NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

Summary A convenient and efficient method of NADPH production from NADP⁺ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP⁺ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a -carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.     

An NAD+-dependent alanine dehydrogenase from a methylotrophic bacterium.

A study was made of the NAD+-dependent alanine dehydrogenase (EC 1.4.1.1) elaborated by the methylotrophic bacterium Pseudomonas sp. strain MA when growing on succinate and NH4Cl. This enzyme was purified 400-fold and was found to be highly specific for NH3 and NAD+; however, hydroxypyruvate and bromopyruvate, but not alpha-oxoglutarate or glyoxylate, could replace pyruvate to a limited extent. The Mr of the native enzyme was shown to be 217,000, and electrophoresis in SDS/polyacrylamide gels revealed a minimum Mr of 53,000, suggesting a four-subunit structure. The enzyme, which has a pH optimum of 9.0, operated almost exclusively in the aminating direction in vitro. It was induced by NH3 or by alanine, and was repressed by growth on methylamine or glutamate. It is suggested that this enzyme has two roles in this organism, namely in NH3 assimilation and in alanine catabolism.     

A new NAD+-dependent opine dehydrogenase from Arthrobacter sp. strain 1C.

A new NAD+-dependent opine dehydrogenase was purified to homogeneity from Arthrobacter sp. strain 1C isolated from soil by an enrichment culture technique. The enzyme has a molecular weight of about 70,000 and consists of two identical subunits with molecular weights of about 36,000. The enzyme catalyzed a reversible oxidation-reduction reaction of opine-type secondary amine dicarboxylic acids. In the oxidative deamination reaction, the enzyme was active toward unusual opines, such as N-[1-R-(carboxyl)ethyl]-S-methionine and N-[1-R-(carboxyl)ethyl]-S-phenylalanine. In the reductive secondary amine-forming reaction with NADH as a cofactor, the enzyme utilized L-amino acids such as L-methionine, L-isoleucine, L-valine, L-phenylalanine, L-leucine, L-alanine, and L-threonine as amino donors and alpha-keto acids such as pyruvate, oxaloacetate, glyoxylate, and alpha-ketobutyrate as amino acceptors. The product enzymatically synthesized from L-phenylalanine and pyruvate in the presence of NADH was identified as N-[1-R-(carboxyl)ethyl]-S-phenylalanine.     

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.     

Nad-dependent formate dehydrogenase from methylotrophic bacteria. Characteristics and stability of soluble and immobilized enzyme]

pH-dependency is studied of kinetic parameters of the reaction catalyzed by NAD-dependent formate dehydrogenase from methylotrophic Bacterium spl strain. Values of Km for NAD and formate, and also of maximum reaction rate are found not to change within the pH range from 6 to 9. Role of SH-groups in the development of the enzyme catalytic activity and the effect of different factors on stability of soluble and immobilized enzyme forms are investigated. Molecular weight of the enzyme (70000), extinction coefficient and catalytical constant (6 s-1) are determined.     

Heterologous production of extreme alkaline thermostable NAD+-dependent formate dehydrogenase with wide-range pH activity from Myceliophthora thermophila

NAD⁺-dependent formate dehydrogenase(s) (EC 1.2.1.2, FDH) catalyzes the interconversion of formate anion to carbon dioxide coupled with the conversion of NAD⁺ or NADH. FDHs attract significant attention in biotechnology due to their potential applications in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, a new FDH from thermophilic fungus Myceliophthora thermophile (MtFDH) was characterized. The gene of the enzyme was synthesised, cloned, expressed in E. coli, as 6His-tagged protein, and purified to homogeneity by metal chelate affinity chromatography. Kinetic analysis suggested that MtFDH exhibits higher catalytic efficiency on NaHCO₃ compared to formate. Notable, recombinant MtFDH displays a pH optimum for the conversion of formate anion to carbon dioxide at extreme alkaline pH (pH 10.5). Thermal stability analysis showed that the enzyme displays good thermostability with T_m 48 °C. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the D-specific 2-hydroxy acid dehydrogenases family. The active-site residues are well conserved compared to other homologous FDHs. The results of the present work provide new knowledge on the structure, function and diversity of FDHs and indicate that MtFDH possess a huge potential for CO₂ reduction or NADH generation and under extreme alkaline conditions.     

