Lactic acid from acid whey permeate in a membrane recycle bioreactor

Whey permeate was obtained by ultrafiltration of cottage cheese whey and supplemented with yeast extract. The lactose in the permeate was converted into lactic acid by Lactobacillus bulgaricus in a high-performance membrane bioreactor configured in the cell recycle mode. At a cell concentration of 10 g l⁻¹, optimum productivity of lactic acid was 35 g l⁻¹ h⁻¹. Increasing the cell concentration to 30 g l⁻¹ enabled the use of a dilution rate of 1 h⁻¹ with complete substrate utilization. At 60 g l⁻¹, productivity was over 80 g l⁻¹ h⁻¹ with complete substrte utilization; this is vastly superior to conventional batch fermentations.     

2-C-Methylaldotetronic acid,a new lactone-forming acid present in plants

2-C-Methylaldotetronic acid (probably the erythro form) was found in considerable amounts in Cannabis sativa, Cereus forbesii, C. peruvianus, Lophophora williamsii, Trichocereus santiaguensis, T. spachianus and T. strigosus. In addition, the acid was present in minor amounts in another five species, all from the Cactaceae. In total, this new plant acid was detected in 12 of 19 investigated species.     

Simple isotope dilution assay for propionic acid and isovaleric acid

A gas chromatographic-mass spectrometric method is described for the assay of propionic acid and of isovaleric acid in physiological fluids by isotope dilution. The acids are derivatized to the pentafluorobenzyl esters to decrease volatility to render them suitable for GC-MS analysis. The following reference values were found. Propionic acid: plasma 0.54 ± 0.38 μmol/1 (n = 13, range 0.03–1.38 μmol/1), urine 1.7±1.6 μmol/mmol creatinine (n = 9, range 0.1–4.9 μmol/mmol creatinine). Isovaleric acid: plasma 0.89+−0.93 μmol/1 (n = 10, range 0.01–3.03 μmol/1), urine 0.38+−0.51 μmol/mmol creatinine (n = 10, range 0.01–1.70 μmol/mmol creatinine).     

Occurrence of rosmarinic acid,chlorogenic acid and rutin in Marantaceae species

In a survey of the higher plants for families with rosmarinic acid-accumulating species we could show for the first time, that some species of the family Marantaceae of the order Zingiberales accumulate rosmarinic acid. Other compounds detected in Marantaceae are chlorogenic acid and rutin (quercetin 3-O-rutinoside). Out of 35 species coming from 9 different genera extracted and analysed, two species of Maranta (Maranta leuconeura, Maranta depressa) and one Thalia species (Thalia geniculata) showed the presence of rosmarinic acid. The two Maranta species additionally contained chlorogenic acid, which was also present in Stromanthe amabilis. Rutin was detected in the genera Calathea, Ctenanthe, Maranta, Pleiostachya and Thalia. For a comparison, species from six other families of the Zingiberales were analysed as well.     

Caldensinic acid,a prenylated benzoic acid from Piper caldense

The CH₂Cl₂ and MeOH extracts from leaves of Piper caldense were subjected to chromatographic separation procedures to afford the new prenylated benzoic acid, caldensinic acid (3-[(2′E,6′E,10′E)-11′-carboxy-3′,7′,15′-trimethylhexadeca-2′,6′,10′,14′-tetraenyl]-4,5-dihydroxybenzoic acid) whose structure was determined by spectral analysis, mainly NMR (¹H, ¹³C, HSQC, HMBC) and ESI-MS. The natural compound and derivatives displayed antifungal activity against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum by direct bioautography.     

Metabolic engineering of Clostridium acetobutylicum for butyric acid production with high butyric acid selectivity

A typical characteristic of the butyric acid-producing Clostridium is coproduction of both butyric and acetic acids. Increasing the butyric acid selectivity important for economical butyric acid production has been rather difficult in clostridia due to their complex metabolic pathways. In this work, Clostridium acetobutylicum was metabolically engineered for highly selective butyric acid production. For this purpose, the second butyrate kinase of C. acetobutylicum encoded by the bukII gene instead of butyrate kinase I encoded by the buk gene was employed. Furthermore, metabolic pathways were engineered to further enhance the NADH-driving force. Batch fermentation of the metabolically engineered C. acetobutylicum strain HCBEKW (pta⁻, buk⁻, ctfB⁻ and adhE1⁻) at pH 6.0 resulted in the production of 32.5 g/L of butyric acid with a butyric-to-acetic acid ratio (BA/AA ratio) of 31.3 g/g from 83.3 g/L of glucose. By further knocking out the hydA gene (encoding hydrogenase) in the HCBEKW strain, the butyric acid titer was not further improved in batch fermentation. However, the BA/AA ratio (28.5 g/g) obtained with the HYCBEKW strain (pta⁻, buk⁻, ctfB⁻, adhE1⁻ and hydA⁻) was 1.6 times higher than that (18.2 g/g) obtained with the HCBEKW strain at pH 5.0, while no improvement was observed at pH 6.0. These results suggested that the buk gene knockout was essential to get a high butyric acid selectivity to acetic acid in C. acetobutylicum.     

