Solvent concentration at 50% protein unfolding may reform enzyme stability ranking and process window identification.

As water miscible organic co-solvents are often required for enzyme reactions to improve e.g., the solubility of the substrate in the aqueous medium, an enzyme is required which displays high stability in the presence of this co-solvent. Consequently, it is of utmost importance to identify the most suitable enzyme or the appropriate reaction conditions. Until now, the melting temperature is used in general as a measure for stability of enzymes. The experiments here show, that the melting temperature does not correlate to the activity observed in the presence of the solvent. As an alternative parameter, the concentration of the co-solvent at the point of 50% protein unfolding at a specific temperature T in short c U 50 T is introduced. Analyzing a set of ene reductases, c U 50 T is shown to indicate the concentration of the co-solvent where also the activity of the enzyme drops fastest. Comparing possible rankings of enzymes according to melting temperature and c U 50 T reveals a clearly diverging outcome also depending on the specific solvent used. Additionally, plots of c U 50 versus temperature enable a fast identification of possible reaction windows to deduce tolerated solvent concentrations and temperature.

Transmembrane Shuttling of Photosynthetically Produced Electrons to Propel Extracellular Biocatalytic Redox Reactions in a Modular Fashion.

Many biocatalytic redox reactions depend on the cofactor NAD(P)H, which may be provided by dedicated recycling systems. Exploiting light and water for NADPH-regeneration as it is performed, e.g. by cyanobacteria, is conceptually very appealing due to its high atom economy. However, the current use of cyanobacteria is limited, e.g. by challenging and time-consuming heterologous enzyme expression in cyanobacteria as well as limitations of substrate or product transport through the cell wall. Here we establish a transmembrane electron shuttling system propelled by the cyanobacterial photosynthesis to drive extracellular NAD(P)H-dependent redox reactions. The modular photo-electron shuttling (MPS) overcomes the need for cloning and problems associated with enzyme- or substrate-toxicity and substrate uptake. The MPS was demonstrated on four classes of enzymes with 19 enzymes and various types of substrates, reaching conversions of up to 99 % and giving products with >99 % optical purity.