γ-Linolenic acid production from Spirulina platensis

Various Spirulina strains were assayed for their productivity of -linolenic acid (Lnn). Spirulina platensis ARM-346 was found to accumulate large amounts of Lnn. Urea as a nitrogen source was found to be most effective giving a yield of 13.5 mg Lnn/g dry cell mass. With increase in temperature the Lnn content was found to increase along with biomass. The optimum temperature for maximum Lnn and biomass production was found to be 35°C. The Lnn content was highest at 0.06% (w/v) NaCl and 0.07% (w/v) K₂HPO₄. Cells cultivated in the orange region of the electromagnetic spectrum as energy source showed a high content of Lnn, and there was less biomass compared to cells grown in red light. When the culture was left in the dark for various times, after 144 h it contained about 26% more Lnn than in conventionally cultivated cells.     

Caffeoylquinic acid derivatives from the roots of Arctium lappa L. (burdock) and their structure–activity relationships (SARs) of free radical scavenging activities

Eight caffeoylquinic acid (CQA) derivatives have been identified from the roots of Arctium lappa L. (burdock), including three new derivatives (1–3) together with five known derivatives (4–8). Their chemical structures were determined by 1D and 2D NMR as well as HR-TOF-MS data. The free radical scavenging activities of each compound were determined experimentally and found to be significant, especially the tri-CQA (8) and di-CQAs (6–7). Besides, our data showed that the number of caffeoyl groups in the caffeoyl derivatives played a crucial role for these physiological functions.     

α-Methylene-γ-aminobutyric acid from Mycena pura

α-Methylene-γ-aminobutyric acid was isolated and characterized from fruit bodies of Mycena pura. It was the decarboxylation product of l-γ-methyleneglutamic acid by l-glutamic acid decarboxylase.     

Enantioselective chemoenzymatic synthesis of (R)-γ-valerolactone from levulinic acid

A number of chemicals with high industrial value can be synthesized from levulinic acid, a feasible building block readily available from cellulosic biomass. Among them, γ-valerolactone is a versatile chemical precursor for the synthesis of value-added products including bio-active molecules, bio-fuels, and carbon-based chemicals. In this study, a novel two-step chemoenzymatic conversion of levulinic acid to (R)-γ-valerolactone via 4-hydroxyvaleric acid was investigated. For that purpose, an engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) with improved catalytic activity toward levulinic acid was employed in the first-step reaction, and dehydration with 1 % (v/v) sulfuric acid was applied for the lactonization of 4-hydroxyvaleric acid to γ-valerolactone in the second step. As a result, enantiomerically pure (R)-γ-valerolactone (>99 % ee) was successfully produced from the free acid form of levulinic acid with the maximum yield of approximately 100 %.     

Biosynthesis of poly(γ-glutamic acid) from l-glutamic acid,citric acid,and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. When l-glutamic acid, citric acid, and ammonium sulfate were used as carbon and nitrogen sources, a large amount of PGA without a by-product such as a polysaccharide was produced. The time courses of cell growth, PGA, glutamic acid, and citric acid concentrations during cultivation were investigated. It was found that glutamic acid added to the medium was apparently not assimilated. It can be presumed that the glutamic acid unit in PGA is mainly produced from citric acid and ammonium sulfate. The PGA productivity was investigated at various concentrations of ammonium sulfate in the media, which caused the depression of cell growth, high productivity of PGA, and the production of PGA with a high relative molecular mass. The yield of PGA determined by gel permeation chromatography (GPC) reached approximately 20 g/l. This yield was the highest value for PGA production by B. subtilis IFO3335, suggesting that B. subtilis IFO3335 was a bacterium that could produce PGA effectively. Time courses relative to the molecular mass of PGA at various concentrations of ammonium sulfate were investigated. It was suggested that B. subtilis IFO3335 excreted a PGA degradation enzyme with the progress of cultivation and that PGA was degraded by this enzyme. Correspondence to: M. Kunioka     

