乳酸杆菌、双歧杆菌代谢产物的气相色谱分析

用气相色谱分析乳酸杆菌、双歧杆菌提取物,结果表明乳酸杆菌提取物中乙酸的含量很高,双歧杆菌提取物挥发性脂肪酸种类较多,有乙酸、丙酸、异丁酸及丁酸等多种短链脂肪酸.分析结果表明短链脂肪酸很可能是乳酸杆菌、双歧杆菌提取物抑菌的主要活性成分.     

Depigmenting activity of new kojic acid derivative obtained as a side product in the synthesis of cinnamate of kojic acid

We synthesized cinnamate derivatives of kojic acid for use as depigmenting agents by various esterification methods. The cinnamate of 5-position of kojic acid (6) was obtained by EDC coupling, DCC coupling, acid chloride, and mixed anhydride methods. To obtain the cinnamate of the 2-position of kojic acid (7), we carried out the nucleophilic addition of the potassium salt of cinnamic acid to kojyl chloride. In this reaction, we discovered the occurrence of a side reaction and identified the structure of the side product thus formed. We evaluated the depigmenting activities of both the side product and the cinnamate derivatives of kojic acid. Interestingly, the side product (11) showed more potent depigmenting activity (IC(50)=23.51μM) than compound 7 (IC(50)>100μM) which is the mother compound of the side product. However, it has no tyrosinase inhibitory activity. Compound 6, the cinnamate of 5-position of kojic acid, also showed moderate depigmenting activity (IC(50)=46.64μM) without tyrosinase inhibitory activity. Production of this side product (11) may have originated from the proton exchange between the potassium salt of cinnamic acid and kojyl chloride. We then efficiently reduced the yield of the side product by controlling the equilibrium of the potassium salt of cinnamic acid. The addition of cinnamic acid greatly reduced the amount of the side product produced.     

Ecological studies on the occurrence of bacteria utilizing lactic acid at pH values below 4.5

Sites within two cheese factories, a silage tower and silage pit and two pet food factories were investigated for lactic acid utilizing bacteria. The bacteria isolated, together with known lactic acid utilizing and aciduric bacteria, were tested for their ability to 'alkalinize' a fermented meat product containing more than 20 g/1 lactic acid at pH > 4.3 a ten-fold dilution of this product and synthetic media, both at pH 4.5. Strains able to alkalinize the diluted product were 13 aerobic isolates (all Acetobacter spp.), nine anaerobic cultures, one strain of Megasphaera elsdenii and two strains of Clostridium tyrobutyricum. Eight of the aerobes could alkalinize undiluted product containing < 1 g/1 residual potassium sorbate, but had no effect on product containing > 3 g/1 sorbate; none of the anaerobic cultures was able to alkalinize undiluted product. Acetobacter spp., therefore, threatened the microbial stability of a fermented product containing < 3 g/1 potassium sorbate. The cultures able to alkalinize diluted product were all from sites contaminated with whey, silage or fermented product for at least 2 d, suggesting substantial accumulation of lactic acid utilizing bacteria at these sites.     

Cell-Free Synthesis of Pectin (Identification and Partial Characterization of Polygalacturonate 4-[alpha]-Galacturonosyltransferase and Its Products from Membrane Preparations of Tobacco Cell-Suspension Cultures)

