Modification of galactitol dehydrogenase from Rhodobacter sphaeroides D for immobilization on polycrystalline gold surfaces.

Galactitol dehydrogenase (GatDH) from Rhodobacter sphaeroides is a multifunctional enzyme that catalyzes in the presence of oxidized beta-nicotinamide adenine dinucleotide (NAD(+)) the interconversion of various multivalent aliphatic alcohols to the corresponding ketones. The recombinant GatDH was provided with an N-terminal His(6)-tag to which distally up to three cysteine residues were attached. This protein construct maintained nearly full enzymatic activity, and it could be covalently immobilized via thiol bonds onto the surface of a gold electrode. Binding of GatDH onto the gold electrode was verified by SPR measurements, and residual enzyme activity was measured by cyclic voltammetry using 1,2-hexanediol as substrate, the cofactor NAD(+) and the redox mediator CTFM (4-carboxy-2,5,7-trinitrofluorenyliden-malonnitrile) in solute form. The results demonstrate the possibility of a directed functional immobilization of proteins on gold surfaces, which represents a proof-of-concept for the development of reactors for electrochemical synthon preparation using dehydrogenases.

Mycobacterium fluoranthenivorans sp. nov., a fluoranthene and aflatoxin B1 degrading bacterium from contaminated soil of a former coal gas plant.

Mycobacterium strain FA4T was isolated with fluoranthene as the single carbon source from soil of a former coal gas plant, polluted with polycyclic aromatic hydrocarbons. The physiological properties, fatty acid pattern, and the 16S ribosomal RNA gene sequence indicated membership to the genus Mycobacterium, but were different from all type strains of Mycobacterium species. Based on comparative 16S rRNA gene sequence analyses strain FA4T could be assigned to the Mycobacterium neoaurum taxon showing 98% sequence similarity to M. diernhoferi as its closest neighbour. The occurrence of epoxymycolate in the cell wall differentiates FA4 from all members of this taxon which synthesize wax-ester mycolates in addition to alpha-mycolates. Strain FA4T is able to degrade aflatoxin B1. This biological attribute might be useful in biological detoxification processes of foods and feeds. From the investigated characteristics it is concluded that strain FA4T represents a new species, for which we propose the name Mycobacterium fluoranthenivorans sp. nov. The type strain of Mycobacterium fluoranthenivorans is FA4T (DSM 44556T = CIP 108203T).

Stereoselective oxidation of aliphatic diols and reduction of hydroxy-ketones with galactitol dehydrogenase from Rhodobacter sphaeroides D.

From the Rhodobacter sphaeroides mutant D a galactitol dehydrogenase (GDH) was isolated and characterized in an earlier investigation (1). The enzyme expressed activity with a wide spread substrate spectrum, like sugars, sugar alcohols, secondary alcohols or the corresponding ketones and it can be used for the production of the rare sugar L-tagatose by regioselective oxidation of galactitol (2). This study focuses on the preparation of optically pure aliphatic diols by oxidation of one enantiomer or stereospecific reduction of keto-alcohols and diketones. The oxidation of 1,2-propanediol, 1,2-butanediol, 1,2-pentanediol and 1,2-hexanediol occurred highly specific with the S-enantiomer leaving the R-enantiomer of the diols in the reaction vessel. Also (S)-1,2,6-hexanetriol was oxidized by GDH to 1,6-dihydroxy-2-hexanone. The Km values of these reactions decreased with increasing length of the carbon chain. Reduction of hydroxyacetone or 1-hydroxy-2-butanone resulted in an excess of 93% (S)-1,2-propanediol and more than 98% of (S)-1,2-butanediol, respectively. The diketone 2,3-hexanedione was only reduced to (2R,3S)-2,3-hexanediol, one of the possible four configurations. The wide substrate spectrum on one hand and the selectivity in the reaction on the other hand make GDH a very interesting enzyme for the production of optically pure building blocks in the chemical synthesis of bioactive compounds.

Phototrophic transformation of phenol to 4-hydroxyphenylacetate by Rhodopseudomonas palustris.