Myosin-cross-reactive antigens from four different lactic acid bacteria are fatty acid hydratases

The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC–MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.     

Enzymatic synthesis of the neuroexcitatory amino acid quisqualic acid by cysteine synthase

Purification of cysteine synthase from the leaves of Quisqualis indica var. villosa reveals the presence of two forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50. Isoenzyme A was purified 10 000-fold and had a specific activity of 10.8 U/mg protein. Isoenzyme B was purified 460-fold with a specific activity of 0.49 U/mg protein. Both isoenzymes have the same M,s (58 000) and dissociate into identical subunits (M_r 29 000). The K_m value of isoenzyme A is 1.9 mM for O-acetyl-L-serine and 59 μM for sulphide, while that of isoenzyme B is 7.1 mM for O-acetyl-L-serine and 4.0 mM for 3,5-dioxo-1,2,4-oxadiazolidine. Both isoenzymes catalyse the formation of cysteine from O-acetyl-L-serine and hydrogen sulphide, but only isoenzyme B catalyses the formation of L-quisqualic acid. Other significant differences occur in the substrate specificity of the two isoenzymes. Some properties of the purified cysteine synthase isoenzymes are also described.     

Cyclopropane fatty acid synthesis affects cell shape and acid resistance in Leishmania mexicana

Cyclopropane fatty acid synthase (CFAS) catalyzes the transfer of a methylene group from S-adenosyl methionine to an unsaturated fatty acid, generating a cyclopropane fatty acid (CFA). The gene encoding CFAS is present in many bacteria and several Leishmania spp. including Leishmania mexicana, Leishmania infantum and Leishmania braziliensis. In this study, we characterised the CFAS-null and -overexpression mutants in L. mexicana, the causative agent for cutaneous leishmaniasis in Mexico and central America. Our data indicate that L. mexicana CFAS modifies the fatty acid chain of plasmenylethanolamine (PME), the dominant class of ethanolamine glycerophospholipids in Leishmania, generating CFA-PME. While the endogenous level of CFA-PME is extremely low in wild type L. mexicana, overexpression of CFAS results in a significant increase. CFAS-null mutants (cfas^?) exhibit altered cell shape, increased sensitivity to acidic pH, and aberrant growth in serum-free media. In addition, the CFAS protein is preferentially expressed during the proliferative stage of L. mexicana and is required for the cell membrane targeting of lipophosphoglycan. Finally, the maturation and localization of CFAS protein are dependent upon the downstream sequence of the CFAS coding region. Without the downstream sequence, the mis-localised CFAS protein cannot fully rescue the defects of cfas^?. Our data suggest that CFA modification of phospholipids can significantly affect the parasite’s response to certain adverse conditions. These findings are distinct from the roles of CFAS in L. infantum, highlighting the functional divergence in lipid modification among Leishmania spp.     

Double-helical structures for polyxanthylic acid

Polyxanthylic acid has been found to exist in two different duplex forms, A_I and A_II. a_I, formed at pH5·7, occurs in a compact lattice with nearest neighbor molecules spaced at 2.11 nm. It has an axial translation per residue, h = 0·301 nm, and a rotation per residue, t = 36·0 °. The intensity distribution in its X-ray fiber diffraction pattern is analogous to that of A-RNA (h = 0·281 nm, t = 32·7 °). On the other hand A_II, formed at pH 8·0, has a less compact, statistically disordered crystal packing with nearest neighbors 2·35 nm apart. It has h = 0·252 nm and t = 32·7 ° and gives an X-ray intensity distribution essentially identical to A-DNA (h = 0·256 nm, t = 32·7 °). Similar right-handed helical duplex models, with flexible C-3′-endo sugar rings, have been developed for each molecular structure. Both have purine purine base-pairs, possibly triply hydrogen-bonded, and certainly with the same symmetry as Watson-Crick pairs but with a 0·2 nm greater C-1′ … C-1′ separation.     

