Stability and reactivity of liposome‐encapsulated formate dehydrogenase and cofactor system in carbon dioxide gas‐liquid flow

Formate dehydrogenase from Candida boidinii (CbFDH) is potentially applicable in reduction of CO₂ through oxidation of cofactor NADH into NAD⁺. For this, the CbFDH activity needs to be maintained under practical reaction conditions, such as CO₂ gas‐liquid flow. In this work, CbFDH and cofactor were encapsulated in liposomes and the liposomal enzymes were characterized in an external loop airlift bubble column. The airlift was operated at 45°C with N₂ or CO₂ as gas phase at the superficial gas velocity U_G of 2.0 or 3.0 cm/s. The activities of liposomal CbFDH/cofactor systems were highly stable in the airlift regardless of the type of gas phase because liposome membranes prevented interactions of the encapsulated enzyme and cofactor molecules with the gas‐liquid interface of bubbles. On the other hand, free CbFDH was deactivated in the airlift especially at high U_G with CO₂ bubbles. The liposomal CbFDH/NADH could catalyze reduction of CO₂ in the airlift giving the fractional oxidation of the liposomal NADH of 23% at the reaction time of 360 min. The cofactor was kept inside liposomes during the reaction operation with less than 10% of leakage. All of the results obtained demonstrate that the liposomal CbFDH/NADH functions as a stable catalyst for reduction of CO₂ in the airlift. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC‐MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate‐coupled cofactor regeneration by oxidation of 2‐propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)‐methyl 3‐hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively. Biotechnol. Bioeng. 2009; 104: 251–260 © 2009 Wiley Periodicals, Inc.     

Unique coenzyme binding mode of hyperthermophilic archaeal sn‐glycerol‐1‐phosphate dehydrogenase from Pyrobaculum calidifontis

A gene encoding an sn‐glycerol‐1‐phosphate dehydrogenase (G1PDH) was identified in the hyperthermophilic archaeon Pyrobaculum calidifontis. The gene was overexpressed in Escherichia coli, and its product was purified and characterized. In contrast to conventional G1PDHs, the expressed enzyme showed strong preference for NADH: the reaction rate (V_max) with NADPH was only 2.4% of that with NADH. The crystal structure of the enzyme was determined at a resolution of 2.45 Å. The asymmetric unit consisted of one homohexamer. Refinement of the structure and HPLC analysis showed the presence of the bound cofactor NADPH in subunits D, E, and F, even though it was not added in the crystallization procedure. The phosphate group at C2’ of the adenine ribose of NADPH is tightly held through the five biased hydrogen bonds with Ser40 and Thr42. In comparison with the known G1PDH structure, the NADPH molecule was observed to be pushed away from the normal coenzyme binding site. Interestingly, the S40A/T42A double mutant enzyme acquired much higher reactivity than the wild‐type enzyme with NADPH, which suggests that the biased interactions around the C2’‐phosphate group make NADPH binding insufficient for catalysis. Our results provide a unique structural basis for coenzyme preference in NAD(P)‐dependent dehydrogenases. Proteins 2016; 84:1786–1796. © 2016 Wiley Periodicals, Inc.     

The mechanism of coupling between oxido‐reduction and proton translocation in respiratory chain enzymes

The respiratory chain of mitochondria and bacteria is made up of a set of membrane‐associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane‐associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co‐operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X‐ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone‐cytochrome c oxidoreductase and NADH‐ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes.     

