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.     

Lipase catalyzed reaction of L-ascorbic acid with cinnamic acid esters and substituted cinnamic acids

To perform the lipase-catalyzed synthesis of L-ascorbic acid derivatives from plant-based compounds such as cinnamic and ferulic acid under mild reaction conditions, the activities of immobilized Candida ntarctica lipase with different cinnamic acid esters and substituted cinnamic acids were compared. As a result, immobilized C. ntarctica lipase was found to prefer vinyl cinnamic acid to other esters such as allyl-, ethyl-, and isobutyl cinnamic acids as well as substituted cinnamic acids such as p-coumaric acid, caffeic acid, ferulic acid, and sinapic acid. Based on these results, large-scale synthesis of 6-O-cinnamyl-L-ascorbic acid ester was performed using immobilized C. ntarctica lipase in dry organic solvent, resulting in 68% yield (493 mg) as confirmed by ¹³C-NMR.     

Biosynthesis of pentahydroxystearic acid of cutin from linoleic acid in Rosmarinus officinalis

The hydroxyfatty acid polymer, cutin, is the structural component of plant cuticle. Combined gas chromatography-mass spectrometry of the hydrogenolysis and deuterolysis products of rosemary cutin (Rosmarinus officinalis) revealed a series of components suggesting the conversion of linoleic acid to 9,10,12,13,18-pentahydroxy-stearic acid. [U-¹⁴C]Linoleic acid was incorporated into the insoluble residue of rapidly expanding rosemary leaves. Depolymerization of the insoluble material followed by isolation of individual components and chemical degradation studies showed that linoleic acid was directly converted into 18-hydroxylinoleic acid, 18-hydroxy-9, 10-epoxyoctadec-12-enoic acid, 9,10,18-trihydroxyoctadec-12-enoic acid, 9,10,18-trihydroxy-12,13-epoxystearic acid, and 9,10,12,13,18-pentahydroxystearic acid. These results strongly suggest that, in the biosynthesis of the phytopolymer, linoleic acid is first converted into 18-hydroxylinoleic acid and that this precursor then undergoes sequential epoxidation-hydration at the Δ⁹ and Δ¹² double bonds to yield 9,10,12,13,18-pentahydroxystearic acid.     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

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.     

An inducible lactone hydrolase yielding 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid from pulvinic acid

The metabolism of vulpinic acid by an unclassified soil micro-organism was studied. A new compound, 2,5-diphenyl-3-hydroxy-4-oxo-2-hexendioic acid (DHOHA) was isolated from the reaction mixture of a cell-free preparation and pulvinic acid. The existence of a hydrolase which catalyses the conversion of vulpinic acid to pulvinic acid was detected in cell-free preparation, and an inducible lactone hydrolase capable of converting pulvinic acid to DHOHA was purified 130-fold and characterized. This enzyme had a MW of ca 34 000, a K_m for pulvinic acid at pH optimum (pH 7.0) less than 10 ^{? 6} M, pI = 5.0, and was inhibited by p-chloromercuriphenylsulfonate and diethylpyrocarbonate. The enzyme was highly specific for pulvinic acid. The initial degradative steps proposed for this organism are vulpinic acid → pulvinic acid → DHOHA.     

Prelunularic acid in liverworts

Prelunularic acid, the first example of an intermediate with a ‘pre-aromatic’ structure in the phenylpropanoid-polymalonate pathway, was isolated from suspension-cultured cells of Marchantia polymorpha. Its structure, including its absolute configuration, was assigned on the basis of spectral properties, direct conversion into lunularic acid, and CD measurements on the bis(p-dimethylaminobenzoate) of the methyl ester. Prelunularic acid was also detected in several liverworts of Marchantiales and Jungermanniales, and appears to be the immediate precursor of lunularic acid instead of the previously postulated hydrangenol or hydrangeic acid.     

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.     

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.     

Rubitic acid,a new triterpene acid from Rubus fruticosus

A new triterpene acid, rubitic acid was isolated from the alcoholic extract of the whole plant of Rubus fruticosus. On the basis of physical methods coupled with chemical investigations, the structure of rubitic acid was shown to be 7α-hydroxy ursolic acid.     

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.     

Preparation of przewalskinic acid A from salvianolic acid B using a crude enzyme from an Aspergillus oryzae strain

Przewalskinic acid A is a rare, water-soluble, and highly biologically active ingredient found, thus far, only in the Salvia przewalskii Maxim herb; however, the content in S. przewalskii herb is very low. In order to obtain useful quantities of przewalskinic acid A, the biotransformatin of salvianolic acid B from Salvia miltiorrhiza root (danshen in Chinese) into przewalskinic acid A was studied using a crude enzyme produced from Aspergillus oryzae D30s strain. The crude enzyme from the A. oryzae strain hydrolyzed salvianolic acid B into przewalskinic acid A and danshensu. The preparation afforded 31.3 g przewalskinic acid A (91.0 % purity) and 13.1 g danshensu (95 % purity) from 75 g salvianolic acid B. The preparation of przewalskinic acid A was therefore very successful with a yield of over 86 %, but the yield of danshensu was only 33 %. The product przewalskinic acid A was identified using ultra-performance liquid chromatography–mass spectrometry (UPLC–MS) and NMR.     