Polygalacturonate 4-[alpha]-galacturonosyltransferase (EC 2.4.1.43) activity has been identified in microsomal membranes isolated from tobacco (Nicotiana tabacum L. cv Samsun) cell-suspension cultures. Incubation of UDP-[14C]galacturonic acid with tobacco membranes results in a time-dependent incorporation of [14C]galacturonic acid into a chloroform-methanol-precipitable and 65% ethanol-insoluble product. The optimal synthesis of product occurs at a pH of 7.8, 25 to 30[deg]C, an apparent Km for UDP-D-galacturonic acid of approximately 8.9 [mu]M, and a Vmax of approximately 150 pmol min-1 mg-1 protein. The product was characterized by scintillation counting, thin-layer chromatography, high-performance anion-exchange chromatography, and gel-filtration chromatography in combination with enzymatic and chemical treatments. The intact product has a molecular mass of approximately 105,000 D based on dextran molecular standards. The product was treated with base to hydrolyze ester linkages (e.g. methyl esters), digested with a homogeneous endopolygalacturonase (EPGase), or base and EPGase treated. Base and EPGase treatment results in cleavage of 34 to 89% of 14C-labeled product into components that co-chromatograph with mono-, di-, and trigalacturonic acid, indicating that a large portion of product contains contiguous 1,4-linked [alpha]-D-galactosyluronic acid residues. Optimal EPGase fragmentation of the product requires base treatment prior to enzymatic digestion, suggesting that 45 to 67% of the galacturonic acid residues in the synthesized homogalacturonan are esterified. At least 40% of the base-sensitive linkages were shown to be methyl esters by comparing the sensitivity of base-treated and pectin methylesterase-treated products to fragmentation by EPGase.     

L-天冬酰胺酶与产物的相互作用

本文从几方面探讨了产物L-天冬氨酸和L-天冬酰胺酶之间的相互作用。产物与珀琥酸对酶荧光影响的比较研究以及产物存在下对碘离子淬灭酶荧光性质的观察表明产物和酶之间存在直接的相互作用。产物保护天冬酰胺酶抗胰蛋白酶水解及抗热失活作用暗示它结合在酶的活性部位附近。     

Identification of the key factors affecting composting of a weathered hydrocarbon-contaminated soil

The batch culture degradation of NASA wastewater containing mixtures of citric acid, methylhydrazine, and their reaction product was studied. The organic contaminants present in the NASA wastewater were degraded by Achromobacter sp., Rhodococcus B30 and Rhodococcus J10. While the Achromobacter sp. showed a preference for the degradation of the citric acid, the Rhodococcus species were most effective in reducing the methylhydrazine and the reaction product. Removals of more than 50% were observed for citric acid, methylhydrazine and the reaction product when the NASA wastewater was inoculated with the microbes in batch cultures. Simulation and chemical characterization of citric acid and hydrazine mixtures show that the interaction is partly of a chemical nature and leads to the formation of a conjugated UV/Visible absorbing compound. An 'azo' carbonyl derivative of the citric acid, consistent with the spectral data obtained from the investigation, has been proposed as the possible product.     

A short-chain aldehyde is a major lipoxygenase product in arachidonic acid-stimulated porcine leukocytes

Porcine leukocytes convert exogenous arachidonic acid to a complex array of products derived via the 5-, 12-, and 15-lipoxygenase pathways of metabolism. The major monohydroxylated metabolite following addition of 100 microM arachidonic acid is 12-hydroxyeicosatetraenoic acid. Of the more polar compounds on reverse-phase high pressure liquid chromatography, the most prominent is a previously uncharacterized arachidonate product which chromatographs near to the omega-oxidized metabolites of leukotriene B4. The structure of this new product was examined by high pressure liquid chromatography, UV, NMR, and also by gas chromatography-mass spectrometry of several derivatives; it was identified as 12-oxododeca-5,8,10-(Z,Z,E)-trienoic acid. It is proposed that this C-12 trienal acid is formed from 12-hydroperoxyeicosatetraenoic acid by a cleavage reaction catalyzed by the leukocyte 12-lipoxygenase in the presence of excess arachidonic acid and under anaerobic conditions. These conditions are satisfied by addition of 100 microM arachidonic acid to the leukocyte suspension (3 X 10(7) cells/ml); 12-hydroperoxyeicosatetraenoic acid is formed as the major product, excess arachidonic acid is available, and the concomitant leukocyte respiratory burst quickly depletes the solution of oxygen. Preliminary experiments indicate that this aldehyde product has significant biological activity in the activation of leukocytes. In the course of an intense inflammatory reaction it is conceivable that the conditions for synthesis of this C-12 trienal acid and related aldehydes could prevail; such aldehydes would constitute an additional class of lipoxygenase product which exacerbates the process of inflammation.     

