BackgroundThe number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds.ResultsThe NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Immobilization of NOX onto agarose activated with glyoxyl groups yielded the most stable enzyme preparation (6-fold more stable than the hyperactivated soluble enzyme). The immobilized derivative was able to be reactivated under physiological conditions after inactivation by high solvent concentrations. The inactivation/reactivation cycle could be repeated at least three times, recovering full NOX activity in all cases after the reactivation step. This immobilized catalyst is presented as a recycling partner for a thermophile alcohol dehydrogenase in order to perform the kinetic resolution secondary alcohols.ConclusionWe have designed, developed and characterized a heterogeneous and robust biocatalyst which has been used as recycling partner in the kinetic resolution of rac-1-phenylethanol. The high stability along with its capability to be reactivated makes this biocatalyst highly re-useable for cofactor recycling in redox biotransformations.

We describe a novel molecular strategy for engendering a strong light-up signal in fluorescence tagging of the genetically encoded HaloTag protein domain. We designed a set of haloalkane-derivatized dyes having twisted internal charge transfer (TICT) structures potentially narrow enough to partially fit into the enzyme’s haloalkane-binding channel. Testing a range of short chain lengths revealed a number of active dyes, with seven carbons yielding optimum light-up signal. The dimethylaminostilbazolium chloroheptyl dye (1d) yields a 27-fold fluorescence emission enhancement (λex = 535 nm; Em(max) = 616 nm) upon reaction with the protein. The control compound with standard 12-atom linkage shows less efficient signaling, consistent with our channel-binding hypothesis. For emission further to the red, we also prepared a chloroheptyl naphthalene-based dye; compound 2 emits at 653 nm with strong fluorescence enhancement upon reaction with the HaloTag domain. The two dyes (1d, 2) were successfully tested in wash-free imaging of protein localization in bacteria, using a HaloTag fusion of the filamenting temperature-sensitive mutant Z (FtsZ) protein in Escherichia coli (E. coli). The new dye conjugates are inexpensive and easily synthesized enzyme substrates with low background and large Stokes shifts, offering substantial benefits over known fluorescent substrates for the HaloTag enzyme.

SummaryTolC is the outer membrane component of tripartite efflux pumps, which expel proteins, toxins and antimicrobial agents from Gram-negative bacteria. Escherichia coli tolC mutants grow well and are slightly elongated in rich media but grow less well than wildtype cells in minimal media. These phenotypes have no physiological explanation as yet. Here, we find that tolC mutants have highly aberrant shapes when grown in M9-glucose medium but that adding iron restores wild-type morphology. When starved for iron, E. coli tolC mutants synthesize but cannot secrete the siderophore enterobactin, which collects in the periplasm. tolC mutants unable to synthesize enterobactin display no growth or morphological defects, and adding exogenous enterobactin recreates these aberrations, implicating this compound as the causative agent. Cells unable to import enterobactin across the outer membrane grow normally, whereas cells that import enterobactin only to the periplasm become morphologically aberrant. Thus, tolC mutants grown in low iron conditions accumulate periplasmic enterobactin, which impairs bacterial morphology, possibly by sequestering iron and inhibiting an iron-dependent reaction involved in cell division or peptidoglycan synthesis. The results also highlight the need to supply sufficient iron when studying TolC-directed export or efflux, to eliminate extraneous physiological effects.

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