Newly isolated and culture collection strains of Rhodopseudomonas palustris were able to transform phenol to 4-hydroxyphenylacetate under phototrophic conditions in the presence of acetate, malate, benzoate, or cinnamate as growth substrates. The reaction was examined with uniformly (14)C-labelled phenol and the product was identified by HPLC retention time, UV-scans, and (1)H- and (13)C-NMR analysis. The transformation reaction was detectable in cell-free extracts in the presence of NAD(+) and acetyl-CoA. For further degradation of 4-hydroxyphenylacetate by R. palustris, low partial pressures of oxygen were essential, presumably for aerobic aromatic ring fission reactions by mono- and di-oxygenases.

Kinetic model discrimination via step-by-step experimental and computational procedure in the enzymatic oxidation of D-glucose.

The enzymatic oxidation of D-glucose to 2-keto-D-glucose (D-arabino-hexos-2-ulose, D-glucosone) is of prospective industrial interest. Pyranose oxidase (POx) from Peniphora gigantea is deactivated during the reaction. To develop a kinetic model including the main reaction and the enzyme inactivation, possible side-reactions of the non-stabilised enzyme with D-glucosone, hydrogen peroxide, and peroxide radicals were considered. A developed step-by-step combined experimental and computational procedure allowed to discriminate among alternative inactivation mechanisms and provides an increased model reliability. The most probable scheme is the enzyme inactivation by hydroxyl radicals formed from produced H2O2 in the presence of Fe2+ ions. This .OH reaction is supported by matrix assisted laser desorption ionisation-mass spectrometry (MALDI-MS) measurement. The estimated kinetic parameter values for the main reaction are of the same order of magnitude as those reported in the literature. The identified model allows a satisfactory process simulation and highlights measures to prevent the enzyme activity loss.

Rare sugars and sugar-based synthons by chemo-enzymatic synthesis.

The unique catalytic potential of the fungal enzyme pyranose oxidase was demonstrated by preparative conversions of a variety of carbohydrates, and by extensive chemical characterization of the reaction products with NMR spectroscopy. The studies revealed that POx not only oxidizes most substrates very efficiently but also that POx possesses a glycosyl-transfer potential, producing disaccharides from beta-glycosides of higher alcohols. Although most substrates are oxidized by POx at the C-2 position, several substrates are converted into the 3-keto-derivatives. On the basis of these products, strategies are developed for the convenient production of sugar-derived synthons, rare sugars and fine chemicals by combining biotechnical and chemical methods.

Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry.

Pyranose oxidases are widespread among lignin-degrading white rot fungi and are localized in the hyphal periplasmic space. They are relatively large flavoproteins which oxidize a number of common monosaccharides on carbon-2 in the presence of oxygen to yield the corresponding 2-keto sugars and hydrogen peroxide. The preferred substrate of pyranose oxidases is D-glucose which is converted to 2-keto-D-glucose. While hydrogen peroxide is a cosubstrate in ligninolytic reactions, 2-keto-D-glucose is the key intermediate of a secondary metabolic pathway leading to the antibiotic cortalcerone. The finding that 2-keto-D-glucose can serve as an intermediate in an industrial process for the conversion of D-glucose into D-fructose has stimulated research on the use of pyranose oxidases in biotechnical applications. Unique catalytic potentials of pyranose oxidases have been discovered which make these enzymes efficient tools in carbohydrate chemistry. Converting common sugars and sugar derivatives with pyranose oxidases provides a pool of sugar-derived intermediates for the synthesis of a variety of rare sugars, fine chemicals and drugs.

Cloning, nucleotide sequence, and overexpression of smoS, a component of a novel operon encoding an ABC transporter and polyol dehydrogenases of Rhodobacter sphaeroides Si4.

The gene coding for sorbitol dehydrogenase (SDH) of Rhodobacter sphaeroides Si4 was located 55 nucleotides upstream of the mannitol dehydrogenase gene (mtlK) within a previously unrecognized polyol operon. This operon probably consists of all the proteins necessary for transport and metabolization of various polyols. The gene encoding SDH (smoS) was cloned and sequenced. Analysis of the deduced amino acid sequence revealed homology to enzymes of the short-chain dehydrogenase/reductase protein family. For structure analysis of this unique bacterial enzyme, smoS was subcloned into the overexpression vector pET-24a(+) and then overproduced in Escherichia coli BL21(DE3), which yielded a specific activity of 24.8 U/mg of protein and a volumetric yield of 38,000 U/liter. Compared to values derived with the native host, R. sphaeroides, these values reflected a 270-fold increase in expression of SDH and a 971-fold increase in the volumetric yield. SDH was purified to homogeneity, with a recovery of 49%, on the basis of a three-step procedure. Upstream from smoS, another gene (smoK), which encoded a putative ATP-binding protein of an ABC transporter, was identified.

Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme.

By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-1 bioreactor at 37 degrees C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing the growth temperature to 27 degrees C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26,000 U/l, which corresponds to 0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammonium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme.

Purification by Immunoaffinity Chromatography, Characterization, and Structural Analysis of a Thermostable Pyranose Oxidase from the White Rot Fungus Phlebiopsis gigantea.

A moderately thermostable pyranose oxidase (PROD) was purified to apparent homogeneity with a yield of 71% from mycelium extracts of the white rot fungus Phlebiopsis gigantea by an efficient three-step procedure that included heat treatment, immunoaffinity chromatography, and gel filtration on Superdex 200. PROD of P. gigantea is a glycoprotein with a pI between pH 5.3 and 5.7. The relative molecular weight (M(infr)) of native PROD is 295,600 (plusmn) 5% as determined by four independent methods. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of PROD revealed two distinct but similar stained bands corresponding to polypeptides with M(infr)s of 77,000 and 70,000, suggesting a heterotetrameric enzyme structure. The tetrameric structure of PROD was confirmed by electron microscopic examinations, which additionally showed the ellipsoidal shape (4.6 by 10 nm) of each subunit. Spectral analyses and direct determinations showed the presence of covalently bound flavin adenine dinucleotide with a stoichiometry of 3.12 mol/mol of enzyme. A broad pH optimum was determined in the range pH 5.0 to 8.0 in 100 mM sodium phosphate, and the activation energy for d-glucose oxidation was 24.7 kJ/mol. The main substrates of PROD are d-glucose, l-sorbose, and d-xylose, for which K(infm) values 1.2, 16.5, and 22.2 mM were determined, respectively. PROD showed high stability during storage. In 100 mM sodium phosphate (pH 6.0 to 8.0), the half-life of PROD activity was >300 days at 40(deg)C, >110 days at 50(deg)C (pH 7.0), and 1 h at 65(deg)C.

Overproduction of mannitol dehydrogenase in Rhodobacter sphaeroides.

Mannitol dehydrogenase (MDH) from Rhodobacter sphaeroides Si4 was overproduced by constructing a strain that overexpresses the MDH gene and by producing high cell concentrations via fed-batch cultivation in a bioreactor. With the gene of mannitol dehydrogenase (mtlK) cloned into the expression vector pKK223-3 expression of MDH in Escherichia coli was obtained, but the specific enzyme activity was lower than in R. sphaeroides Si4. In order to overexpress mtlK in R. sphaeroides, plasmid pAK82 was constructed by cloning a DNA fragment carrying mtlK into the broad-host-range expression vector pRK415. When pAK82 was introduced into R. sphaeroides Si4 the specific mannitol dehydrogenase activity in the strain obtained was 0.48 unit (U)mg-1,3.4-fold higher than in the wild type. In this way the enzyme yield from cultivation in a bioreactor could be improved from 110 Ul-1 to 350 Ul-1. A further increase in productivity was obtained by fed-batch cultivation of R. sphaeroides Si4 [pAK82]. Using this cultivation method an optical density of 27.6 was reached in the bioreactor, corresponding to a dry mass of 16.6 g l-1. Since MDH formation correlated with biomass production, the MDH yield could be raised to 918 Ul-1, an 8.3-fold increase in comparison to batch cultivation of the wild-type strain.

Cloning, nucleotide sequence and characterization of the mannitol dehydrogenase gene from Rhodobacter sphaeroides.