Failure of 3-hydroxy-3-phenylpropanoic acid and cinnamic acid to serve as precursors of tropic acid in Datura innoxia

Datura innoxia plants were fed the R- and S-isomers of [3-¹⁴C]-3-hydroxy-3-phenylpropanoic acid, and [3-¹⁴C]cinnamic acid along with dl-[4-³H]phenylalanine. The hyoscyamine and scopolamine isolated from the plants 7 days later were labeled with tritium, but devoid of ¹⁴C, indicating that 3-hydroxy-3-phenylpropanoic acid and cinnamic acid are not intermediates between phenylalanine and tropic acid. The [³H] tropic acid obtained by hydrolysis of the hyoscyamine was degraded and shown to have essentially all its tritium located at the para position of its phenyl group, a result consistent with previous work.     

Enhancement of acid resistance of Scenedesmus dimorphus by acid adaptation

When using flue gas as carbon source for microalgae cultivation, the resulting acidic environment caused by SO _X and NO _X can inhibit microalgal growth. In this study, Scenedesmus dimorphus acquired increased acid resistance by prior exposure to sublethal acid stress; a process defined as acid adaptation. Among the five algal species tested, S. dimorphus showed the highest level of acid tolerance to extreme acid challenge (exposure to pH 3.0). Non-adapted and acid-adapted exponential algal cells were used as inocula for tubular photobioreactors aerated with 2 % CO₂. Previously adapted at pH 4.0 for 1 h, S. dimorphus developed highest growth rate under extreme acidic condition, and the maximum biomass concentration and specific growth rate at pH 3.0 (3.638?±?0.074 g?L^?1 and 1.037?±?0.008 d^?1, respectively) were respectively 14.22 and 10.79 % higher than those of non-adapted cells. Moreover, acid-adapted cells could tolerate lower pH of 2.5, at which the growth of non-adapted cells was totally inhibited. All the results indicated that acid adaptation was an effective approach for the acid resistance enhancement of microalgae.     

Jasmonic acid interacts with abscisic acid to regulate plant responses to water stress conditions

Phytohormones are key players in signaling environmental stress conditions. Hormone profiling together with proline accumulation were studied in leaves and roots of different mutant lines of Arabidopsis. Regulation of proline accumulation in this system seems complex and JA-deficient (jar1-1) and JA-insensitive (jai1) lines accumulating high levels of proline despite their very low ABA levels seems to discard an ABA-dependent response. However, the pattern of proline accumulation in jai1 seedlings parallels that of ABA. Under stress conditions, there is an opposite pattern of ABA accumulation in roots of jar1-1/coi1-16 (in which ABA only slightly increase) and jai1 (in which ABA increase is even higher than in WT plants). This also makes JA-ABA crosstalk complex and discards any lineal pathway that could explain this hormonal interaction.     

Hydroxycinnamic acid esters of isocitric acid: Accumulation and enzymatic synthesis in Amaranthus cruentus

New hydroxycinnamic acid esters have been isolated from the cotyledons of Amaranthus cruentus. (E)-Caffeoylisocitric acid was identified as the major constituent and p-coumaroyl-and feruloylisocitric acids as minor ones on the basis of ¹H and ¹³C NMR spectroscopy. FAB mass spectrometry, high-performance liquid and thin-layer chromatography. The structure of caffeoylisocitric acid was confirmed by chromatographic comparison with synthetic material. The accumulation of the new hydroxycinnamoylisocitric acids and their enzymatic synthesis via the hydroxycinnamoyl-CoA thioesters as acyl donors are described. The caffeoyl-CoA-dependent acyltransferase showed a rapid transient increase in activity reaching ca 5 pkat per cotyledon pair (i.e. ca 390 pkat per mg protein) at day six of seedling development.     

Interactions of a 5-nitroxide-labeled poly(uridylic acid) with poly(adenylic acid)

