Nicotinamide adenine dinucleotide (NAD) and its 2′-phosphorylated form NADP are crucial cofactors for a large array of biocatalytically important redox enzymes. Their high cost and relatively poor stability, however, make them less attractive electron mediators for industrial processes. Nicotinamide cofactor biomimetics (NCBs) are easily synthesized, are inexpensive, and are also generally more stable than their natural counterparts. A bottleneck for the application of these artificial hydride carriers is the lack of efficient cofactor recycling methods. Therefore, we engineered the thermostable F420:NADPH oxidoreductase from Thermobifida fusca (Tfu-FNO), by structure-inspired site-directed mutagenesis, to accommodate the unnatural N1 substituents of eight NCBs. The extraordinarily low redox potential of the natural cofactor F420H2 was then exploited to reduce these NCBs. Wild-type enzyme had detectable activity toward all selected NCBs, with Km values in the millimolar range and kcat values ranging from 0.09 to 1.4 min-1. Saturation mutagenesis at positions Gly-29 and Pro-89 resulted in mutants with up to 139 times higher catalytic efficiencies. Mutant G29W showed a kcat value of 4.2 s-1 toward 1-benzyl-3-acetylpyridine (BAP+), which is similar to the kcat value for the natural substrate NADP+. The best Tfu-FNO variants for a specific NCB were then used for the recycling of catalytic amounts of these nicotinamides in conversion experiments with the thermostable ene-reductase from Thermus scotoductus (TsOYE). We were able to fully convert 10 mM ketoisophorone with BAP+ within 16 h, using F420 or its artificial biomimetic FOP (FO-2′-phosphate) as an efficient electron mediator and glucose-6-phosphate as an electron donor. The generated toolbox of thermostable and NCB-dependent Tfu-FNO variants offers powerful cofactor regeneration biocatalysts for the reduction of several artificial nicotinamide biomimetics at both ambient and high temperatures. In fact, to our knowledge, this enzymatic method seems to be the best-performing NCB-recycling system for BNAH and BAPH thus far.

Bubbly turbulent flow in a channel is investigated using interface-resolved direct numerical simulation. An efficient coupled level-set volume-of-fluid solver based on a fast Fourier transform algorithm is implemented to enable a high resolution and fast computation at the same time. Up to 384 bubbles are seeded in the turbulent channel flow corresponding to 5.4% gas volume fraction. Bubbles are clustered in the channel center due to the downward flow direction. The bubbles induce additional pseudo-turbulence in the channel center and are also able to attenuate the energy in the boundary layer by reducing the shear production. Turbulent kinetic energy budget indicates a significant buoyancy production in the channel center. A local equilibrium between buoyancy production and dissipation is observed here besides the shear production peak in the boundary layer. Comparing the local production and dissipation indicates a coexistence of boundary layer turbulence near the wall and bubble-induced pseudo-turbulence in the channel center. The liquid phase and gas phase are coupled through the complex liquid-gas interface. Local flow topology analysis is depicted in the liquid phase around the bubbles as well as in the gas phase. The flow topology of the liquid phase and the gas phase differs from each other significantly. Local dissipation is more dominant in the liquid phase near the bubble interface, whereas local enstrophy is preferred in the gas phase. In the liquid phase, a high dissipation event is preferred close to the interface, whereas a high enstrophy event is dominant away from the interface.