A chemo-enzymatic route to synthesize (S)-γ-valerolactone from levulinic acid

Levulinic acid is a feasible platform chemical derived from acid-catalyzed hydrolysis of lignocellulose. The conversion of this substrate to (S)-γ-valerolactone ((S)-GVL) was investigated in a chemo-enzymatic reaction sequence that benefits from mild reaction conditions and excellent enantiomeric excess of the desired (S)-GVL. For that purpose, levulinic acid was chemically esterified over the ion exchange resin Amberlyst 15 to yield ethyl levulinate (LaOEt). The keto ester was successfully reduced by (S)-specific carbonyl reductase from Candida parapsilosis (CPCR2) in a substrate-coupled cofactor regeneration system utilizing isopropanol as cosubstrate. In classical batch experiments, a maximum conversion of 95 % was achieved using a 20-fold excess of isopropanol. Continuous reduction of LaOEt was carried out for 24 h, and a productivity of more than 5 mg (S)-ethyl-4-hydroxypentanoate (4HPOEt) per μg CPCR2 was achieved. Afterwards (S)-4HPOEt (>99%ee) was substituted to lipase-catalyzed lactonization using immobilized lipase B from Candida antarctica to yield (S)-GVL in 90 % overall yield and >99%ee.     

Improving poly-(γ-glutamic acid) production from a glutamic acid-independent strain from inulin substrate by consolidated bioprocessing

Bioprocess and Biosystems Engineering - To excavate the application of Jerusalem artichoke on poly(γ-glutamic acid) (γ-PGA) production, a γ-PGA producing strain Bacillus...     

First partial synthesis of α-boswellic acid from oleanolic acid

While β-boswellic acid is very readily available by extraction from frankincense resin, the accessibility of α-boswellic from the resin involved great effort and tedious purification procedures. Alternatively, a partial synthesis from readily available oleanolic acid was developed, the key steps of which are a reduction of the carboxyl group C-28 furnishing a methyl group, followed by palladium-assisted oxidation of the methyl group C-24, and configurational inversion at C-3.     

γ-Aminobutyric acid system in isolated dorsal and ventral horn neurons from bovine spinal cord

In order to search for the relationship between the structure and the function of the nervous system, the spinal cord provides suitable material. We devised a procedure for isolation of large ventral and small dorsal horn neurons. Then we examined the -aminobutyric acid (GABA) system in both neurons isolated using our procedure. Glutamic decarboxylase (GAD) activity in dorsal horn neurons was much higher than that in ventral horn neurons. Further, GABA uptake activity by the dorsal horn neurons was also somewhat higher than that by the ventral horn neurons, although some properties of GABA uptake were found to be almost the same in both neuronal fractions. However, we could not find any difference of GABA--ketoglutarate transaminase (GABA-T) activity between the dorsal and ventral horn neurons. These results suggest that GAD and GABA uptake may be indicators for cell specificity to some extent.     

Biosynthesis and Hydrolysis of Poly(γ-glutamic acid) from Bacillus subtilis IF03335

Poly(γ-glutamic acid) (PGA) production in Bacillus subtilis IF03335 was studied. When citric acid as a carbon source was added to a glutamic acid medium containing L-glutamic acid and ammonium sulfate, a large amount of pure PGA was produced. On the other hand, when glucose was added to the glutamic acid medium, a by-product was produced, which seemed to be a polysaccharide. Moreover, the mode of hydrolysis was investigated with PGA in aqueous solutions at 80, 100, and 120°C by monitoring the time-dependent changes in the molecular weights. Hydrolytic degradation of PGA was found to proceed through a random chain scission.     

Biosynthesis of poly (γ-glutamic acid) from l-glutamine,citric acid and ammonium sulfate in Bacillus subtilis IFO3335

Poly(-glutamic acid) (PGA) production in Bacillus subtilis IFO3335 was studied. PGA was only slightly produced from medium (100 ml) containing 2 g citric acid and 0.5 g ammonium sulfate in B. subtilis IFO3335. When 0.01 g/100 ml l-glutamine was added to this medium, a large amount of PGA (0.45 g/100 ml), without any by-products such as polysaccharides, was produced. The changes in cell growth, and PGA, glutamic acid, citric acid and ammonium sulfate concentrations in this medium during cultivation were investigated. It was found that PGA was effectively produced for the short time of 20 h after an induction period and that glutamic acid was scarcely excreted during PGA production. PGA could be effectively produced using this medium containing l-glutamine, citric acid and ammonium sulfate. It is suggested that a small amount of l-glutamine added to the medium activated enzymes in the pathway of PGA synthesis in B. subtilis IFO3335. It can be presumed that the enzyme catalyzing the reaction from 2-oxoglutaric acid to l-glutamic acid was glutamate synthase in this bacterium.     