Transposon mutagenesis and antibiotic enrichment were employed to isolate a mutant of Rhodobacter sphaeroides Si4 designated strain M22, that had lost the ability to grow on D-mannitol and to produce the enzyme mannitol dehydrogenase (MDH). DNA flanking the transposon in the mutant strain was used as a probe for the identification and cloning of the MDH gene (mtlK). A 5.5 kb EcoRI/BglII fragment from R. sphaeroides Si4 was isolated and shown to complement the mutation in R. sphaeroides M22. Successful complementation required that a promoter of the vector-plasmid pRK415 be present, suggesting that the mtlK gene is part of a larger operon. Using oligonucleotides derived from the N-terminal sequence of MDH as probes mtlK was located on the complementing fragment and the gene was sequenced. The mtlK open reading frame encodes a protein of 51,404 Da with an N-terminal sequence identical to that obtained from amino acid analysis of the purified MDH. The MDH of R. sphaeroides Si4 exhibits distant similarity to the mannitol-1-phosphate dehydrogenases from Escherichia coli and Enterococcus faecalis, with 28.1% and 26.3% identity, respectively. Mutant strains deficient in MtlK displayed substantial levels of sorbitol dehydrogenase activity, originally thought to be only a minor activity associated with the MDH enzyme. It is likely that we have uncovered an additional polyol dehydrogenase with activity for sorbitol. The mtlK gene can be used for overexpression of MDH in E. coli in order to obtain sufficient amounts of enzyme for further investigations and applications.

Purification and characterization of a pyranose oxidase from the basidiomycete Peniophora gigantea and chemical analyses of its reaction products.

A pyranose oxidase was isolated from mycelium extracts of the basidiomycete Peniophora gigantea. This enzyme was purified 104-fold to apparent homogeneity with a yield of about 75% by steps involving fractionated ammonium sulphate precipitation, chromatography on DEAE-Sephacel, Sephacryl S 300, S Sepharose and Q Sepharose. The native pyranose oxidase has a relative molecular mass (M(r)) of 322,800 +/- 18,300 as determined on the basis of its Stokes' radius (rs = 6.2 nm) and sedimentation coefficient (S20,w = 10.6), dynamic light-scattering experiments, gradient-gel electrophoresis and cross-linking studies. SDS/PAGE resulted in one single polypeptide band of M(r) 76,000 indicating that the enzyme consists of four subunits of identical size. The pyranose oxidase was shown to be an extremely stable glycoprotein with an isoelectric point of pH 5.3. It contains covalently bound FAD with an estimated stoichiometry of 3.6 molecules FAD/molecule enzyme. Pyranose oxidase was active with the substrates D-glucose, D-xylose, L-sorbose, D-galactose, methyl beta-D-glucoside, maltose and D-fucose. Regioselective oxidation of D-glucose, L-sorbose and D-xylose to 2-keto-D-glucose, 5-keto-D-fructose and 2-keto-D-xylose, was demonstrated by identifying the reaction products by mass spectroscopy 13C-NMR spectroscopy and 1H-NMR spectroscopy after purification and derivatization. The pH optimum of the pyranose oxidase was in the range pH 6.0-6.5 in 0.1 M potassium phosphate, and its activation energy (delta H degree) for the conversion of D-glucose was 34.6 kJ/mol. The reactions with the sugars exhibited Michaelis-Menten kinetics, and the Km values determined for D-glucose, L-sorbose, D-xylose and oxygen were 1.1 mM, 50.0 mM, 29.4 mM and 0.65 mM, respectively. The activity of pyranose oxidase was only slightly affected by chelating reagents, thiol reagents, reducing reagents and bivalent cations each at 1 mM.

Pentitol metabolism of Rhodobacter sphaeroides Si4: purification and characterization of a ribitol dehydrogenase.

The phototrophic bacterium Rhodobacter sphaeroides strain Si4 induced ribitol dehydrogenase (EC 1.1.1.56) when grown on ribitol- or xylitol-containing medium. This ribitol dehydrogenase was purified to apparent homogeneity by ammonium sulphate precipitation, affinity chromatography on Procion red, and chromatography on Q-Sepharose. For the native enzyme an isoelectric point of pH 6.1 and an apparent M(r) of 50,000 was determined. SDS-PAGE yielded a single peptide band of M(r) 25,000 suggesting a dimeric enzyme structure. The ribitol dehydrogenase was specific for NAD+ but unspecific as to its polyol substrate. In order of decreasing activity ribitol, xylitol, erythritol, D-glucitol and D-arabitol were oxidized. The pH optimum of substrate oxidation was 10, and that of substrate reduction was 6.5. The equilibrium constant of the interconversion of ribitol to D-ribulose was determined to be 0.33 nM at pH 7.0 and 25 degrees C. The Km-values determined for ribitol, ribulose, xylitol and NAD+ (in the presence of ribitol) were 6.3, 12.5, 77 and 0.077 mM, respectively. Because of the favourable Km for ribitol, a method for quantitative ribitol determination was elaborated.