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO₂) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO₂ for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO₂ and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO₂ into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO₂ reduction. Structural analysis of the enzyme through homology modeling is also presented.     

Improving the purification of NAD+-dependent formate dehydrogenase from Candida methylica

The Candida methylica (cm) recombinant wild type formate dehydrogenase (FDH) gene has been cloned into the pQE-2 TAGZyme expression vector and the 6xHis-tagged FDH gene has been overexpressed in JM105 cells to purify the FDH protein more efficiently, by the use of exopeptidases, TAGZyme Purification System, which has allowed the complete removal of the small N-terminal His-tag. After the purification procedure, 1.2 mg/mL cmFDH protein of >95% purity was obtained. The kinetic parameters of cmFDH have been determined by observing the oxidation of the nicotinamide coenzyme at 340 nm. The results have also been compared to the yield of standard vs. affinity purification of FDH.     

NAD-dependent formate dehydrogenase from methylotrophic bacteria. Isolation and properties]

NAD-Dependent formate dehydrogenase (FDH) has been isolated from methylotrophyc strain Bacterium sp 1 by (NH4)2SO4 fractionation of cell extract, ion-exchange chromatography and preparative isotachophoresis. Preparation of FDH is homogeneous in analytical polyacrylamide gel electrophoresis and under ultracentrifugation. Sedimentation coefficient of FDH is 4.9S. Mikhaelis constants are 1.1-10(-4) M for NAD and 1.5-10(-2) M for formate. In the absence of sulfhydril compounds FDH is unstable, but it is stable in the presence of mercaptoethanol or ditiotreitol.     

Characterization of a thermally stable and organic solvent-adaptative NAD+ -dependent formate dehydrogenase from Bacillus sp. F1

Aims: To characterize a robust NAD⁺‐dependent formate dehydrogenase firstly obtained from a nonmethylotroph, Bacillus sp. F1. Methods and Results: The Bacillus sp. F1 NAD⁺‐dependent formate dehydrogenase (BacFDH) gene was cloned by TAIL‐PCR and heterologous expressed in Escherichia coli. BacFDH was stable at temperatures below 55°C, and the half‐life at 60°C was determined as 52·9 min. This enzyme also showed a broad pH stability and retained more than 80% of the activities after incubating in buffers with different pH ranging from 4·5 to 10·5 for 1 h. The activity of BacFDH was significantly enhanced by some metal ions. Moreover, BacFDH exhibited high tolerance to 20% dimethyl sulfoxide, 60% acetone, 10% methanol, 20% ethanol, 60% isopropanol and 20% n‐hexane. Like other FDHs, BacFDH displayed strict substrate specificity for formate. Conclusion: We isolated a robust formate dehydrogenase, designated as BacFDH, which showed excellent thermal stability, organic solvent stability and a broad pH stability. Significance and Impact of the Study: The multi‐aspect stability makes BacFDH a competitive candidate for coenzyme regeneration in practical applications of chiral chemicals and pharmaceuticals synthesis with a relatively low cost, especially for the catalysis performed in extreme pH conditions and organic solvents.     

N-arylazido-beta-alanyl-NAD+, a new NAD+ photoaffinity analogue. Synthesis and labeling of mitochondrial NADH dehydrogenase