Computational design of Candida boidinii xylose reductase for altered cofactor specificity

In this study we introduce a computationally‐driven enzyme redesign workflow for altering cofactor specificity from NADPH to NADH. By compiling and comparing data from previous studies involving cofactor switching mutations, we show that their effect cannot be explained as straightforward changes in volume, hydrophobicity, charge, or BLOSUM62 scores of the residues populating the cofactor binding site. Instead, we find that the use of a detailed cofactor binding energy approximation is needed to adequately capture the relative affinity towards different cofactors. The implicit solvation models Generalized Born with molecular volume integration and Generalized Born with simple switching were integrated in the iterative protein redesign and optimization (IPRO) framework to drive the redesign of Candida boidinii xylose reductase (CbXR) to function using the non‐native cofactor NADH. We identified 10 variants, out of the 8,000 possible combinations of mutations, that improve the computationally assessed binding affinity for NADH by introducing mutations in the CbXR binding pocket. Experimental testing revealed that seven out of ten possessed significant xylose reductase activity utilizing NADH, with the best experimental design (CbXR‐GGD) being 27‐fold more active on NADH. The NADPH‐dependent activity for eight out of ten predicted designs was either completely abolished or significantly diminished by at least 90%, yielding a greater than 10⁴‐fold change in specificity to NADH (CbXR‐REG). The remaining two variants (CbXR‐RTT and CBXR‐EQR) had dual cofactor specificity for both nicotinamide cofactors.     

Rapid method for the assessment of cell lysis in microalgae cultures

A solid understanding of the effect of hydrodynamic forces encountered by microalgae in bioprocesses would benefit existing bioprocesses, eventually allowing an increase in their productivity. For this purpose, a sensitive method able to quantify cell lysis is crucial. Most of the available protocols and methods intended for this purpose were developed for animal or insect cells. In the case of microalgae, the commercial kits tested were unable to determine the cell lysis extension. The method proposed here was developed to relate the release of a cytoplasmic component (enzyme lactate dehydrogenase (LDH)) with cell lysis by measuring the NADH (reduced form of nicotinamide cofactor adenine dinucleotide) produced by LDH. Although different commercial kits based on similar processes are available, they are more complicated to use and not applicable to microalgae nor when longer-term tests are to be performed.     

Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Luciferase and fluorescent protein as dual reporters analyzing the effect of <Emphasis Type="Italic">n</Emphasis>-dodecyltrimethylammonium bromide on the physiology of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

With the growing interest in using surfactants to improve microbial cell performance for whole-cell biocatalysis and bioremediation, understanding the interactions between surfactants and bacteria is of great importance. By using cyanine fluorescent protein (CFP) and bacterial luciferase (LUX) as dual bioreporters, the effects of n-dodecyltrimethylammonium bromide (DTAB) on the whole cells and intracellular proteins in Pseudomonas putida cultures were quantitatively and systematically studied. The dual reporter system was shown to be a useful indicator to assess the effect of DTAB treatment on whole-cell metabolic activity, membrane permeability, and cellular enzyme activity. CFP was useful to assess the leakage of intracellular enzymes and the lysis of cells and was able to reflect the activities of most cellular enzymes, while LUX reflected the permeability of cell membranes and cellular metabolic activity. The validity of CFP–LUX dual bioreporters was further confirmed by detecting changes in extracellular proteins, membrane potential, oxygen consumption rate (OUR), and intracellular catechol 2,3-dioxygenase (C23O) activity with the addition of DTAB. The dual LUX–CFP bioreporter is a useful tool for analyzing the surfactant–bacterium interactions for biotechnological applications.     

The cytosolic or the mitochondrial glutathione peroxidase‐type tryparedoxin peroxidase is sufficient to protect procyclic Trypanosoma brucei from iron‐mediated mitochondrial damage and lysis

