Astaxanthin production using Paracoccus carotinifaciens: a way forward?

Paracoccus carotinifaciens could be considered a key microbial factory for obtaining healthier natural products such as astaxanthin (AXT), thus contributing to a bioeconomy. Short cultivation time, high production titers, and thin cell wall are the main advantages that make this bacterium promising in the development of sustainable third-generation biorefineries.     

Efficient and environmentally friendly method for carotenoid extraction from Paracoccus carotinifaciens utilizing naturally occurring Z-isomerization-accelerating catalysts

This study aimed to improve the efficiency of Paracoccus carotinifaciens-derived carotenoid (astaxanthin, adonirubin, adonixanthin) extraction using environmentally friendly Z-isomerization-accelerating catalysts. Adding naturally occurring catalysts such as isothiocyanates and polysulfides to the extraction solvent significantly improved the efficiency of carotenoid extraction, likely because of enhanced solubility of carotenoid Z-isomers compared with all-E-isomers. Indeed, addition of the catalysts markedly increased the content of carotenoid Z-isomers in the resulting extract. The use of a catalyst allyl isothiocyanate, which is abundantly included in Brassicaceae plant family, at high extraction temperature and long extraction time led to increased carotenoid recovery and Z-isomer content. These findings will enhance the efficiency of organic solvent-based extraction of carotenoids from carotenoid-rich sources. Numerous studies have reported that the Z-isomers of carotenoids exhibit greater bioavailability and antioxidant capacity than the all-E-isomers. Hence, the method proposed here utilizing Z-isomerization-accelerating catalysts could enhance both the extraction efficiency and beneficial health effects of carotenoids.     

Functional expression of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis

The astaxanthin biosynthesis gene cluster in Paracoccus haeundaensis consists of six genes: crtW, crtZ, crtY, crtI, crtB, and crtE contain 726, 486, 1158, 1503, 912, and 879 base pairs, respectively. Individual carotenoid biosynthesis genes of P. haeundaensis have now been expressed in E. coli and each gene product has been purified to homogeneity. Their molecular characteristics, including enzymatic activities, are reported here.     

ATP-AMP phosphotransferase from Paracoccus denitrificans

1H NMR is used to study the solution structure of vitamin-D-induced bovine intestinal calcium-binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80 s-1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.     

The effect of oxygen on denitrification during steady-state growth of Paracoccus halodenitrificans

Dimethylsulphoxide (DMSO) and trimethylamine oxide (TMAO) sustained anaerobic growth of Proteus vulgaris with the non-fermentable substrate lactate. Cytoplasmic membrane vesicles energized by electron transfer from formate to DMSO displayed anaerobic uptake of serine, which was hindered by metabolic inhibitors known to destroy the proton motive force. This showed that DMSO reduction was coupled with a chemiosmotic mechanism of energy conversion; similar data for TMAO respiration have been presented previously. All biochemical tests applied indicated that the oxides were reduced by the same reductase system. The DMSO and TMAO reductase activities showed the same mobility on ion-exchange chromatography, and polyacrylamide disc gel electrophoresis (pH 8.9), gradient gel electrophoresis, and gel isoelectric focusing; mol. wt. and pI determined were 95,000 and 4.6, respectively. DMSO inhibited reduction of [¹⁴C]TMAO in vesicles. The reductase was inducible to a certain extent; both oxides being equally efficient as inducers. TMAO was reduced at a higher rate than DMSO, explaining faster growth of cells and increased uptake of serine in vesicles with TMAO as electron acceptor. Comparative studies with Escherichia coli also gave evidence for common TMAO and DMSO reductase systems.Abbreviations TMAO trimethylamine oxide - DMSO dimethylsulphoxide     

The effect of oxygen on denitrification in Paracoccus denitrificans and Pseudomonas aeruginosa

Denitrification by Paracoccus denitrificans and Pseudomonas aeruginosa was studied using quadrupole membrane-inlet mass spectrometry to measure simultaneously and continuously dissolved gases. Evidence was provided for aerobic denitrification by both species: in the presence of O2, N2O production increased in Pa. denitrificans, while that of N2 decreased; with Ps. aeruginosa, the concentrations of both N2 and N2O increased on introducing O2 into the gas phase. Disappearance of NO-3 was monitored in anaerobically and aerobically grown cells which were maintained either anaerobically or aerobically: the rate and extent of NO-3 utilization by both species depended on growth and maintenance conditions. The initial rate of disappearance was most rapid under completely anaerobic conditions, and lowest rates occurred when cells were grown anaerobically and maintained aerobically. In nitrogen balance experiments both species converted over 87% of the added NO-3 to N2 and N2O under both anaerobic and aerobic maintenance conditions.     

The preparation of tightly coupled membrane vesicles from Paracoccus denitrificans

Nonequilibrium sedimentation of membrane vesicles from Paracoccus denitrificans through Ficoll gradients results in a separation into two fractions. The fraction which pellets through dense Ficoll is uncoupled. The second fraction, retarded by dense Ficoll, shows both improved concentrative transport activity and greater uncoupler stimulation of respiration as compared to the original vesicle preparation.     

The Met99Gln mutant of amicyanin from Paracoccus versutus

The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus. Dynamic and structural characteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR, cyclic voltammetry, and isomorphous metal replacement. M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin. In addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been assessed and compared to values for M121Q azurin. For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M. These values are very similar to the midpoint potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin. A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.     

The peroxidase activity of cytochrome c-550 from Paracoccus versutus.

