Obligatory intermolecular electron-transfer from FAD to FMN in dimeric P450BM-3.

Cytochromes P450 typically catalyze the monooxygenation of hydrophobic compounds resulting in the insertion of one atom of dioxygen into the organic substrate and the reduction of the other oxygen atom to water. The two electrons required for the reaction are normally provided by another redox active protein, for example cytochrome P450 reductase (CPR) in mammalian endoplasmic reticulum membranes. P450BM-3 from Bacillus megaterium is a widely studied P450 cytochrome in which the P450 is fused naturally to a diflavin reductase homologous to CPR. From the original characterization of the enzyme by Fulco's laboratory, the enzyme was shown to have a nonlinear dependence of reaction rate on enzyme concentration. In recent experiments we observed enzyme inactivation upon dilution, and the presence of substrate can diminish this inactivation. We therefore carried out enzyme kinetics, cross-linking experiments, and molecular weight determinations that establish that the enzyme is capable of dimerizing in solution. The dimer is the predominant form at higher concentrations under most conditions and is the only form with significant activity. Further experiments selectively knocking out the activity of individual domains with site-directed mutagenesis and measuring enzyme activity in heterologous dimers establish that the electron-transfer pathway in P450BM-3 passes through both protein molecules in the dimer during a single turnover, traversing from the FAD domain of one molecule into the FMN domain of the other molecule before passing to the heme domain. Analysis of our results combined with other analyses in the literature suggests that the heme domain of either monomer may accept electrons from the reduced FMN domain.

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein.

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.

Redox properties of the isolated flavin mononucleotide- and flavin adenine dinucleotide-binding domains of neuronal nitric oxide synthase.

Electron transfer through neuronal nitric oxide synthase (nNOS) is regulated by the reversible binding of calmodulin (CaM) to the reductase domain of the enzyme, the conformation of which has been shown to be dependent on the presence of substrate, NADPH. Here we report the preparation of the isolated flavin mononucleotide (FMN)-binding domain of nNOS with bound CaM and the electrochemical analysis of this and the isolated flavin adenine dinucleotide (FAD)-binding domain in the presence and absence of NADP(+) and ADP (an inhibitor). The FMN-binding domain was found to be stable only in the presence of bound CaM/Ca(2+), removal of which resulted in precipitation of the protein. The FMN formed a kinetically stabilized blue semiquinone with an oxidized/semiquinone reduction potential of -179 mV. This is 80 mV more negative than the potential of the FMN in the isolated reductase domain, that is, in the presence of the FAD-binding domain. The FMN semiquinone/hydroquinone redox couple was found to be similar in both constructs. The isolated FAD-binding domain, generated by controlled proteolysis of the reductase domain, was found to have similar FAD reduction potentials to the isolated reductase domain. Both formed a FAD-hydroquinone/NADP(+) charge-transfer complex with a long-wavelength absorption band centered at 780 nm. Formation of this complex resulted in thermodynamic destabilization of the FAD semiquinone relative to the hydroquinone and a 30 mV increase in the FAD semiquinone/hydroquinone reduction potential. Binding of ADP, however, had little effect. The possible role of the nicotinamide/FADH(2) stacking interaction in controlling electron transfer and its likely dependence on protein conformation are discussed.

Ultraviolet sealing and poly(dimethylacrylamide) modification for poly(dimethylsiloxane)/glass microchips.

Simple sealing methods for poly(dimethylsiloxane) (PDMS)/glass-based capillary electrophoresis (CE) microchips by UV irradiation are described. Further, we examined the possibility to modify the inner surface of separation channels, using polymethylacrylamide (PDMA) as a dynamic coating reagent. The surface properties of native PDMS, UV-irradiated PDMS, and PDMA-coated PDMS were systematically studied by atomic force microscopy (AFM), infrared absorption by attenuated total reflection infrared (ATR-IR) spectroscopy, and contact angle measurement. We found that PDMA forms a stable coating on PDMS and glass surfaces, eliminating the nonhomogeneous electroosmotic flow (EOF) in channels on PDMS/glass microchips, and improving the hydrophilicity of PDMS surfaces. Mixtures of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and fluorescein were separated in 35 s using PDMA-coated PDMS/glass microchips. A high efficiency of theoretical plates with at least 1365 (105 000 N/m) and a good reproducibility with relative standard deviations (RSD) below 4% in five successive separations were achieved.