African trypanosomes express three virtually identical glutathione peroxidase (Px)‐type enzymes that occur in the cytosol (Px I and II) and mitochondrion (Px III) and detoxify fatty acid‐derived hydroperoxides. Selective deletion of the genes revealed that procyclic Trypanosoma brucei lacking either the cytosolic or mitochondrial enzyme proliferate nearly as wild‐type parasites, whereas the knockout of the complete genomic locus is lethal. Flow cytometry and immunofluorescence analyses revealed that the Px I‐III‐deficient parasites lose their mitochondrial membrane potential, which is followed by a loss of the lysosomal signal but not the glycosomal one. Mitochondrial damage and cell lysis are prevented by Trolox, ubiquinone derivatives and the iron chelator deferoxamine, whereas starch‐deferoxamine is inefficient. In glucose‐rich medium, cell death is attenuated suggesting that oxidants generated by the respiratory chain contribute to the lethal phenotype. Thus, the Px‐type peroxidases protect procyclic cells from an iron‐mediated oxidative membrane damage that originates at the mitochondrion. This contrasts with the situation in bloodstream cells, where the lysosome is the primarily affected organelle. Strikingly, either the cytosolic or the mitochondrial form of the peroxidases is required and sufficient to protect the mitochondrion and prevent cell lysis.     

The Role of Yeast VDAC Genes on the Permeability of the Mitochondrial Outer Membrane

In addition to the POR1 gene, which encodes the well-characterized voltage dependent anion-selective channel (YVDAC1) of the mitochondrial outer membrane, the yeast Saccharomyces cerevisiae contains a second gene (POR2) encoding a protein (YVDAC2) with 50% sequence identity to YVDAC1. Mitochondria isolated from yeast cells deleted for the POR1 gene (Δpor1) had a profoundly reduced outer membrane permeability as measured by the ability of an intermembrane space dehydrogenase to oxidize exogenously added NADH. Mitochondria missing either YVDAC1 or both YVDAC1 and YVDAC2 showed a 2-fold increase in the rate of NADH oxidation when the outer membrane was deliberately damaged. Mitochondria from parental cells showed only a 10% increase indicating that the outer membrane is highly permeable to NADH. In the absence of YVDAC1, we calculate that the outer membrane permeability to NADH is reduced 20-fold. The low NADH permeability in the presence of YVDAC2 was not due to the low levels of YVDAC2 expression as mitochondria from cells expressing levels of YVDAC2 comparable to those of YVDAC1 in parental cells showed no substantial increase in NADH permeability, indicating a minimal role of YVDAC2 in this permeability. The residual permeability may be due to other pathways because cells missing both genes can still grow on nonfermentable carbon sources. However, YVDAC1 is clearly the major pathway for NADH flux through the outer membrane in these mitochondria. Received: 23 May 1997/Revised: 3 October 1997     

Purification and characterization of NADH oxidase from Serpulina (Treponema) hyodysenteriae.

NADH oxidase (EC 1.6.99.3) was purified from cell lysates of Serpulina (Treponema) hyodysenteriae B204 by differential ultracentrifugation, ammonium sulfate precipitation, and chromatography on anion-exchange, dye-ligand-affinity, and size-exclusion columns. Purified NADH oxidase had a specific activity 119-fold higher than that of cell lysates and migrated as a single band during denaturing gel electrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]). The enzyme was a monomeric protein with an estimated molecular mass of 47 to 48 kDa, as determined by SDS-PAGE and size-exclusion chromatography. Optimum enzyme activity occurred in buffers with a pH between 5.5 and 7.0. In the presence of oxygen, beta-NADH but not alpha-NADH, alpha-NADPH, or beta-NADPH was rapidly oxidized by the enzyme (Km = 10 microM beta-NADH; Vmax = 110 mumol beta-NADH min-1 mg of protein-1). Oxygen was the only identified electron acceptor for the enzyme. On isoelectric focusing gels, the enzyme separated into three subforms, with isoelectric pH values of 5.25, 5.35, and 5.45. Purified NADH oxidase had a typical flavoprotein absorption spectrum, with peak absorbances at wavelengths of 274, 376, and 448 nm. Flavin adenine dinucleotide was identified as a cofactor and was noncovalently associated with the enzyme at a molar ratio of 1:1. Assays of the enzyme after various chemical treatments indicated that a flavin cofactor and a sulfhydryl group(s), but not a metal cofactor, were essential for activity. Hydrogen peroxide and superoxide were not yielded in significant amounts by the S. hyodysenteriae NADH oxidase, indirect evidence that the enzyme produces water from reduction of oxygen with NADH. The N-terminal amino acid sequence of the NADH oxidase was determined to be MKVIVIGCHGAGTWAAK. In its biochemical properties, the NADH oxidase of S. hyodysenteriae resembles the NADH oxidase of another intestinal bacterium, Enterococcus faecalis.     