Microbial production of poly-γ-glutamic acid

Poly-γ-glutamic acid (γ-PGA) is a natural, biodegradable and water-soluble biopolymer of glutamic acid. This review is focused on nonrecombinant microbial production of γ-PGA via fermentation processes. In view of its commercial importance, the emphasis is on l-glutamic acid independent producers (i.e. microorganisms that do not require feeding with the relatively expensive amino acid l-glutamic acid to produce γ-PGA), but glutamic acid dependent production is discussed for comparison. Strategies for improving production, reducing costs and using renewable feedstocks are discussed.     

Ab-initio SCF investigation ofγ-aminobutyric acid

Summary The potential energy surface of the neutral form of-aminobutyric acid was investigated by means of ab-initio 4-31 G SCF calculations. Geometries, energies, and selected wave numbers of all 62 symmetry unique local minima are reported. Intramolecular interactions and all reactions, which involve the intramolecular hydrogen bond, are discussed and compared with those present in the homologues-alanine and glycine.Abbreviations GABA -aminobutyric acid - PES potential energy surface - RHF Roothaan-Hartree-Fock - SCF self consistent field     

γ-Carboxyglutamic acid content of hepatocellular carcinoma-associated des-γ-carboxy prothrombin

Serum des-γ-carboxy prothrombin (DCP) is a useful marker for the diagnosis of hepatocellular carcinoma (HCC), but the exact mechanism of its synthesis and its structural properties in liver diseases are unknown. DCP is measured by the monoclonal antibody MU-3. The purpose of this study was to examine the epitope of MU-3 and to characterize the differences in DCP between HCC and benign liver diseases. The epitope of MU-3 was examined by ELISA using prothrombin Gla domain polypeptides and was determined to be amino acid residues 17–27 of the prothrombin Gla domain, which has four γ-carboxyglutamic acid residues (Gla) at positions 19, 20, 25 and 26. Peptides having a glutamic acid residue (Glu) at these positions reacted strongly to MU-3 but lost reactivity when Glu 19 or 20 was changed to Gla. In the order of γ-carboxylation, MU-3 reacted strongly to DCP containing 0–1 Gla, weakly to 2–4 Gla and not at all to DCP containing more than five Gla. After adsorbing normal prothrombin with barium carbonate, DCP reaction to MU-3 was measured by determining the amount of DCP that was adsorbed by MU-3-coated beads. The proportion of DCP reacting to MU-3 in HCC was 41.0–76.8%, whereas in patients with benign liver diseases, only 0–42.1% reacted to MU-3. These results indicate that DCP variants preferentially synthesized in HCC have less than four Gla, which are restricted to positions 16, 25, 26 and 29, whereas DCP variants in benign liver diseases have more than five Gla.     

Production of arachidonic acid and dihomo-γ-linolenic acid from glycerol by oil-producing filamentous fungi, Mortierella in the ARS culture collection

