Substrate inhibition by the blockage of product release and its control by tunnel engineering.

Substrate inhibition is the most common deviation from Michaelis-Menten kinetics, occurring in approximately 25% of known enzymes. It is generally attributed to the formation of an unproductive enzyme-substrate complex after the simultaneous binding of two or more substrate molecules to the active site. Here, we show that a single point mutation (L177W) in the haloalkane dehalogenase LinB causes strong substrate inhibition.

FireProtASR: A Web Server for Fully Automated Ancestral Sequence Reconstruction.

There is a great interest in increasing proteins' stability to widen their usability in numerous biomedical and biotechnological applications. However, native proteins cannot usually withstand the harsh industrial environment, since they are evolved to function under mild conditions. Ancestral sequence reconstruction is a well-established method for deducing the evolutionary history of genes.

A Haloalkane Dehalogenase from Saccharomonospora viridis Strain DSM 43017, a Compost Bacterium with Unusual Catalytic Residues, Unique ()-Enantiopreference, and High Thermostability.

Haloalkane dehalogenases can cleave a carbon-halogen bond in a broad range of halogenated aliphatic compounds. However, a highly conserved catalytic pentad composed of a nucleophile, a catalytic base, a catalytic acid, and two halide-stabilizing residues is required for their catalytic activity. Only a few family members, e.

An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation.

Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium-dependent chloroperoxidase from , forming hypohalous acids that are detected using aminophenyl fluorescein.

Baeyer-Villiger monooxygenases from Yarrowia lipolytica catalyze preferentially sulfoxidations.

Nine new putative Baeyer-Villiger monooxygenase encoding genes were identified in the eukaryote Yarrowia lipolytica and eight were subsequently cloned and expressed. These enzymes, Yarrowia monooxygenases A-H (YMOA-H), were used in biocatalysis reactions with ketones, sulfides and sulfoxides as substrates. YMOB converts ketones and sulfides, albeit with low activities.

Hot spots for the protein engineering of Baeyer-Villiger monooxygenases.

Baeyer-Villiger monooxygenases (BVMOs) are versatile biocatalysts for the conversion of ketones to lactones or esters while also being able to efficiently oxidize sulfides to sulfoxides. However, there are limitations for the application of BVMOs in synthesis. In this review we provide an overview of the protein engineering studies aiming at optimizing different properties of BVMOs.

Switch in Cofactor Specificity of a Baeyer-Villiger Monooxygenase.

Baeyer-Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable.

Metabolism of alkenes and ketones by Candida maltosa and related yeasts.

Knowledge is scarce about the degradation of ketones in yeasts. For bacteria a subterminal degradation of alkanes to ketones and their further metabolization has been described which always involved Baeyer-Villiger monooxygenases (BVMOs). In addition, the question has to be clarified whether alkenes are converted to ketones, in particular for the oil degrading yeast Candida maltosa little is known.