Design of a cytochrome P450BM3 reaction system linked by two‐step cofactor regeneration catalyzed by a soluble transhydrogenase and glycerol dehydrogenase

A cytochrome P450BM3‐catalyzed reaction system linked by a two‐step cofactor regeneration was investigated in a cell‐free system. The two‐step cofactor regeneration of redox cofactors, NADH and NADPH, was constructed by NAD⁺‐dependent bacterial glycerol dehydrogenase (GLD) and bacterial soluble transhydrogenase (STH) both from Escherichia coli. In the present system, the reduced cofactor (NADH) was regenerated by GLD from the oxidized cofactor (NAD⁺) using glycerol as a sacrificial cosubstrate. The reducing equivalents were subsequently transferred to NADP⁺ by STH as a cycling catalyst. The resultant regenerated NADPH was used for the substrate oxidation catalyzed by cytochrome P450BM3. The initial rate of the P450BM3‐catalyzed reaction linked by the two‐step cofactor regeneration showed a slight increase (approximately twice) when increasing the GLD units 10‐fold under initial reaction conditions. In contrast, a 10‐fold increase in STH units resulted in about a 9‐fold increase in the initial reaction rate, implying that transhydrogenation catalyzed by STH was the rate‐determining step. In the system lacking the two‐step cofactor regeneration, 34% conversion of 50 μM of a model substrate (p‐nitrophenoxydecanoic acid) was attained using 50 μM NADPH. In contrast, with the two‐step cofactor regeneration, the same amount of substrate was completely converted using 5 μM of oxidized cofactors (NAD⁺ and NADP⁺) within 1 h. Furthermore, a 10‐fold dilution of the oxidized cofactors still led to approximately 20% conversion in 1 h. These results indicate the potential of the combination of GLD and STH for use in redox cofactor recycling with catalytic quantities of NAD⁺ and NADP⁺. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Analyzing Thermal Stability of Cell Membrane of Salmonella Using Time-Multiplexed Impedance Sensing

Heat treatment is one of the most widely used methods for inactivation of bacteria in food products. Heat-induced loss of bacterial viability has been variously attributed to protein denaturation, oxidative stress, or membrane leakage; indeed, it is likely to involve a combination of these processes. We examine the effect of mild heat stress (50–55°C for ≤12 min) on cell permeability by directly measuring the electrical conductance of samples of Salmonella enterica serovar Typhimurium to answer a fundamental biophysical question, namely, how bacteria die under mild heat stress. Our results show that when exposed to heat shock, the cell membrane is damaged and cells die mainly due to the leakage of small cytoplasmic species to the surrounding media without lysis (confirmed by fluorescent imaging). We measured the conductance change, ΔY, of wild-type versus genetically modified heat-resistant (HR) cells in response to pulse and ramp heating profiles with different thermal time constants. In addition, we developed a phenomenological model to correlate the membrane damage, cytoplasmic leakage, and cell viability. This model traces the differential viability and ΔY of wild-type and HR cells to the difference in the effective activation energies needed to permeabilize the cells, implying that HR cells are characterized by stronger lateral interactions between molecules, such as lipids, in their cell envelope.     