Sequential enzymes of linoleic acid oxidation in corn germ: lipoxygenase and linoleate hydroperoxide isomerase

Linoleic acid oxidation catalyzed by lipoxygenase (lipoxidase) activity in extracts of defatted corn germ does not terminate in the product, linoleic acid hydroperoxide, unless the lipoxygenase is first partially purified. If purification is not attempted, the hydroperoxide product exists only as a barely detectable intermediate in the synthesis of three products. One of these was identified as 9-hydroxy-10-oxo-cis-12-octadecenoic acid formed from the hydroperoxide by the enzyme, linoleate hydroperoxide isomerase. Another product, 13-hydroxy-10-oxo-trans-11-octadecenoic acid, is believed to be formed by an isomerase also. The third product was the linoleate ester of one of the hydroxy-oxo-fatty acids, 9-(cis-9,cis-12-octadecadienoyl)-10-oxo-cis-12-octadecenoic acid. It is not known if the synthesis of the ester is enzyme-catalyzed. When a mixture of 13-hydroperoxy-cis-9,trans-11-octa-decadienoic acid and 9-hydroperoxy-trans-10,cis-12-octa-decadienoic acid from soybean lipoxygenase oxidation of linoleic acid was used as a substrate, 13-hydroxy-12-oxo-cis-9-octadecenoic acid and 9-hydroxy-12-oxo-trans-10-octadecenoic acid were formed as the major products of catalysis by linoleate hydroperoxide isomerase(s) from corn. Smaller quantities of 9-hydroxy-10-oxo-cis-12-octadecenoic acid and 13-hydroxy-10-oxo-trans-11-octadecenoic acid were also formed.     

Effect of Kojic Acid on the Oxidation of DL-DOPA,Norepinephrine, and Dopamine by Mushroom Tyrosinase

Kojic acid inhibits effectively the rate of formation of pigmented product(s) and of oxygen uptake when DL-DOPA, norepinephrine and dopamine are oxidized by mushroom tyrosinase. In addition to the direct effect of kojic acid on the enzyme, kojic acid also affects the UV-VIS spectrum of the final product(s) formed, this being due to the ability of the o-quinones of these substrates to oxidize kojic acid to a yellow product(s). Kojic acid can thus prevent the conversion of the o-quinones of DL-DOPA, norepinephrine and dopamine to their corresponding melanin.     

A New Keto Acid, 5-Acetoxy-4-ketohexanoic Acid,Formed from Ethanol by Yeast

Of five strains of yeast tested, three which produced 5-hydroxy-4-ketohexanoic acid (HKH) were also found to form a new keto acid, when grown on medium containing ethanol as the sole carbon source. The new product was isolated in pure form and the structure was investigated by elementary analysis, infrared spectral analysis, NMR analysis and determination of its degradation product.

The compound was shown to be 5-acetoxy-4-ketohexanoic acid. 5-Acetoxy-4-keto-hexanoic acid (AKH) was synthesized and found to be identical with the isolated product.     

The Sulphation of p-hydroxyphenylpyruvic acid and related compounds by the rat liver cytosol.

Cytosol preparations of rat liver and kidney were examined for their ability to transfer sulphate from adenosine 3'-phosphate 5'-sulphatophosphate to p-hydroxyphenylpyruvic acid. Little activity towards this substrate was observed, and the main product detected in the reaction mixtures was identified as p-hydroxybenzyl alcohol O-sulphate. This was not formed from p-hydroxybenzaldehyde, a spontaneous oxidation product of p-hydroxyphenylpyruvic acid, by sulphation followed by a rapid enzyme-catalysed reduction of the intermediate phydroxybenzaldehyde O-sulphate. This product was formed mainly by this sequence of reactions, but the reverse, reduction followed by sulphation, also appeared possible. p-Hydroxybenzyl alcohol itself was very readily sulphated by both preparations, and the liver also produced a sulpho-conjugate of homogentisic acid. These observations are important in calculating the turnover of L-tyrosine O-sulphate in the mammalian system, and establish that p-hydroxyphenylpyruvic acid O-sulphate is an end product of its metabolism, rather than an intermediate in its synthesis by reversed transamination.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Production of Tricarboxylic Acid Anhydrides from Fatty Acids by a Lipase-producing Strain of Pseudomonas cepacia

