Enzymic synthesis of caffeoylglucaric Acid from chlorogenic Acid and glucaric Acid by a protein preparation from tomato cotyledons

The phenylpropane metabolism of tomato (Lycopersicon esculentum Mill) cotyledons was investigated. The HPLC analysis revealed two hydroxycinnamic-acid conjugates as major components, identified as chlorogenic acid (5-O-caffeoylquinic acid) and caffeoylglucaric acid (2-O- or 5-O-caffeoyl-glucaric acid). Quantitative analyses indicated a precursor-product relationship between the chlorogenic and caffeoylglucaric acids. Protein preparations from tomato cotyledons were found to catalyze the formation of caffeoylglucaric acid with chlorogenic acid as acyl donor and free glucaric acid as acceptor molecule. This enzyme activity, possibly to be classified as hydroxycinnamoylquinic acid:glucaric acid hydroxycinnamoyltransferase, acts together with hydroxycinnamoyl-CoA: quinic acid hydroxycinnamoyltransferase.     

Secoiridoid and triterpenic acids from the stems of Nauclea diderrichii

The stem bark of Nauclea diderrichii has yielded diderroside, a new secoiridoid glucoside, as well as quinovic acid, 3-oxoquinovic acid and 3-O-glucosylquinovic acid. The hydrocarbon fraction was dominated by n-heptacosane and n-nonacosane, which accords with the predominance of n-octacosanoic acid in the alkanoic acid fraction.     

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.     

Oxalic acid production by citric acid-producing Aspergillus niger overexpressing the oxaloacetate hydrolase gene oahA

The filamentous fungus Aspergillus niger is used worldwide in the industrial production of citric acid. However, under specific cultivation conditions, citric acid-producing strains of A. niger accumulate oxalic acid as a by-product. Oxalic acid is used as a chelator, detergent, or tanning agent. Here, we sought to develop oxalic acid hyperproducers using A. niger as a host. To generate oxalic acid hyperproducers by metabolic engineering, transformants overexpressing the oahA gene, encoding oxaloacetate hydrolase (OAH; EC 3.7.1.1), were constructed in citric acid-producing A. niger WU-2223L as a host. The oxalic acid production capacity of this strain was examined by cultivation of EOAH-1 under conditions appropriate for oxalic acid production with 30 g/l glucose as a carbon source. Under all the cultivation conditions tested, the amount of oxalic acid produced by EOAH-1, a representative oahA-overexpressing transformant, exceeded that produced by A. niger WU-2223L. A. niger WU-2223L and EOAH-1 produced 15.6 and 28.9 g/l oxalic acid, respectively, during the 12-day cultivation period. The yield of oxalic acid for EOAH-1 was 64.2 % of the maximum theoretical yield. Our method for oxalic acid production gave the highest yield of any study reported to date. Therefore, we succeeded in generating oxalic acid hyperproducers by overexpressing a single gene, i.e., oahA, in citric acid-producing A. niger as a host.     

Diterpenoids and a sesquiterpene lactone from viguiera ladibractate

Eight known diterpene acids, ent-12-oxokaur-9(11),16-dien-19-oic acid, ent-12β-hydroxykaur-9(11),16-dien-19-oic acid, ent-isokaur-15(16)-en-17,19-dioic acid, ent-15α,16-epoxy-17-hydroxykaura-19-oic acid, ent-kaura-17,19-dioic acid, ent-kaur-16-en-19-oic acid, grandifloric acid, angeloyloxygrandifloric acid, as well as a new sesquiterpene lactone, ladibranolide, were isolated from Viguiera ladibractate. The stereochemistry of the sesquiterpene lactone was established by NOE experiments.     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

Production of ω-hydroxyundec-9-enoic acid and n-heptanoic acid from ricinoleic acid by recombinant Escherichia coli-based biocatalyst

ω-Hydroxyundec-9-enoic acid and n-heptanoic acid are valuable building blocks for the production of flavors and antifungal agents as well as bioplastics such as polyamides and polyesters. However, a biosynthetic process to allow high productivity and product yield has not been reported. In the present study, we engineered an Escherichia coli-based biocatalytic process to efficiently produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid from a renewable fatty acid (i.e., ricinoleic acid). Expression systems for catalytic enzymes (i.e., an alcohol dehydrogenase of Micrococcus luteus, a Baeyer–Villiger monooxygenase of Pseudomonas putida KT2440, an esterase of Pseudomonas fluorescens SIK WI) and biotransformation conditions were investigated. Biotransformation during stationary growth phase of recombinant E. coli in a bioreactor allowed to produce ω-hydroxyundec-9-enoic acid and n-heptanoic acid at a rate of 3.2 mM/h resulting in a final product concentration of ca. 20 mM. The total amount of ω-hydroxyundec-9-enoic acid and n-heptanoic acid produced reached 6.5 g/L (4.0 g/L of ω-hydroxyundec-9-enoic acid and 2.5 g/L of n-heptanoic acid). These results indicate that the high value carboxylic acids ω-hydroxyundec-9-enoic acid and n-heptanoic acid can be produced from a renewable fatty acid via whole-cell biotransformation.     