Physiology of Aspergillus niger in oxygen‐limited continuous cultures: Influence of aeration,carbon source concentration and dilution rate

In industrial production of enzymes using the filamentous fungus Aspergillus niger supply of sufficient oxygen is often a limitation, resulting in the formation of by‐products such as polyols. In order to identify the mechanisms behind formation of the different by‐products we studied the effect of low oxygen availability, at different carbon source concentrations and at different specific growth rates, on the metabolism of A. niger, using continuous cultures. The results show that there is an increase in the production of tricarboxylic acid (TCA) cycle intermediates at low oxygen concentrations. Indeed, at these conditions, a decrease in the mitochondrial respiratory chain activity leads to an accumulation of NADH and to a decreased ATP production which uncouples catabolism and anabolism, influences the intracellular pH and leads to production and excretion of organic acids. Moreover, mannitol is being produced in order to ensure reoxidation of NADH, and this is the main cellular response to balance the ratio NADH/NAD at low oxygen availability. Mannitol production is also coupled to low specific growth rate, which suggests a control of carbon catabolite repression on the mannitol pathway. The roles of two other polyols, erythritol and glycerol, were also investigated. Both compounds are known to accumulate intracellularly, at high osmotic pressure, in order to restore the osmotic balance, but we show that the efficiency of this system is affected by a leakage of polyols through the membrane. Biotechnol. Bioeng. Biotechnol. Bioeng. 2009;103: 956–965. © 2009 Wiley Periodicals, Inc.     

Evidence for electron transport across the plasma membrane of Zea mays root cells

Exogenous ferricyanide is reduced by roots of Z. mays. In contrast to oxidation of exogenous electron donors, ferricyanide reduction occurs mostly at the apical 5 mm of the root. Using just this portion of the root, it is shown that the activity is neither a consequence of uptake of ferricyanide followed by excretion of its reduced form, nor of leakage of a reductant. Addition of ferricyanide for 40 s or 5 min results in an apparent oxidation of NADPH but not of NADH; rates of ferricyanide reduction vary together with levels of NADPH but not of NADH in the presence or absence of oxygen. It is concluded that an enzyme which can oxidize cytoplasmic NADPH and transfer the electrons to an external acceptor exists at the cell surface of maize roots. This finding extends the results of others who showed similar redox activity at the surface of Fe-depleted dicotyledonous roots, and indicates that an energy source other than ATP exists at the cell surface of a variety of plants under unstressed conditions.     

The Sulfonylurea-Inhibited NADH Oxidase Activity of HeLa Cell Plasma Membranes has Properties of a Protein Disulfide–Thiol Oxidoreductase with Protein Disulfide–Thiol Interchange Activity

Plasma membrane vesicles of HeLa cells are characterized by a drug-responsive oxidation of NADH. The NADH oxidation takes place in an argon or nitrogen atmosphere and in samples purged of oxygen. Direct assay of protein thiols by reaction with 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent), suggests that protein disulfides may be the natural electron acceptors for NADH oxidation by the plasma membrane vesicles. In the presence of NADH, protein disulfides of the membranes were reduced with a concomitant stoichiometric increase in protein thiols. The increase in protein thiols was inhibited in parallel to the inhibition of NADH oxidation by the antitumor sulfonylurea LY181984 with an EC₅₀ of ca. 30 nM. LY181984, with an EC₅₀ of 30 nM, also inhibited a protein disulfide–thiol interchange activity based on the restoration of activity to inactive (scrambled) RNase and thiol oxidation. The findings suggest that thiol oxidation, NADH-dependent disulfide reduction (NADH oxidation), and protein disulfide–thiol interchange in the absence of NADH all may be manifestations of the same sulfonylurea binding protein of the HeLa plasma membrane. A surface location of the thiols involved was demonstrated using detergents and the impermeant thiol reagent p-chloromercuriphenylsulfonic acid (PCMPS). The surface location precludes a physiological role of the protein in NADH oxidation. Rather, it may carry out some other role more closely related to a function in growth, such as protein disulfide–thiol interchange coupled to cell enlargement.     