The filamentous fungi of the genus Mortierella are known to produce arachidonic acid from glucose, and the species alpina is currently used in industrial production of arachidonic acid in Japan. In anticipation of a large excess of the co-product glycerol from the national biodiesel program, we are trying to find new uses for bioglycerin. We screened 12 Mortierella species: M. alpina NRRL 6302, M. claussenii NRRL 2760, M. elongata NRRL 5246, M. epigama NRRL 5512, M. humilis NRRL 6369, M. hygrophila NRRL 2591, M. minutissima NRRL 6462, M. multidivaricata NRRL 6456, M. nantahalensis NRRL 5216, M. parvispora NRRL 2941, M. sepedonioides NRRL 6425, and M. zychae NRRL 2592 for their production of arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA) from glycerol. With glucose as substrate all of the strains tested produced AA and DGLA. The total fatty acid content of 125 mg/g cell dry weight (CDW) and fatty acid composition for AA (19.63%) and DGLA (5.95%) in the mycelia of M. alpina grown on glucose were comparable with those reported by Takeno et al. (Appl Environ Microbiol 71:5124–5128, 2005). With glycerol as substrate all species tested grew on glycerol and produced AA and DGLA except M. nantahalensis NRRL 5216, which could not grow on glycerol. The amount of AA and DGLA produced were comparable with those obtained with glucose-grown mycelia. The top five AA producers (mg AA/CDW) from glycerol were in the following order: M. parvispora > M. claussenii > M. alpina > M. zychae > M. minutissima. The top five dry mycelia weights were: M. zychae > M. epigama > M. hygrophila > M. humilis > M. minutissima. The top five species for total fatty acids production (mg /g CDW) were: M. claussenii > M. parvispora > M. minutissima > M. hygrophila > M. maltidivaricata. We selected two species, M. alpina and M. zychae for further studies with glycerol substrate. Their optimum production conditions were determined. Time course studies showed that the maximum cell growth and AA production for both species were at 6 days of incubation. Therefore, glycerol can be considered for industrial use in the production of AA and DGLA.     

N-γ-L-glutamyl-β-D-aminophenylpropanoic acid,a dipeptide from the aquatic fern,azolla caroliniana

A novel dipeptide, γ-L-glutamyl-β-D-phenylpropanoic acid has been isolated from extracts of the water fern, Azolla caroliniana.     

Stimuli-responsive materials prepared from carboxymethyl chitosan and poly(γ-glutamic acid) for protein delivery

The development of stimuli-responsive materials in response to the molecules involved in biological processes has gained increased attentions. In this work, carboxymethyl chitosan (CM-chitosan) and poly(γ-glutamic acid) (pGlu) were reacted with a naturally occurring compound, genipin, leading to the formation of genipin-crosslinked CM-chitosan/pGlu conjugates with fluorescence emissions. The genipin-conjugated polymers were sensitive to the oxidation product of glucose, gluconic acid and hydrogen peroxide (H₂O₂). Fluorescence emissions of the polymers were quenched by gluconic acid and H₂O₂. An increase in the hydrodynamic diameter together with the quenching of fluorescence indicated that the genipin-conjugated polymers were self-aggregated into nanoparticles, in response to the stimulus of gluconic acid (but not for H₂O₂). Bovine serum albumin (BSA) could be loaded in the self-aggregated nanoparticles, and the incorporated BSA slowly released from the nanoparticles under hyper-gluconic acid conditions. This material is hence proposed as a stimuli-responsive material for optical sensing and protein delivery purposes.     

α-Lipoic acid dependent regeneration of ascorbic acid from dehydroascorbic acid in rat liver mitochondria

Rat liver mitochondria were examined for their ability to reduce dehydroascorbic acid to ascorbic acid in an -lipoic acid dependent or independent manner. The a-lipoic acid dependent reduction was stimulated by factors that increased the NADH dependent reduction of -lipoic acid to dihydrolipoic acid in coupled reactions. Optimal conditions for dehydroascorbic acid reduction to ascorbic acid were achieved in the presence of pyruvate, -lipoic acid, and ATP. Electron transport inhibitors, rotenone and antimycin A, further enhanced the dehydroascorbic acid reduction. The reactions were strongly inhibited by 1 mM iodoacetamide or sodium arsenite. Mitoplasts were qualitatively similar to intact mitochondria in dehydroascorbate reduction activity. Pyruvate dehydrogenase and -ketoglutarate dehydrogenase reduced dehydroascorbic acid to ascorbic acid in an -lipoic acid, coenzyme A, and pyruvate or -ketoglutarate dependent fashion. Dehydroascorbic acid was also catalytically reduced to ascorbic acid by purified lipoamide dehydrogenase in an -lipoic acid (K _0.5=1.4±0.8 mM) and lipoamide (K _0.5=0.9±0.3 mM) dependent manner.