‘Uncommon’ amino acids in the seeds of 64 species of Caesalpinieae

Characteristic associations of free amino acids occur in the seeds of various groups of species within the Caesalpinieae. Guilandina species are distinctive in accumulating 4-ethylideneglutamic acid in their seeds, Gymnocladus and Gleditzia species in accumulating isomers of 3-hydroxy-4-methylglutamic acid, Bussea species in accumulating azetidine-2-carboxylic acid, Peltophorum species in accumulating a previously undescribed imino acid tentatively identified as a derivative of 4-hydroxypipecolic acid.     

Conjugated Linoleic Acid Accumulation via 10-Hydroxy-12-Octadecaenoic Acid during Microaerobic Transformation of Linoleic Acid by Lactobacillus acidophilus

Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells of Lactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- or trans-9, cis-11-octadecadienoic acid and trans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.     

Anaerobic fermentation for n-caproic acid production: A review

Anaerobic fermentation-based technologies are used for treating organic residues, and producing high value-added products, such as solvents, gases, and organic acids. Among several organic acids, n-caproic acid can be used as antimicrobial agent, additive in animal feed, flavor additive, and feedstock for chemical and biofuel industries. n-Caproic acid formation occurs through a carboxylic acid chain elongation process, which uses reverse β-oxidation of acetic and/or n-butyric acid, and ethanol or lactic acid as an electron donor. This review addresses important issues in commercial n-caproic acid production: metabolic pathways, kinetics and thermodynamics, substrates, reactors, inhibition of competing biological activities, pH, and acid extraction. Additionally, a mathematical model to describe the reverse β-oxidation kinetics was evaluated from existing literature. Current investigations show a wide range of n-caproic acid production rates (3.0–55.8 g/(L·d)), using different open cultures, fermentation conditions, and methods for inhibiting the methanogenesis. Clostridium kluyveri presence and a dominance of the Clostridium spp. were identified as determinant when ethanol was provided as electron donor. Continuous n-caproic acid extraction through pertraction is a promising technology, which combines selective extraction and enhanced production rates. However, confirming the industrial feasibility of this process requires further investigation.     

An efficient one-pot synthesis of polyphenolic amino acids and evaluation of their radical-scavenging activity

A simple and efficient procedure for the synthesis of N-acyl 4-hydroxy, 4-hydroxy-3-methoxy and 3,4-dihydroxy phenylglycine amides by a strategy based on the multicomponent Ugi reaction is proposed. Hydroxybenzaldehyde derivatives were reacted with 4-methoxybenzylamine, cyclohexyl isocyanide and benzoic acid or 2-naphthylacetic acid to give Ugi adducts that were treated with trifluoroacetic acid yielding N-acyl hydroxyphenylglycine amides in good yields. The same procedure using as acid component protocatechuic acid or hydrocaffeic acid gave N-catechoyl 3,4-dihydroxyphenylglycine amides. The use of N-benzyloxycarbonylglycine as acid component allowed the preparation of a 3,4-dihydroxyphenylglycyl dipeptide derivative. Radical-scavenging activity studies of the polyphenolic amino acid derivatives showed a sharp increase in activity with the increase in number of hydroxyl or catechol groups present. Cyclic voltammetry experiments established a correlation between oxidation peak potentials and the radical-scavenging activity.     

Molecular characterization of a venom acid phosphatase Acph-1-like protein from the Asiatic honeybee Apis cerana

Bee venom contains a variety of peptides and enzymes, including acid phosphatases. An acid phosphatase has been identified from European honeybee (Apis mellifera) venom. However, although the amino acid sequence is known, no functional information is currently available for bee venom acid phosphatase Acph-1-like proteins. In this study, an Asiatic honeybee (Apis cerana) venom acid phosphatase Acph-1-like protein (AcAcph-1) was identified. The analysis of the predicted AcAcph-1 amino acid sequence revealed high levels of identity with other bee venom acid phosphatase Acph-1-like proteins. Recombinant AcAcph-1 was expressed as a 64-kDa protein in baculovirus-infected insect cells. The enzymatic properties of recombinant AcAcph-1, determined using p-nitrophenyl phosphate (p-NPP) as a substrate, showed the highest activity at 45 °C and pH 4.8. Northern and western blot analyses showed that AcAcph-1 was expressed in the venom gland and was present as a 64-kDa protein in bee venom. In addition, N-glycosylation of AcAcph-1 was revealed by tunicamycin treatment of recombinant virus-infected insect Sf9 cells and by glycoprotein staining of purified recombinant AcAcph-1. Our findings show that AcAcph-1 functions as a venom acid phosphatase. This paper provides the first evidence of the role of a bee venom acid phosphatase Acph-1-like protein.     