Rv2074 is a novel F420H2‐dependent biliverdin reductase in Mycobacterium tuberculosis

Bilirubin is a potent antioxidant that is produced from the reduction of the heme degradation product biliverdin. In mammalian cells and Cyanobacteria, NADH/NADPH‐dependent biliverdin reductases (BVRs) of the Rossmann‐fold have been shown to catalyze this reaction. Here, we describe the characterization of Rv2074 from Mycobacterium tuberculosis, which belongs to a structurally and mechanistically distinct family of F₄₂₀H₂‐dependent BVRs (F‐BVRs) that are exclusively found in Actinobacteria. We have solved the crystal structure of Rv2074 bound to its cofactor, F₄₂₀, and used this alongside molecular dynamics simulations, site‐directed mutagenesis and NMR spectroscopy to elucidate its catalytic mechanism. The production of bilirubin by Rv2074 could exploit the anti‐oxidative properties of bilirubin and contribute to the range of immuno‐evasive mechanisms that have evolved in M. tuberculosis to allow persistent infection.     

Protective effect of edaravone against PrP106‐126–induced PC12 cell death

The prion protein peptide PrP106‐126 induces cell apoptosis through mechanisms involving production of intracellular reactive oxygen species. The present study investigated the effects of edaravone, a potent free radical scavenger in clinical use, on cell cytotoxicity induced by PrP106‐126. Results showed that PrP106‐126 decreased PC12 cell viability in a dose‐ and time‐dependent manner. Edaravone significantly antagonized the cytotoxic effects of PrP106‐126. Mechanistically, PrP106‐126 decreased PC 12 intracellular glutathione (GSH) concentrations, decreased superoxide dismutase (SOD) enzyme activity, increased concentrations of the oxidation end product malondialdehyde (MDA), depolarized the mitochondrial membrane, and increased caspase‐3 activity. Edaravone alone did not affect GSH, SOD, or MDA but did effectively reverse all of the intracellular prooxidant effects induced by PrP106‐126 and inhibit induced apoptosis in PC12 cells. In conclusion, edaravone may be a viable candidate for the treatment of oxidative stress‐induced neurodegenerative disease. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:235–241, 2010; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.20330     

Biotransformation of R-2-hydroxy-4-phenylbutyric acid by D-lactate dehydrogenase and Candida boidinii cells containing formate dehydrogenase coimmobilized in a fibrous bed bioreactor

R-2-hydroxy-4-phenylbutyric acid (R-HPBA) is an important intermediate in the manufacture of angiotensin converting enzyme inhibitors. In this work, a recombinant D-lactate dehydrogenase (LDH) was used to transform 2-oxo-4-phenylbutyric acid (OPBA) to R-HPBA, with concomitant oxidation of beta-nicotinamide adenine dinucleotide (NADH) to NAD(+). The cofactor NADH was regenerated by formate dehydrogenase (FDH) present in whole cells of Candida boidinii, which were pre-treated with toluene to make them permeable. The whole cells used in the process were more stable and easier to prepare as compared with the isolated FDH from the cells. Kinetic study showed that the reaction rate was dependent on the concentration of cofactor, NAD(+), and that both R-HPBA and OPBA inhibited the reaction. A novel method for co-immobilization of whole cells and LDH enzyme on cotton cloth was developed using polyethyleneimine (PEI), which induced the formation of PEI-enzyme-cell aggregates and their adsorption onto cotton cloth, leading to multilayer co-immobilization of cells and enzyme with high loading (0.5 g cell and 8 mg LDH per gram of cotton cloth) and activity yield ( > 95%). A fibrous bed bioreactor with co-immobilized cells and enzyme on the cotton cloth was then evaluated for R-HPBA production in fed-batch and repeated batch modes, which gave relatively stable reactor productivity of 9 g/L . h and product yield of 0.95 mol/mol OPBA when the concentrations of OPBA and R-HPBA were less than 10 g/L.