Next to their natural electron transport capacities, c-type cytochromes possess low peroxidase and cytochrome P-450 activities in the presence of hydrogen peroxide. These catalytic properties, in combination with their structural robustness and covalently bound cofactor make cytochromes c potentially useful peroxidase mimics. This study reports on the peroxidase activity of cytochrome c-550 from Paracoccus versutus and the loss of this activity in presence of H2O2. The rate-determining step in the peroxidase reaction of cytochrome c-550 is the formation of a reactive intermediate, following binding of peroxide to the haem iron. The reaction rate is very low compared to horseradish peroxidase (approximately one millionth), because of the poor accessibility of the haem iron for H2O2, and the lack of a base catalyst such as the distal His of the peroxidases. This is corroborated by the linear dependence of the reaction rate on the peroxide concentration up to at least 1 M H2O2. Steady-state conversion of a reducing substrate, guaiacol, is preceded by an activation phase, which is ascribed to the build-up of amino-acid radicals on the protein. The inactivation kinetics in the absence of reducing substrate are mono-exponential and shown to be concurrent with haem degradation up to 25 mM H2O2 (pH 8.0). At still higher peroxide concentrations, inactivation kinetics are biphasic, as a result of a remarkable protective effect of H2O2, involving the formation of superoxide and ferrocytochrome c-550.     

The effect of monoterpenes on astaxanthin production by a mutant ofPhaffia rhodozyma

Summary The total pigment and astaxanthin content ofPhaffia rhodozyma increased with increasing concentrations -pinene up to 500 l -pinene/l. Above this concentration the total pigment and astaxanthin content as well as the biomass production decreased. The addition of 500 l -pinene/l increased the total pigment content from 1652 g/g to 2201 g/g and the astaxanthin content from 1554 g/g to 1883 g/g. A sharp decrease in maximum specific growth rate occurred above 150 l -pinene/l.     

Dimeric porin from Paracoccus denitrificans.

Paracoccus denitrificans was shown to contain a 33,000-dalton porin, which produced pores of large (1.6 to 1.8 nm) diameter. Cross-linking studies showed that the porin existed as dimers in the outer membrane.     

Cytochrome c1 from Paracoccus denitrificans

Cytochrome c1 was purified from the bacterium Paracoccus denitrificans. It is an acidic, hydrophobic polypeptide with an apparent molecular weight of around 65000 and a single, covalently attached heme; it cross-reacts immunologically with cytochrome c1 from yeast mitochondria. The amino acid sequence of the tryptic heme peptide of the bacterial cytochrome c1 shows extensive homology to the corresponding region of beef heart cytochrome c1 [Wakabayashi, S. et al. (1982) J. Biol. Chem. 257, 9335-9344]. Positive evidence for a stable association of the Paracoccus cytochrome c1 with other polypeptides and b-type heme components ('bc1-complex') has not yet been obtained.     

The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans

We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al. [(1999) J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans. Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca(2+), or higher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formation with redox partners were performed in the presence of Ca(2+) in order to simplify the equilibria that had to be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K(d) values of 0.8 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface. For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range. We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase.     

Antidepressant-like effect of saponins extracted from Chaihu-jia-longgu-muli-tang and its possible mechanism

In this study, we investigated the antidepressant-like effect of saponins (SCLM) extracted from a traditional Chinese medicine, Chaihu-jia-longgu-muli-tang (CLM), in mice and rats using the tail suspension test (TST) and forced swimming test (FST). Subchronic administration of 100 and 200 mg/kg (p.o.) SCLM for 7 days reduced immobility time in the TST and FST in mice and also decreased immobility time at 70 and 140 mg/kg (p.o.) in the FST in rats. The results also showed that the anti-immobility activity of SCLM in these two tests is dose-dependent, without accompanying significant effects on locomotor activity. In addition, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and lactic dehydrogenase (LDH) assays showed that 25, 50 and 100 microg/ml SCLM or 10 microM fluoxetine (FLU), protected PC12 cells from the lesion induced by 10 microM corticosterone (Cort) treatment for 48 h. In the fura-2/AM (acetoxymethyl ester) labeling assay, 50 and 100 microg/ml SCLM, 10 microM FLU attenuated the intracellular Ca2+ overloading induced by 200 microM Cort treatment for 48 h in PC12 cells. Using RT-PCR, the mRNA level of nerve growth factor (NGF) was also detected. Treatment with SCLM (50, 100 microg/ml) for 48 h elevated the NGF mRNA expression in PC12 cells. In summary, these results suggest that SCLM possesses an antidepressant-like activity in behavioral models that might be mediated via the cytoprotective action shown in PC12 cells.     

The membrane-bound hydrogenase from Paracoccus denitrificans. Purification and molecular characterization

The membrane-bound hydrogenase from Paracoccus denitrificans was purified 68-fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 mumol H2 min-1 (mg protein)-1 (methylene blue reduction). Purification involved solubilization by Triton X-114, phase separation, chromatography on DEAE-Sephacel, ammonium-sulfate precipitation and chromatography on Procion-red HE-3B-Sepharose. Gel electrophoresis under denaturing conditions revealed two non-identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100 kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane-bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half-life for inactivation under air at 20 degrees C was 8 h. The Paracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c, FMN, 2,6-dichloroindophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue (V = 161.9 U/mg; Km = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross-reacted with purified antibodies raised against the membrane-bound hydrogenase from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.     

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa₃-type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa₃-type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem-containing oxidase, called cytochrome bb₃, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::Km^R) an alternative cyto-chrome c oxidase has been characterized; this cbb₃-type oxidase has been partially purified. Both cytochrome aa₃ and cytochrome bb₃ are redox-driven proton pumps. The proton-pumping capacity of cytochrome cbb₃ has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.