The physicochemical properties of a high-molecular-weight spin-labeled nucleic acid, (RUGT,U)_n, synthesized by enzymatic copolymerization, were evaluated by uv and ESR spectroscopy. It was shown earlier that spin labeling of nucleic acids by chemical modification to an extent which gives a nitroxide-to-nucleotide ratio greater than 0.002 can cause noticeable lattice perturbations (A. M. Bobst, A. Hakam, P. W. Langemeier, and S. Kouidou (1979), Arch. Biochem. Biophys. 187, 339–345). The presence of RUGT, a 5-nitroxide-labeled uridine residue, in a (U)_n lattice at a $\text{RUGT}\text{U}$ ratio of 0.01 is shown here not to affect the complexation with (A)_n, since the uv melting temperature (T₀^OD) of the 2 → 1 transition and the hypochromicity changes were the same for (RUGT,U)_n· (A)_n and (U)_n·(A)_n. ESR measurements indicated that the nitroxide radical reflects the transition accurately within the error limit, although a slight destabilization of the spinlabeled segment could not be excluded. Computer simulations showed conclusively that the spin melting temperature (T_m^sp) corresponds to the temperature at which half of the spin-labeled segments are no longer complexed, for the ESR spectrum at T_m^spcan be simulated with equal contributions from the line shapes of ESR spectra taken before and after the transition. Arrhenius plots obtained by using two different approaches for computing correlation times were qualitatively the same. Computer analysis also revealed that the formation of a (RUGT,U)_n·(A)_n complex can be described by a two-state model, in contrast to results obtained with chemically spin-labeled (U)_n. Thus, using (RUGT,U)_n over chemically spin-labeled (U)_n can offer distinct advantages.     

The structural dependence of amino acid hydrophobicity parameters

Amino acid hydrophobicity parameters, G_hp log P (partition coefficient) values, free energies of solution, G_sol and hydration numbers, are well correlated by equations derived from the relationship O_X = Aα_X + Lσ_IX + Sν_X + I_iX + H₁n_HX + H₂n_nX + b₀ where O is the quantity correlated; X denotes the amino acid side chain; α is a polarizability parameter; σ_I, a localized electrical effect parameter; ν, a steric parameter; i, an indicator variable which accounts for an ionic X ; n_H and n_n the number of OH or NH bonds and of full nonbonding orbitals in X, respectively, and b₀ is the intercept. The equation is based on the assumption that Δ_hp log P and ΔG_sol are all functions of the difference in intermolecular forces between the amino acid and some medium, and the amino acid and water. The parameters were chosen to model the intermolecular forces of interest.Generally the most important factor is α_x. This is followed by ν, i, and n_H. Least important is σ_I. ΔG_sol depends on α, n_H and n_n. Hydration numbers depend on i, n_H and n_n. The hydrophobicity of amino acid side chains is the result of a preference for a nonpolar medium as a increases and for a polar medium as i, n_H and σ_I increase. It is quantitatively accounted for by the model, and no special “hydrophobic bond” need be involved. The results show that log P values for amino acids are composite quantities whose composition is variable.     

Deoxyribonucleic acid hybridization among strains of lactobacilli

Hybridization of deoxyribonucleic acid (DNA) from Lactobacillus bulgaricus (ATCC 11842) with DNA of L. lactis (ATCC 12315), L. helveticus (ATCC 15009), and L. jugurt (ATCC 521) showed 86.0% reassociation with L. lactis, 4.8% with L. helveticus, and none with L. jugurt.     

Modified method for determination of hippuric acid and methylhippuric acid in urine by gas chromatography

A modified method for the simultaneous determination of hippuric acid (HA) and o-, m- and p-methylhippuric acids (o-, m- and p-MHAs) in urine is described. These metabolites were extracted, derivatized into their methyl ester derivatives and analyzed using a gas chromatograph equipped with flame ionization detector and a DB-1 capillary column. The derivatives of HA, o-, m- and p-MHAs were well separated within 11 min. The accuracy and precision in the present method were sufficient for quantitative analysis, and the results obtained by the GC method were highly correlated with those by the HPLC method (NIOSH 8301).     

The biohydrogenation of α-linoleic acid and oleic acid by rumen micro-organisms

1. α-[U-¹⁴C]Linolenic acid was incubated with the rumen contents of sheep and the metabolic products were characterized by thin-layer chromatography, gas–liquid chromatography and absorption spectroscopy in the ultraviolet and infrared. 2. A tentative scheme for the biohydrogenation route to stearic acid is presented. The main pathway is through diconjugated cis–cis–cis-octadecatrienoic acid, non-conjugated trans–cis (cis–trans)-octadecadienoic acid and trans-octadecenoic acid, but other pathways are apparent. 3. Washed rumen micro-organisms possessed only a limited capacity to hydrogenate α-linolenic acid and oleic acid but the rate was greatly stimulated by a factor(s) present in the supernatant rumen liquor. 4. Pure cultures of Clostridium perfringens, Streptococcus faecalis, Escherichia coli and a coliform organism isolated from sheep faeces possessed negligible ability to hydrogenate unsaturated fatty acids compared with a mixed population of rumen micro-organisms. Butyrivibrio fibrisolvens slowly converted linoleic acid into octadecenoic acid.