Optimizing separation conditions for riboflavin, flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection.

A method was developed for the quantitative determination of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), using free solution capillary zone electrophoresis in uncoated fused-silica capillaries with laser-induced fluorescence (LIF) detection. Various factors influencing the separation and detection of flavin vitamers were investigated, including pH (5.5-10.5), concentration and nature of the run buffer (phosphate, borate and carbonate), applied voltage (15-30 kV), temperature (15-30 degrees C) and injection time. Optimal resolution and detection were obtained with a pH 9.8, 30 mM aqueous phosphate buffer at 15 degrees C and 30 kV of applied voltage. LIF detection was obtained with a He-Cd laser source using an excitation wavelength at 442 nm and lambda(em) > or = 515 nm. Riboflavin could be determined in the concentration ranges 0.5-350 microg/l with a rather low detection limit (LOD) down to 50 amol. The LODs of FAD and FMN were slightly higher, 300 and 350 amol, respectively. Combined with a simple clean-up procedure, the practical utility of this method is illustrated by the measurements of flavin derivates in foods and beverages, such as wines, milk, yoghurt and raw eggs.

Determination of riboflavin, flavin mononucletide and flavin adenine dinucleotide in biological tissues by capillary zone electrophoresis and laser-induced fluorescence detection.

The separation of riboflavin, flavin mononucleotide and flavin adenine dinucleotide was investigated by capillary zone electrophoresis using laser-induced fluorescence detection. In the systematic approach developed, the differential electrophoretic mobilities were first maximized by adjusting the pH. Increasing the buffer concentration improved the separation at the expense of migration times. A buffer consisting of 50 mM phosphate adjusted to pH 8.5 was found to provide a very efficient and stable electrophoretic system. Responses were linear within the range 0.1-100 micromol L(-1), and the detection limits of B2 vitamers were 0.23 nmol L(-1) or less. The method was successfully applied to a variety of biological tissues from different animals.

Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains.

The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].

Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases.

The kinetics of flavin reduction in two mutant forms of human cytochrome P450 reductase have been studied by stopped-flow spectroscopy with absorption and fluorescence detection. The mutant enzymes were altered at the position of Trp-676, which, by analogy with the structure of rat CPR, is close to the isoalloxazine ring of the enzyme-bound FAD. We show that mutant CPRs in which Trp-676 has been changed to histidine (W676H) and alanine (W676A) can be reduced by NADPH only to the two-electron level in single mixing stopped-flow experiments. The concentration dependence of the rate of hydride transfer indicates that the second, noncatalytic NADPH-binding site present in wild-type CPR is retained in the mutant enzymes. Detailed studies of W676H CPR indicate that further reduction of the enzyme beyond the two electron level is prevented due to the slow release of NADP(+) from the active site following the first hydride transfer from NADPH, owing to the stability of a reduced enzyme-NADP(+) charge-transfer complex. Reduction to the four-electron level is achieved in a sequential mixing stopped-flow experiment. In this procedure, W676H CPR is reacted first with a stoichiometric amount of NADPH, and then, following a delay of 100 ms, with excess NADPH. The data indicate that occupancy of the noncatalytic coenzyme site also hinders NADP(+) release from reduced enzyme. Fluorescence stopped-flow studies of the W676H and wild-type CPR enzymes reveal that the complex signals associated with reduction of wild-type CPR by NADPH are attributable to changes in the environment of residue W676. From these studies, a model is proposed for nicotinamide binding in wild-type CPR. In this model W676 serves as a trigger to release NADP(+) from the active site following hydride transfer. In the W676H enzyme, the slow release of NADP(+) is a consequence of the combined effects of (i) removing W676 by mutagenesis (thus removing the trigger for displacement) and (ii) the binding of NADPH in the noncatalytic site, thus trapping NADP(+) in the catalytic site.

On a new artificial mediator accepting NADP(H) oxidoreductase from Clostridium thermoaceticum.

