cascade presents the first example of the successful de novo design of a single whole-cell biocatalyst for formal stereoselective C-H amination.