Purification and properties of a polyol dehydrogenase from the phototrophic bacterium Rhodobacter sphaeroides.

A polyol dehydrogenase was detected in cell extracts of the facultative phototrophic bacterium Rhodobacter sphaeroides strain Si 4 grown on D-glucitol (sorbitol) as the sole carbon source. The enzyme was purified 150-fold to apparent homogeneity by steps involving fractionated (NH4)2SO4 precipitation, chromatography on Q-Sepharose and phenyl-Sepharose, and FPLC on Superose 12. The relative molecular mass (Mr) of the native polyol dehydrogenase was 47,200 as calculated from its Stokes' radius (rs = 2.76 nm) and sedimentation coefficient (s20, w = 4.15 S). SDS/PAGE resulted in one single band representing a polypeptide with a Mr of 52,200, indicating that the native protein is a monomer. The isoelectric point of the polyol dehydrogenase was determined to be pH 4.3. The enzyme was specific for NAD+ and oxidized both D-glucitol and D-mannitol to D-fructose, as well as D-arabinitol to D-ribulose. The pH optimum of substrate oxidation was pH 9.0 in 0.1 M Tris/HCl and that of substrate reduction was pH 6.5 in 0.1 M potassium phosphate. The reactions exhibited normal Michaelis-Menten kinetics allowing the estimation of KM values for NAD+ (0.18 mM) in the presence of D-glucitol, and for D-glucitol (31.8 mM), D-mannitol (0.29 mM) and D-arabinitol (1.8 mM), respectively. The KM value for D-fructose was 16.3 mM and that for NADH 0.02 mM. The equilibrium constants determined for the conversion of D-mannitol, D-glucitol and D-arabinitol were 4.5 nM, 0.58 nM and 80 pM, respectively. Based on the catalytic preference of the polyol dehydrogenase for D-mannitol, an enzymatic assay for D-mannitol was elaborated.

Susceptibility of Rhodobacter sphaeroides to beta-lactam antibiotics: isolation and characterization of a periplasmic beta-lactamase (cephalosporinase).

Thirteen strains of the gram-negative, facultative phototrophic bacterium Rhodobacter sphaeroides were examined fro susceptibility to beta-lactam antibiotics. All strains were sensitive to the semisynthetic penicillins ampicillin, carbenicillin, oxacillin, cloxacillin, and methicillin, but 10 of the 13 strains were resistant to penicillin G, as well as a number of cephalosporins, such as cephalothin, cephapirin, and cephalosporin C. A beta-lactamase (EC 3.5.2.6) with strong cephalosporinase activity was detected in all of the resistant strains of R. sphaeroides. With strain Y-1 as a model, it was shown that the beta-lactamase was inducible by penicillin G, cephalosporin C, cephalothin, and to some minor extent, cephapirin. The beta-lactamase was located in the periplasmic space, from which it could be extracted by osmotic shock disruption. By using this fraction, the beta-lactamase was purified 34-fold to homogeneity by steps involving batch adsorption to and elution from DEAE-Sephadex A50, chromatography on Q-Sepharose, and preparative polyacrylamide gel electrophoresis. The molecular masses of the native and denatured enzymes were determined to be 38.5 kilodaltons by gel filtration and 40.5 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating a monomeric structure. The isoelectric point was estimated to be at pH 4.3. In Tris hydrochloride buffer, optimum enzyme activity was measured at pH 8.5. The beta-lactamase showed high activity in the presence of the substrates cephalothin, cephapirin, cephalosporin C, and penicillin G, for which the apparent Km values were 144, 100, 65, and 110 microM, respectively. Cephalexin, cepharidine, and cephaloridine were poor substrates. The beta-lactamase was strongly inhibited by cloxacillin and oxacillin but only slightly inhibited by phenylmethylsulfonyl fluoride or thiol reagents such as iodoacetate and p-chloromercuribenzoate.

Physical map of the Rhodobacter sphaeroides bacteriophage phi RsG1 genome and location of the prophage on the host chromosome.

We constructed a physical map of the 50-kilobase-pair (kb) DNA of the temperate Rhodobacter sphaeroides bacteriophage phi RsG1, with the relative positions of the cleavage sites for the nine restriction endonucleases KpnI, HindIII, XbaI, ClaI, BclI, EcoRV, EcoRI, BglII, and BamHI indicated. Using biotinylated phi RsG1 DNA as a probe in hybridization studies, we detected homologies with virus DNA and fragments of restriction endonuclease-digested host chromosomal DNA but not with plasmid DNA. This indicates that the prophage is integrated into the host chromosome. In addition, the use of specific probes such as the 10.4-kb BglII A fragment and the 2.65-kb BamHI H fragment allowed the determination of the position of phage attachment site (attP).