A lipase-producing strain of Pseudomonas cepacia isolated from a soil sample was found to produce five compounds when oleic acid was added to the culture medium as lipase inducer. The five compounds were isolated by solvent extraction, silicagel column chromatography and preparative HPLC, and their structural elucidation was performed by mass spectrometry, and infrared and nuclear magnetic resonance spectroscopies. The products were identified as dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 1 ), undec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (product 2 ), dodec-3-ene-I,3,4-tricarboxylic acid 3,4-anhydride (product 3 ), dodec-3,8-diene-1,3,4-tricarboxylic acid 3,4-anhydride (product 4 ) and dodec-3,6-diene-I,3,4-tricarboxylic acid 3,4-anhydride (product 5 ). Accumulation of these compounds in the culture medium started after oleic acid consumption and followed a pattern similar to that found for cell growth and for lipase production. The five compounds were radioactively labeled when [U- 14 C]oleic acid was supplied to the culture medium, thus showing that they were produced by transformation of the acid. When isolated from cultures containing [1,2- 13 C]acetic acid and oleic acid as the sole sources of carbon, the compounds showed to contain the 13 C isotope only in the first five atoms of carbon of the molecule. Several long chain fatty acids also acted as precursors of these compounds, with maximal yields for chain lengths between 11 and 18 atoms of carbon. None of the five compounds acted as lipase inducer when added to the culture medium instead of oleic acid. The compounds showed moderate antibacterial and antifungal activities when tested in solid media bioassays.     

Identification of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, 4-hydroxy-2-oxohexanoic acid, and 2-hydroxyhexa-2,4-dienoic acid and related enzymes involved in testosterone degradation in Comamonas testosteroni TA441

Comamonas testosteroni TA441 utilizes testosterone via aromatization of the A ring followed by meta-cleavage of the ring. The product of the meta-cleavage reaction, 4,5-9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-dien-4-oic acid, is degraded by a hydrolase, TesD. We directly isolated and identified two products of TesD as 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid and (2Z,4Z)-2-hydroxyhexa-2,4-dienoic acid. The latter was a pure 4Z isomer. 2-Hydroxyhexa-2,4-dienoic acid was converted by a hydratase, TesE, and the product isolated from the reaction solution was identified as 2-hydroxy-4-hex-2-enolactone, indicating the direct product of TesE to be 4-hydroxy-2-oxohexanoic acid.     

A novel ring-expanded product with enhanced tyrosinase inhibitory activity from classical Fe-catalyzed oxidation of rosmarinic acid, a potent antioxidative Lamiaceae polyphenol

The iron-ion catalyzed oxidation of the ethanol solution of rosmarinic acid, a potent antioxidant polyphenol of Lamiaceae (Labiatae) plants, afforded a highly tyrosinase-inhibitory active product. The structure of the active product in the oxidation product mixture was determined using extensive NMR spectroscopy to have a novel oxygen-containing seven-membered ring system. The formation mechanism of the unique ring structure from the catechol part of the rosmarinic acid was proposed.     