The purification and partial characterisation of an NADP(H) dependent artificial mediator accepting pyridine nucleotide oxidoreductase (AMAPOR) from the anaerobic Clostridium thermoaceticum is described. Depending on the redox potential of the artificial mediators the AMAPOR is able to regenerate NADP+ or NADPH rendering the enzyme useful for preparative work applying NADP(H) dependent oxidoreductases. At 37 degrees C crude extracts of C. thermoaceticum have an AMAPOR activity of 5-7 U mg(-1). This is 28 degrees under the optimal growth temperature of this microrganism. Out of apparently more than 10 AMAPOR active proteins in the crude cell extracts visible after electrophoresis and activity staining on the gel, two of these proteins were isolated. They seem to be two different oligomers. According to gel electrophoresis they show apparent molecular masses of about 200 and 400 kDa. These two forms showed after SDS gel electrophoresis two monomers with apparent molecular masses of 42 and 56 kDa which we call alpha and beta. The two oligomers may have the compositions alpha2beta2 and alpha4beta4. They contain Fe/S cluster and FAD. Various amounts of the FAD were lost during the purification procedure. This loss is partially reversible after addition of FAD. The AMAPOR reacts with rather different artificial mediators such as viologens, quinones e.g. 1,4-benzoquinone or anthraquinone-2,6-disulphonate, 2,6-dichloro-indophenol and clostridial rubredoxin. Two different ferredoxins from C. thermoaceticum, oxygen or lipoamide are no substrates indicating the here described AMAPOR is not a diaphorase in the usual sense.

Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans. Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli.

The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M). The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein.

Sequencing and preliminary characterization of the Na+-translocating NADH:ubiquinone oxidoreductase from Vibrio harveyi.

The Na(+)-translocating NADH: ubiquinone oxidoreductase (Na(+)-NQR) generates an electrochemical Na(+) potential driven by aerobic respiration. Previous studies on the enzyme from Vibrio alginolyticus have shown that the Na(+)-NQR has six subunits, and it is known to contain FAD and an FeS center as redox cofactors. In the current work, the enzyme from the marine bacterium Vibrio harveyi has been purified and characterized. In addition to FAD, a second flavin, tentatively identified as FMN, was discovered to be covalently attached to the NqrC subunit. The purified V. harveyi Na(+)-NQR was reconstituted into proteoliposomes. The generation of a transmembrane electric potential by the enzyme upon NADH:Q(1) oxidoreduction was strictly dependent on Na(+), resistant to the protonophore CCCP, and sensitive to the sodium ionophore ETH-157, showing that the enzyme operates as a primary electrogenic sodium pump. Interior alkalinization of the inside-out proteoliposomes due to the operation of the Na(+)-NQR was accelerated by CCCP, inhibited by valinomycin, and completely arrested by ETH-157. Hence, the protons required for ubiquinol formation must be taken up from the outside of the liposomes, which corresponds to the bacterial cytoplasm. The Na(+)-NQR operon from this bacterium was sequenced, and the sequence shows strong homology to the previously reported Na(+)-NQR operons from V. alginolyticus and Haemophilus influenzae. Homology studies show that a number of other bacteria, including a number of pathogenic species, also have an Na(+)-NQR operon.

Sources and nature of heterogeneity in recombinant phenol hydroxylase derived from the basidiomycetous soil yeast Trichosporon cutaneum.

Preparations of the dimeric flavoenzyme phenol hydroxylase derived from Trichosporon cutaneum were found to contain an active tetrameric form when the enzyme was produced in Escherichia coli. The relative content of the tetramer was estimated from scans of silver-stained native PAGE gels and/or size-exclusion chromatography (SEC). Proportions of up to 22% of the enzyme protein, depending on the growth temperature and the level of added inducer, were observed in independent cultures as well as in purified preparations. No tetramer was ever seen in cell extracts or purified preparations from T. cutaneum. Traces of higher multimers and of possibly deamidated species were also detected in preparations of the recombinant enzyme. The rate of enzyme production seems to be the major factor in promoting formation of the tetramer, whereas the specific growth rate of the fermentor culture appears to be of minor importance. The dimeric and the tetrameric forms were purified using either SEC or ion-exchange chromatography as a final step. The two purified species did not interchange under a variety of conditions, indicating that they are not undergoing rapid equilibria. The FAD of either form, as isolated by SEC, was present to a lower-than-expected extent of 2 equiv/dimer. However, by removing FAD and reconstituting the resulting apoproteins with the cofactor, the FAD content could be increased to 2 equiv. in the dimer and 3 equiv. in the tetramer. Both reconstituted forms exhibited absorption spectra identical with that of phenol hydroxylase from T. cutaneum as well as that of the recombinant enzyme. All spectra were equally perturbed by one equivalent of phenol per enzyme-attached FAD. The ratio of specific activities of the dimeric and the tetrameric forms was, however, lower than expected from the ratio of their FAD contents. The results are compatible with the notion that the tetramer consists of a native phenol hydroxylase dimer associated with a non-native one with a decreased ability to bind FAD, either in one or both of its constituent 'monomers'.