Identification by gas-liquid chromatography-mass spectrometry of 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid,a new sialic acid from horse submandibular gland

The novel sialic acid 4-O-acetyl-9-O-lactyl-N-acetylneuraminic acid has been identified as a constituent of horse submandibular gland glycoproteins in addition to the already know equine sialic acids, N-acetylneuraminic acid, 4-O-acetyl-N-acetylneuraminic acid, 9-O-acetyl-N-acetylneuraminic acid, 4,9-di-O-acetyl-N-acetylneuraminic acid, N-glycolylneuraminic acid, 4-O-acetyl-N-glycolylneuraminic acidand 9-O-acetyl-N-glycolylneuraminic acid. The structure has been established by combined gas-liquid chromatography-mass spectrometry.     

Decarboxylation of Sorbic Acid by Spoilage Yeasts Is Associated with the PAD1 Gene

The spoilage yeast Saccharomyces cerevisiae degraded the food preservative sorbic acid (2,4-hexadienoic acid) to a volatile hydrocarbon, identified by gas chromatography mass spectrometry as 1,3-pentadiene. The gene responsible was identified as PAD1, previously associated with the decarboxylation of the aromatic carboxylic acids cinnamic acid, ferulic acid, and coumaric acid to styrene, 4-vinylguaiacol, and 4-vinylphenol, respectively. The loss of PAD1 resulted in the simultaneous loss of decarboxylation activity against both sorbic and cinnamic acids. Pad1p is therefore an unusual decarboxylase capable of accepting both aromatic and aliphatic carboxylic acids as substrates. All members of the Saccharomyces genus (sensu stricto) were found to decarboxylate both sorbic and cinnamic acids. PAD1 homologues and decarboxylation activity were found also in Candida albicans, Candida dubliniensis, Debaryomyces hansenii, and Pichia anomala. The decarboxylation of sorbic acid was assessed as a possible mechanism of resistance in spoilage yeasts. The decarboxylation of either sorbic or cinnamic acid was not detected for Zygosaccharomyces, Kazachstania (Saccharomyces sensu lato), Zygotorulaspora, or Torulaspora, the genera containing the most notorious spoilage yeasts. Scatter plots showed no correlation between the extent of sorbic acid decarboxylation and resistance to sorbic acid in spoilage yeasts. Inhibitory concentrations of sorbic acid were almost identical for S. cerevisiae wild-type and Δpad1 strains. We concluded that Pad1p-mediated sorbic acid decarboxylation did not constitute a significant mechanism of resistance to weak-acid preservatives by spoilage yeasts, even if the decarboxylation contributed to spoilage through the generation of unpleasant odors.     

Ferulic acid esters of sugar carboxylic acids from primary leaves of rye (secale cereale)

New hydroxycinnamic acid esters have been isolated from primary leaves of rye and identified as positional isomers of (E)-0-feruloylgluconic acids on the basis of TLC, HPLC, FAB mass spectrometry, ¹H NMR and ¹³C NMR spectroscopy. The 2-(E)-0-feruloylgluconic acid was the major isomer. A second ferulic acid ester, a very minor constituent in rye, was identified as 2(E)-0-feruloyl-4-methoxyaldaric acid, which is probably a glucaric acid derivative.     

Stereospecificity of plant microsomal cinnamic acid 4-hydroxylase

Cinnamic acid 4-hydroxylase from parsley cell suspension cultures is specific for trans-cinnamic acid as substrate. cis-Cinnamic acid is a competitive inhibitor of the enzyme with a K_i of approximately 0.34 mm. Hydrocinnamic acid is neither a substrate nor an inhibitor.     

Characterization of hydroxycinnamoyltransferase from rice and its application for biological synthesis of hydroxycinnamoyl glycerols

Hydroxycinnamoyltransferases (HCTs) catalyze the transfer of the cinnamoyl moiety from hydroxycinnamoyl-CoA to various acceptors such as shikimic acid, quinic acid, hydroxylated acid, and glycerol. Four rice HCT homologues (OsHCT1–4) to tobacco HST were cloned, and OsHCT4 was expressed in Escherichia coli as a glutathione S-transferase fusion protein. Using the purified recombinant protein and biotransformation techniques, whether OsHCT4 shows hydroxycinnamoyltransferase activity with a variety of acyl group acceptors was investigated. The results of high performance liquid chromatography (HPLC) and mass spectrometry (MS) established that OsHCT4 mediated the trans-esterification of glycerol as well as shikimic acid in the presence of hydroxycinnamoyl-CoA. The structure of the reaction product was determined using nuclear magnetic resonance spectroscopy (NMR). E. coli cells co-expressing 4CL (4-coumarate:coenzyme A ligase) and OsHCT4 converted p-coumaric acid, ferulic acid, and caffeic acid into the corresponding glycerides. While this conversion is very efficient in vitro, the physiological significant in rice is currently unknown.     

Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase From Tomato (Lycopersicon esculentum)

A novel acyltransferase from cotyledons of tomato (Lycopersicon esculentum Mill.), which catalyzes the transfer of caffeic acid from chlorogenic acid (5-O-caffeoylquinic acid) to glucaric and galactaric acids, was purified with a 2400-fold enrichment and a 4% recovery. The enzyme showed specific activities (theoretical V_max per milligram of protein) of 625 nanokatals (caffeoylglucaric acid formation) and 310 nanokatals (caffeoylgalactaric acid formation). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis it gave an apparent M_r of 40,000, identical to the value obtained by gel filtration column chromatography. Highest activity was found at pH 5.7, which was constant over a range of 20 to 120 millimolar K-phosphate. The isoelectric point of the enzyme was at pH 5.75. The reaction temperature optimum was at 38°C and the apparent energy of activation was calculated to be 57 kilojoules per mole. The apparent K_m values were 0.4 millimolar for glucaric acid, 1.7 millimolar for galactaric acid, and with both acceptors as second substrates 20 millimolar for chlorogenic acid. The relative ratio of the V_max/K_m values for glucaric acid and galactaric acid was found to be 100:12. Substrate-competition experiments support the conclusion that one single enzyme is responsible for both the glucaric and galactaric acid ester formation with marked preference for glucaric acid. It is proposed that the enzyme be called chlorogenic acid:glucaric acid O-caffeoyltransferase (EC 2.3.1.-). The three caffeic acid-dependent enzyme activities involved in the formation of the glucaric and galactaric acid esters, the chlorogenic acid:glucaric acid caffeoyltransferase as the key activity as well as the caffeic acid:CoA ligase and the caffeoyl-CoA:quinic acid caffeoyltransferase as the preceding activities, were determined. The time course of changes in these activities were followed during development of the seedling in the cotyledons and growth of the young plant in the first and second leaf. The results from tomato seedlings suggest a sequential appearance of these enzymes.     

The preparation and microbiological evaluation of the inhibitory effects of some acrylic acid derivatives

The following acrylic acid derivatives have been prepared and microbiologically evaluated as possible inhibitors of the growth of lactobacilli; indoleacrylic acid, β-(2-quinolyl)-, β-(3-quinolyl)-, β-(4-quinolyl) acrylic acids, cinnamic acid, p-hydroxycinnamic acid, p-dimethylaminocinnamic acid, p-diethylaminocinnamic acid, thienylacrylic acid, furylacrylic acid, and α-ethylacrylic acid.The utilization of tryptophan by Leuconostoc mesenteroides P-60 and Lactobacillus arabinosus was inhibited by the isomeric quinolylacrylic acid derivatives as well as by indoleacrylic acid. With this latter compound and the β-(3-quinolyl)acrylic acid, competitive inhibition was shown.p-Hydroxycinnamic acid inhibited the utilization of phenylalanine and tyrosine by all the organisms tested. At similar concentrations neither cinnamic acid nor phenol exerted any inhibitory effect.The effects of all inhibitors could be at least partially reversed by the addition of larger quantities of the corresponding amino acids.     

Biosynthesis of Lipoic Acid in Arabidopsis: Cloning and Characterization of the cDNA for Lipoic Acid Synthase

Lipoic acid is a coenzyme that is essential for the activity of enzyme complexes such as those of pyruvate dehydrogenase and glycine decarboxylase. We report here the isolation and characterization of LIP1 cDNA for lipoic acid synthase of Arabidopsis. The Arabidopsis LIP1 cDNA was isolated using an expressed sequence tag homologous to the lipoic acid synthase of Escherichia coli. This cDNA was shown to code for Arabidopsis lipoic acid synthase by its ability to complement a lipA mutant of E. coli defective in lipoic acid synthase. DNA-sequence analysis of the LIP1 cDNA revealed an open reading frame predicting a protein of 374 amino acids. Comparisons of the deduced amino acid sequence with those of E. coli and yeast lipoic acid synthase homologs showed a high degree of sequence similarity and the presence of a leader sequence presumably required for import into the mitochondria. Southern-hybridization analysis suggested that LIP1 is a single-copy gene in Arabidopsis. Western analysis with an antibody against lipoic acid synthase demonstrated that this enzyme is located in the mitochondrial compartment in Arabidopsis cells as a 43-kD polypeptide.