Enzymatic aromatization of 4-ketocyclohexanecarboxylic acid to p-hydroxybenzoic acid

$\text{Corynebacterium cyclohexanicum}$ grows well on cyclohexane carboxylic acid as the sole carbon source, and accumulates a large amount of 4-ketocyclohexanecarboxylic acid in the culture medium at an early growth phase. When this keto acid was aerobically incubated with a crude extract of the organism, an acidic product was produced. Under anaerobic conditions, the same product was obtained using K₃Fe(CN)₆ as electron acceptor. The product was identified as p-hydroxybenzoic acid by means of gas-liquid chromatography, infrared spectroscopy, and mass spectroscopy. Two pathways are proposed for the aromatization of 4-ketocyclohexanecarboxylic acid.     

An NMR method for the study of proton transport across phospholipid vesicles

We have identified [1-¹⁴C]-oxindole-3-acetic acid as a catabolic product of [1-¹⁴C]-indole-3-acetic acid metabolism in Zea mays seedlings. The isolation, and chemical and mass spectral characterization of oxindole-3-acetic acid from corn kernel tissue is described together with data suggesting oxindole-3-acetic acid to be a major catabolic product of indole-3-acetic acid.     

Amino acid consumption in naïve and recombinant CHO cell cultures: producers of a monoclonal antibody

Most commercial media for mammalian cell culture are designed to satisfy the amino acid requirements for cell growth, but not necessarily those for recombinant protein production. In this study, we analyze the amino acid consumption pattern in naïve and recombinant Chinese hamster ovary (CHO) cell cultures. The recombinant model we chose was a CHO-S cell line engineered to produce a monoclonal antibody. We report the cell concentration, product concentration, and amino acid concentration profiles in naïve and recombinant cell cultures growing in CD OptiCHO™ medium with or without amino acid supplementation with a commercial supplement (CHO CD EfficientFeed™ B). We quantify and discuss the amino acid demands due to cell growth and recombinant protein production during long term fed batch cultivation protocols. We confirmed that a group of five amino acids, constituting the highest mass fraction of the product, shows the highest depletion rates and could become limiting for product expression. In our experiments, alanine, a non-important mass constituent of the product, is in high demand during recombinant protein production. Evaluation of specific amino acid demands could be of great help in the design of feeding/supplementation strategies for recombinant mammalian cell cultures.     

Influence of substrate supply on the production of sophorose lipids by Candida bombicola ATCC 22214

Summary Candida (Torulopsis) bombicola ATCC 22214 produces 180 g/l sophorose lipids using glucose and oleic acid (technical grade) as combined substrates in an extended fed-batch cultivation. Excess of oleic acid generated a paste-like product. However, only when oleic acid was not detectable during the whole run of cultivation, a microcrystalline product precipitated. The unsaturated C-18 fatty acids of the technical grade oleic acid were transferred unchanged into the sophorose lipid.     

Production of polymalic acid and malic acid by Aureobasidium pullulans fermentation and acid hydrolysis

Malic acid is a dicarboxylic acid widely used in the food industry and also a potential C4 platform chemical that can be produced from biomass. However, microbial fermentation for direct malic acid production is limited by low product yield, titer, and productivity due to end‐product inhibition. In this work, a novel process for malic acid production from polymalic acid (PMA) fermentation followed by acid hydrolysis was developed. First, a PMA‐producing Aureobasidium pullulans strain ZX‐10 was screened and isolated. This microbe produced PMA as the major fermentation product at a high‐titer equivalent to 87.6 g/L of malic acid and high‐productivity of 0.61 g/L h in free‐cell fermentation in a stirred‐tank bioreactor. Fed‐batch fermentations with cells immobilized in a fibrous‐bed bioreactor (FBB) achieved the highest product titer of 144.2 g/L and productivity of 0.74 g/L h. The fermentation produced PMA was purified by adsorption with IRA‐900 anion‐exchange resins, achieving a ～100% purity and a high recovery rate of 84%. Pure malic acid was then produced from PMA by hydrolysis with 2 M sulfuric acid at 85°C, which followed the first‐order reaction kinetics. This process provides an efficient and economical way for PMA and malic acid production, and is promising for industrial application. Biotechnol. Bioeng. 2013; 110: 2105–2113. © 2013 Wiley Periodicals, Inc.