Purification and properties of alcohol oxidase from Candida methanosorbosa M-2003.

Alcohol oxidase from Candida methanosorbosa was purified about sixfold with a yield of 37.6% from the culture broth of Candida methanosorbosa M-2003. The enzyme preparation was homogeneous on slab gel electrophoresis. The purified enzyme had an optimal pH from 6.0 to 9.0 and was stable in the range 6.0-8.5. Its optimal temperature of reaction was 50 degrees C, and it was stable below 50 degrees C. In the presence of NaN3, the enzyme retained its initial activity at 30 degrees C for 35 days, indicating stability for a long term, so far. The isoelectric point was pH 4.3. Its molecular weight was 620, 000 by gel filtration chromatography and 80,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results indicate that the enzyme consists of 8 subunits.

Crystallization and preliminary X-ray analysis of cholesterol oxidase from Brevibacterium sterolicum containing covalently bound FAD.

Single crystals of cholesterol oxidase from Brevibacterium sterolicum containing a covalently bound form of the FAD cofactor have been obtained. The crystals are grown by vapor diffusion using the hanging drop technique from 12% polyethylene glycol, Mr 8000, and 75 mM MnSO4 as the precipitant at pH 5.2. In order to obtain large diffraction quality crystals, nucleation must occur at 22 degrees C with subsequent growth at 17 degrees C. The crystals belong to the monoclinic space group P21 with cell dimensions a = 78. 5 A, b = 126.7 A, c = 82.4 A and beta = 108.9 degrees with two protein molecules per asymmetric unit. Diffraction of these crystals has been observed to at least 2.2 A resolution and they are suitable for an X-ray structure analysis.

Discovery of a third coenzyme in sarcosine oxidase.

Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 degrees C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.

On-line capillary electrophoresis-electrospray ionization mass spectrometry of nucleotides.

Capillary zone electrophoresis (CZE) and reversed anionic capillary isotachophoresis (CITP) conditions have been developed for the separation of mixtures comprised of monophosphate nucleosides, pyridine and flavin dinucleotides, and monophosphate dinucleosides. Results for the on-line coupling of CZE and CITP with electrospray ionization mass spectrometry (MS) are presented. CITP-tandem MS is utilized to provide both molecular weight and structural information of monophosphate dinucleotides. The fragmentation pattern of dinucleotides in the low collision energy range is described. The resulting mass spectra are readily interpreted in terms of dinucleotide structures. These results demonstrate the new capability for applications for the study of DNA and RNA.

Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.

Purification and semienzymic synthesis of flavin adenine dinucleotide-3'-phosphate.

Nonradioactive immunoassays incorporating an element of amplification in their detection system require the use of components that are highly purified. Flavin adenine dinucleotide-3'-phosphate (FADP) is the primary substrate used in such an amplification assay. For incorporation into a simple, single-pot assay system, the concentration of contaminating flavin adenine dinucleotide (a prosthetic group for the enzyme D-aminoacid oxidase used in the amplification cascade assay) in this primary substrate must be minimized to achieve maximum sensitivity. Production of the substrate to a high degree of purity has been achieved using apo-glucose oxidase to specifically remove contaminating flavin adenine dinucleotide from solution and hydrolysis of a cyclic intermediate as a final production protocol by ribonuclease T2 to give the product in high yield. The use of continuous ultrafiltration reactors at each stage is described and compared to a final production step utilizing immobilized ribonuclease T2. These reactors allow large volumes of material to be handled and assist in the scale-up of these processes. The suitability of each protocol is assessed for the commercial production of FADP.