Purification and characterization of a bifunctional L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) from Rhodopseudomonas sphaeroides Y.

A bifunctional enzyme, L-(+)-tartrate dehydrogenase-D-(+)-malate dehydrogenase (decarboxylating) (EC 1.1.1.93 and EC 1.1.1. . . , respectively), was discovered in cells of Rhodopseudomonas sphaeroides Y, which accounts for the ability of this organism to grow on L-(+)-malate. The enzyme was purified 110-fold to homogeneity with a yield of 51%. During the course of purification, including ion-exchange chromatography and preparative gel electrophoresis, both enzyme activities appeared to be in association. The ratio of their activities remained almost constant [1:10, L-(+)-tartrate dehydrogenase/D-(+)-malate dehydrogenase (decarboxylating)] throughout all steps of purification. Analysis by polyacrylamide gel electrophoresis revealed the presence of a single protein band, the position of which was coincident with both L-(+)-tartrate dehydrogenase and D-(+)-malate dehydrogenase (decarboxylating) activities. The apparent molecular weight of the enzyme was determined to be 158,000 by gel filtration and 162,000 by ultracentrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis yielded a single polypeptide chain with an estimated molecular weight of 38,500, indicating that the enzyme consisted of four subunits of identical size. The isoelectric point of the enzyme was between pH 5.0 and 5.2. The enzyme catalyzed the NAD-linked oxidation of L-(+)-tartrate as well as the oxidative decarboxylation of D-(+)-malate. For both reactions, the optimal pH was in a range from 8.4 to 9.0. The activation energy of the reaction (delta Ho) was 71.8 kJ/mol for L-(+)-tartrate and 54.6 kJ/mol for D-(+)-malate. NAD was required as a cosubstrate, and optimal activity depended on the presence of both Mn2+ and NH4+ ions. The reactions followed Michaelis-Menten kinetics, and the apparent Km values of the individual reactants were determined to be: L-(+)-tartrate, 2.3 X 10(-3) M; NAD, 2.8 X 10(-4) M; and Mn2+, 1.6 X 10(-5) M with respect to L-(+)-tartrate; and D-(+)-malate, 1.7 X 10(-4) M; NAD, 1.3 X 10(-4); and Mn2+, 1.6 X 10(-5) M with respect to D-(+)-malate. Of a variety of compounds tested, only meso-tartrate, oxaloacetate, and dihydroxyfumarate were effective inhibitors. meso-Tartrate and oxaloacetate caused competitive inhibition, whereas dihydroxyfumarate caused noncompetitive inhibition. The Ki values determined for the inhibitors were, in the above sequence, 1.0, 0.014, and 0.06 mM with respect to L-(+)-tartrate and 0.28, 0.012, and 0.027 mM with respect to D-(+)-malate.

Adaptation of Rhodopseudomonas sphaeroides to Growth on d-(-)-Tartrate and Large-Scale Production of a Constitutive d-(-)-Tartrate Dehydratase During Growth on dl-Malate.

Of 10 strains of the purple non-sulfur bacterium Rhodopseudomonas sphaeroides, 8 acquired the ability to grow on d-(-)-tartrate; however, growth occurred only after extended lag phases ranging from 2 to 14 days. These lag phases occurred because only a small number of inoculum cells were able to grow by forming the enzyme d-(-)-tartrate dehydratase [d-(-)-tartrate hydro-lyase; EC number not yet available]. Once cells had grown on d-(-)-tartrate, d-(-)-tartrate dehydratase was formed constitutively. Therefore, mass cultivation of R. sphaeroides for production of large quantities of enzyme was possible on substrates much cheaper than d-(-)-tartrate. When 0.38 mol of dl-malate was used as a substrate in a chemotrophic fed-batch culture, a final biomass of 15 g (dry weight) liter and 1,500 U of d-(-)-tartrate dehydratase liter of culture were formed. The enzyme can be used for selective cleavage of racemic tartaric acid and for quantitative determination of d-(-)-tartrate.