N-Arylazido-beta-alanyl-NAD+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] has been prepared by alkaline phosphatase treatment of arylazido-beta-alanyl-NADP+ [N3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NADP+]. This NAD+ analogue was found to be a potent competitive inhibitor (Ki = 1.45 microM) with respect to NADH for the purified bovine heart mitochondrial NADH dehydrogenase (EC 1.6.99.3). The enzyme was irreversibly inhibited as well as covalently labeled by this analogue upon photoirradiation. A stoichiometry of 1.15 mol of N-arylazido-beta-alanyl-NAD+ bound/mol of enzyme, at 100% inactivation, was determined from incorporation studies using tritium-labeled analogue. Among the three subunits, 0.85 mol of the analogue was bound to the Mr = 51,000 subunit, and each of the two smaller subunits contained 0.15 mol of the analogue when the dehydrogenase was completely inhibited upon photolysis. Both the irreversible inactivation and the covalent incorporation could be prevented by the presence of NADH during photolysis. These results indicate that N-arylazido-beta-alanyl-NAD+ is an active-site-directed photoaffinity label for the mitochondrial NADH dehydrogenase, and are further evidence that the Mr = 51,000 subunit contains the NADH binding site. Previous studies using A-arylazido-beta-alanyl-NAD+ [A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+] demonstrated that the NADH binding site is on the Mr = 51,000 subunit [Chen, S., & Guillory, R. J. (1981) J. Biol. Chem. 256, 8318-8323]. Results are also presented to show that N-arylazido-beta-alanyl-NAD+ binds the dehydrogenase in a more effective manner than A-arylazido-beta-alanyl-NAD+.     

NAD-dependent formate dehydrogenase from methylotrophic bacteria Pseudomonas sp. 101. I. Amino acid sequence

The primary structure of NAD-dependent formate dehydrogenase from methylotrophic bacterium Pseudomonas sp. 101 is determined. The enzyme is composed of two identical subunits, each comprising 393 amino acid residues, and has a molecular weight of 43.1 kD. To elucidate the protein's amino acid sequence, four types of digestion were used: cyanogen bromide cleavage at methionine residues, endoproteinase Lys-C digestion at lysine residues, endoproteinase Glu-C cleavage at glutamic acid residues, and tryptic digestion. The peptides obtained were purified to homogeneity and characterized.     

Evidence for the presence of a new NAD+-dependent formate dehydrogenase in Pseudomonas sp. 101 cells grown on a molybdenum-containing medium

The facultatively methylotrophic bacterium Pseudomonas sp. 101, grown on methanol in presence of molybdate, contains a new formate dehydrogenase (N-FDH) catalyzing NAD+-dependent oxidation of formate. The activity of this N-FDH could also be measured in presence of artificial electron acceptors, ferricyanide and 2,6-dichlorophenol indophenol. This new enzyme is absent in cells grown on a methanol-containing medium with tungstate, where only another two, previously described formate dehydrogenases, which are active only with NAD+ or only with artificial acceptors, respectively, were determined. The N-FDH was partially purified by a combination of ion-exchange and gel-filtration chromatography, and was shown to differ in its properties from the known NAD+-dependent counterpart.     

Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD(+)-dependent formate dehydrogenase

Metabolic engineering studies have generally focused on manipulating enzyme levels through either the amplification, addition, or deletion of a particular pathway. However, with cofactor-dependent production systems, once the enzyme levels are no longer limiting, cofactor availability and the ratio of the reduced to oxidized form of the cofactor can become limiting. Under these situations, cofactor manipulation may become crucial in order to further increase system productivity. Although it is generally known that cofactors play a major role in the production of different fermentation products, their role has not been thoroughly and systematically studied. However, cofactor manipulations can potentially become a powerful tool for metabolic engineering. Nicotinamide adenine dinucleotide (NAD) functions as a cofactor in over 300 oxidation-reduction reactions and regulates various enzymes and genetic processes. The NADH/NAD+ cofactor pair plays a major role in microbial catabolism, in which a carbon source, such as glucose, is oxidized using NAD+ producing reducing equivalents in the form of NADH. It is crucially important for continued cell growth that NADH be oxidized to NAD+ and a redox balance be achieved. Under aerobic growth, oxygen is used as the final electron acceptor. While under anaerobic growth, and in the absence of an alternate oxidizing agent, the regeneration of NAD+ is achieved through fermentation by using NADH to reduce metabolic intermediates. Therefore, an increase in the availability of NADH is expected to have an effect on the metabolic distribution. This paper investigates a genetic means of manipulating the availability of intracellular NADH in vivo by regenerating NADH through the heterologous expression of an NAD(+)-dependent formate dehydrogenase. More specifically, it explores the effect on the metabolic patterns in Escherichia coli under anaerobic and aerobic conditions of substituting the native cofactor-independent formate dehydrogenase (FDH) by and NAD(+)-dependent FDH from Candida boidinii. The over-expression of the NAD(+)-dependent FDH doubled the maximum yield of NADH from 2 to 4 mol NADH/mol glucose consumed, increased the final cell density, and provoked a significant change in the final metabolite concentration pattern both anaerobically and aerobically. Under anaerobic conditions, the production of more reduced metabolites was favored, as evidenced by a dramatic increase in the ethanol-to-acetate ratio. Even more interesting is the observation that during aerobic growth, the increased availability of NADH induced a shift to fermentation even in the presence of oxygen by stimulating pathways that are normally inactive under these conditions.     

