The cell-free biocatalytic production of fine chemicals by oxidoreductases has continuously grown over the past years. Since especially dehydrogenases depend on the stoichiometric use of nicotinamide pyridine cofactors, an integrated efficient recycling system is crucial to allow process operation under economic conditions. Lately, the variety of cofactors for biocatalysis was broadened by the utilization of totally synthetic and cheap biomimetics. Though, to date the regeneration has been limited to chemical or electrochemical methods. Here, we report an enzymatic recycling by the flavoprotein NADH-oxidase from Lactobacillus pentosus (LpNox). Since this enzyme has not been described before, we first characterized it in regard to its optimal reaction parameters. We found that the heterologously overexpressed enzyme only contained 13% FAD. In vitro loading of the enzyme with FAD, resulted in a higher specific activity towards its natural cofactor NADH as well as different nicotinamide derived biomimetics. Apart from the enzymatic recycling, which gives water as a by-product by transferring four electrons onto oxygen, unbound FAD can also catalyze the oxidation of biomimetic cofactors. Here a two electron process takes place yielding H2O2 instead. The enzymatic and chemical recycling was compared in regard to reaction kinetics for the natural and biomimetic cofactors. With LpNox and FAD, two recycling strategies for biomimetic cofactors are described with either water or hydrogen peroxide as by-product.

The increasing demand for chiral compounds supports the development of enzymatic processes. Dehydrogenases are often the enzymes of choice due to their high enantioselectivity combined with broad substrate acceptance. However, their requirement on costly NAD­(P)/H as cofactor has sparked interest in the development of biomimetic derivatives that are easy to synthesize and, therefore, less expensive. Until now, few reactions with biomimetics have been described and regeneration is limited to nonenzymatic means, which are not suitable for incorporation and in situ approaches. Herein, we describe a regeneration enzyme, glucose dehydrogenase from Sulfolobus solfataricus (SsGDH), and demonstrate its activity with different biomimetics with the structure nicotinamide ring-alkyl chain-phenyl ring. Subsequent enzyme engineering resulted in the double mutant SsGDH Ile192Thr/Val306Ile, which had a 10-fold higher activity with one of the biomimetics compared with the wild-type enzyme. Using this engineered variant in combination with an enoate reductase from Thermus scotoductus resulted in the first enzyme-coupled regeneration process for biomimetic cofactor without ribonucleotide or ribonucleotide analogue and full conversion of 10 mM 2-methylbut-2-enal with 1-phenethyl-1,4-dihydropyridine-3-carboxamide as cofactor.

The search for nitric oxide cheletropic traps (NOCTs) of the 7,7,8,8-tetraalkyl-o-quinodimethane type which would have properties appropriate for monitoring the formation of nitric oride in cell cultures and in vivo by magnetic resonance techniques is described. In addition to the necessary condition that a NOCT reacts rapidly with NO to yield a persistent nitroxide radical, two additional properties were sought: (i) thermal stability at the temperature of interest (37 "C) and (ii) water solubility. To these ends, a number of 1 ,1,3,3-tetraailcyIyl-2-indanones (and a related naphthalene derivative) were synthesized and subjected to UV photolysis in solution, a procedure which generally (though not in all cases) caused the elimination of carbon monoxide and formation of the corresponding o-quinodimethane. The thermal instability of many of these compounds is due to a 1,S-sigmatropic hydrogen atom transfer which, for example, converts 7,7,8,8-tetramethyl-o-quinodimethane (1) to o-isopropyl-a-methylstyrene ( 1 4 with a half-life of only ca. 140 s at 37 "C. Several o-quinodimethanes were discovered which were, for all practical purposes, completely stable at 37 OC. The most suitable lipid-soluble NOCT discovered was 7-(2-indenyl)-7,8,8-trimethyl-o-quinodimethane (5),which is stable and reacts very rapidly with NO to form a persistent nitroxide. Various derivatives of 5 were also examined and found to be equally, or almost equally, effective NOCTs. Water solubility was explored by addition of water-solubilizing groups to the ring of 1. The carboxylic acid group, 13, was found to be particularly suitable, since the carboxylate anion 14 conferred excellent water solubility without interfering with either the nitric oxide trapping reaction or the necessary photoelimination of carbon monoxide frpjn the starting indanone. Of even greater importance, the carboxylate group had no apparent effect on the rate of the thermal 1,s-sigmatropic rearrangement; Le., the rates of decay of 14 and 1 were equal within experimental error. It is concluded that NOCTs of the o-quinodimethane class having long lifetimes and a high reactivity toward NO can now be prepared with appropriate lipophilic, hydrophilic, or amphiphilic properties. These NOCTs should prove suitable for exploratory use in biological systems.