Jessic acid and related acid triterpenoids from combretum elaeagnoides

A novel triterpenoid acid, jessic acid, was extracted from the leaves of Combretum elaeagnoides, where it was found together with its methyl ester and its α-L-arabinopyranoside, all three compounds occurring in significantly large amounts. Jessic acid is 1α,3β-dihydroxy-23-oxo-24-methylenecycloartan-30-oic acid.     

Adaptive evolution improves acid tolerance and succinic acid production in Actinobacillus succinogenes

Fermentation at low pH is an efficient way to improve the competitiveness of biological succinic acid-producing process. Actinobacillus succinogenes shows good performance of succinic acid production under anaerobic conditions, but its succinic acid production capability at the low-pH is inefficient due to the poor acid resistance. Herein, a mutant A. succinogenes BC-4 with improved cell growth and succinic acid production under weak acid conditions was obtained by adaptive evolution. The specific growth rate and succinic acid production of BC-4 reached 0.13 g/L/h and 20.77 g/L, which were increased by 3.25- and 2.95- fold, respectively compared with the parent strain under anaerobic condition at pH 5.8. The activities of specific enzymes with ATP generation were significantly enhanced under weak acidic conditions, resulting in 1.28-fold increase in the maximum ATP level. Membrane fatty acid composition analysis demonstrated that the ratio of saturated to unsaturated fatty acids was decreased from 1.62 to 1.44 in mutant BC-4, leading to improved intracellular pH homeostasis. Furthermore, the change from long-chain to median-chain fatty acid might lower the permeability of H⁺ into cytoplasm for survival under acid stress. These results indicated that A. succinogenes BC-4 is a promising candidate for succinic acid production under weak acid condition.     

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.     

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.     

Incorporation of cis-parinaric acid,a fluorescent fatty acid,into synaptosomal phospholipids by an acyl-CoA acyltransferase

The cis-isomer of parinaric acid, a naturally occurring C-18 polyene fatty acid, was incubated with brain subcellular fractions and the polarization of fluorescence increased in a time dependent manner. Greatest increases occurred in synaptosomal and microsomal membranes. This increase in polarization of fluorescence was found with the cis, but not the trans, isomer of parinaric acid and required Mg²⁺ or Ca²⁺ and was stimulated by coenzyme A and ATP. Synaptosomes were incubated with cis-parinaric acid and lipids were extracted and examined by high performance liquid chromatography. The highest incorporations of cis-parinaric acid were found in phosphatidylcholine (71%) and phosphatidylethanolamine (20%) while only traces were found in phosphatidylserine and phosphatidylinositol. [³H]Oleic acid was also incorporated into membrane phospholipids and unlabeled oleic acid blocked incorporation of cis-parinaric acid. It is proposed that cis-parinaric acid, like fatty acids normally found in brain, is incorporated into membrane phospholipids by an acyl-CoA acyltransferase. The presence of this enzyme in nervous tissue may make it possible to easily introduce fluorescent fatty acid probes into membrane phospholipids and to thereby facilitate study of membrane-mediated processes.     

Dienoic acid contents of rats with linoleic acid and pyridoxine deficiencies

Low dienoic acid content of the tissues of rats has been produced in two ways: (a) withdrawal of essential fatty acid from the diet, and (b) exclusion of pyridoxine or thiamine from the diet or severe restriction of food (caloric deficiency).By the first method dienoic acid content of the tissues fell to lower levels, and typical symptoms of the deficiency were produced. By the second procedure, the fall was more rapid in the acute vitamin deficiencies but no symptoms characteristic of linoleic acid deficiency occurred. A similar but less rapid course was followed in caloric restriction.Since no essential difference in dienoic acid content of the tissues of rats was found under these several dietary conditions, it was concluded that pyridoxine under the conditions of these experiments does not exert a special supplementary action in the production or conservation of polyenoic fatty acids.     

GC-MS identification of abscisic acid and abscisic acid metabolites in seed of Vigna unguiculata

Abscisic acid, phaseic acid and 4′-dihydrophaseic acid were identified by GC-MS of derivatized (Me, MeTMSi) extracts from immature fruits of Vigna unguiculata. The fruits also contained some other ABA-related compounds, one of which might be epi-4′-dihydrophaseic acid while another was tentatively identified as 6′-hydroxymethylabscisic acid.     

A resolution of glutamic acid

N-Carbobenzoxy-dl-glutamic acid was asymmetrically hydrolyzed by hog kidney acylase to l-glutamic acid ([α]_D = +31.9 °) and N-carbobenzoxy-d-glutamic acid ([α]_d = + 7.5 °). On catalytic hydrogenation of the latter, d-glutamic acid ([α]_d = ?31.5 °) was obtained readily and in high yield.