Regeneration of NADH with immobilized systems of alanine dehydrogenase and hydrogen dehydrogenase

Summary An alternative approach to the regeneration of coenzymes using immobilized hydrogen dehydrogenase (hydrogenase) is described. Hydrogenase isolated from Alcaligenes Eutrophus was immobilized to porous glass particles and used in combination with alanine dehydrogenase for formation of alanine, while the NADH consumed was regenerated by molecular hydrogen. Different physical arrangements of the two enzymes were compared. Alanine was conveniently assayed with a specially designed enzyme thermistor method.     

Pilot scale production and isolation of recombinant NAD+- and NADP+-specific formate dehydrogenases.

The expression of the recombinant wild-type NAD+- and mutant NADP+-dependent formate dehydrogenases (EC 1.2.1.2., FDH) from the methanol-utilizing bacterium Pseudomonas sp. 101 in Escherichia coli cells has been improved to produce active and soluble enzyme up to the level of 50% of total soluble proteins. The cultivation process for E. coli/pFDH8a and E. coli/pFDH8aNP cells was optimized and scaled up to a volume of 100 L. A downstream purification process has been developed to produce technical grade NAD+- and NADP+-specific formate dehydrogenases in pilot scale, utilizing extraction in aqueous two-phase systems.     

Enzymatic production of d-3-phenyllactic acid by Pediococcus pentosaceus d-lactate dehydrogenase with NADH regeneration by Ogataea parapolymorpha formate dehydrogenase

3-Phenyllactic acid (PLA) is an antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. Enzymatic production of PLA can be carried out from phenylpyruvic acid by lactate dehydrogenase (LDH); however, the enzymatic reaction is accompanied by NADH oxidation that inhibits PLA biotransformation. Here, NADH regeneration was achieved using the formate dehydrogenase from Ogataea parapolymorpha and introduced into the d-PLA production process using the d-LDH from Pediococcus pentosaceus. Optimum PLA production by dual enzyme treatment was at pH 6.0 and 50 °C with both enzymes at 0.4 μM. Using 0.2 mM NADH, d-PLA production by NADH regeneration system reached 5.5 mM, which was significantly higher than that by a single-enzyme reaction.     

Transition metal requirement to express high level NAD+-dependent formate dehydrogenase from a serine-type methylotrophic bacterium

Abstract Formate dehydrogenase (EC 1.2.1.2) from an aerobic organism was found to be metal-dependent. This NAD⁺-dependent enxyme required the presence of tungsten or molybdenum to express high enzyme levels in the facultative methylotrophic Methylobacterium sp. RXM. The apparent V _max of the reaction increased 22-fold in a tungstate-containing medium when compared with a non-metal-supplemented growth medium. The absence of those metals in the culture medium resulted in the partial loss of an energy-yielding step and approx. 50% decrease in the cell yield was observed. Moreover, formate accumulated in the extracellular medium and culture pH dropped. Tungsten produced a higher stimulation of formate dehydrogenase activity than that obtained with molybdenum for batch cultivation of Methylobacterium sp. RXM.