液膜体系中氨基酸迁移的研究

用Ａｌｉｑｕａｔ３３６－Ｓｐａｎ８０－甲苯制成液体表面活性剂的膜体系,对氨基酸的迁移行为进行了研究,确定了苯丙氨酸完全及快速迁移的制乳和适宜条件,２ｍｉｎ的迁移率可达９０％以上。在苯丙氨酸的最佳迁移条件下,其它氨基酸如天冬氨酸、精氨酸、甘氨酸等都能有较高的迁移率。此法适用于微量氨基酸的提取和分离。     

Extraction of organic acids from kiwifruit juice using a supported liquid membrane process

In order to extract or remove organic acids from kiwifruit juice, we evaluated their separation and transport rates through supported liquid membranes (SLMs). The liquid membrane consisted of an organic solution composed of a carrier (Aliquat 336/Alamine 336) and a linear alcohol (oleyl alcohol) and was loaded on a microporous polypropylene support (commercial grade Celgard 2500/2400). These SLMs were evaluated (i) in a batch cell to determine the permeability and (ii) in a continuous spiral membrane module to study the effects of various process parameters – flow of feed and strip solutions, membrane composition, recycling mode of operation and kiwifruit juice at natural pH. It was observed that there exists an optimum for each system: pH?2.5–?3.0 for Alamine 336/oleyl alcohol and pH?4.5 for Aliquat 336/oleyl alcohol. At this pH?the flux rates of citric acid and malic acid was greater (6–8 times) than that of quinic acid. The flux rates decreased (greatly for citric acid) with the flow rate of feed and strip solutions and increased (considerably for citric acid) with the SLM composition . The recycling of feed and strip solutions significantly improved the removal efficiency. The SLM system retained its performance over a period of a few days. The SLM process allowed extraction of the above three organic acids (ascorbic acid was removed in trace amounts) from kiwifruit juice at a rate of a few percent (5%) in a single-pass processing.     

Separation of L-valine from fermentation broths using a supported liquid membrane

A carrier-mediated counter transport process is proposed to separate and to purify an amino acid produced by microbial fermentation. The case of L-valine permeation through a liquid membrane, constituted by a solution of Aliquat 336 in decanol and supported by a hydrophobic microporous membrane, is reported. A mathematical model was developed to estimate distribution coefficients and permeabilities and to predict the influence of hydrodynamic and pH conditions on supported liquid membrane (SLM) performances. Optimum conditions for the transport and the concentration of valine were achieved with synthetic aqueous valine solutions. Series of experiments on fermentation broths, where molasses and biomass contents were varied, permitted pointing out the role of the broth composition on the kinetics and yields of separation. The selectivity of transport of valine by an Aliquat 336/decanol liquid membrane was about 10 toward molasses dyes, 100 toward glucose, and beyond 1000 toward sucrose. This allowed us to achieve the recovery and one step of purification of the product in a single operation. The stability of the Aliquat 336/decanol liquid membrane was sufficient to ensure a selective transport of valine during a continuous run lasting 18 days.     

The Effect of a Quaternary Amine (Aliquat 336) on Growth and Photosynthesis of the Green Alga Chlorella emersonii, and the Effect on Photosynthesis in Isolated Spinach Chloroplasts

A quaternary amine, Aliquat 336, inhibits the growth of the green alga Chlorella emersonii, ¹⁴C-fixation of the alga is also inhibited. The effect and the site of action of the compound was studied by using isolated spinach chloroplasts. The carbon dioxide dependent oxygen evolution of the chloroplasts is inhibited directly upon the addition of the amine and the oxygen evolution is replaced by an oxygen uptake. By investigating some electron transport reactions in the chloroplasts we were able to show that Aliquat 336 affects the electron transport on the level of photophosphorylation. The results from the in vivo and the in vitro experiments thus show that the quaternary amine affects the photosynthetic process. Aliquat 336 is a solvent extractant used in several industrial processes for extraction of metals from aqueous solutions. Aliquat 336 could be considered a presumptive water pollutant as the compound could enter a recipient water body and thus affect photosynthesis.     

Uptake of N-acetylneuraminic acid by Escherichia coli K-235. Biochemical characterization of the transport system.

Kinetic measurement of the uptake of N-acetyl[4,5,6,7,8,9-14C]neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0. Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM. The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation [2,4-dinitrophenol (100%); NaN3 (66%]) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%). The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar. N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect. Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake. The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E. coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system. Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation. Protein synthesis is not needed for sialic acid transport.     

Determination of N-acetyl- and N-glycolylneuraminic acids in gangliosides by combination of neuraminidase hydrolysis and fluorometric high-performance liquid chromatography using a GM3 derivative as an internal standard

A highly sensitive method for quantification of sialic acids in gangliosides was developed. The sialic acids, released by hydrolysis of gangliosides, were converted to fluorescent derivatives with 1,2-diamino-4,5-(methylenedioxy)benzene (DMB) and separated on a reversed-phase C18 column with an isocratic elution. As little as 0.1-1.0 nmol of sialic acid in ganglioside was quantified. The use of acetate buffer instead of water in the mobile phase could prevent damage on the column and reduce background peaks derived from the reagents. When gangliosides were subjected to acid hydrolysis, the velocity of hydrolysis varied depending on their structures and a part of the sialic acid liberated decomposed with prolonged heating time. Therefore gangliosides were hydrolyzed by Arthrobacter ureafaciens neuraminidase in the presence of sodium cholate after addition of an internal standard. For the internal standard, GM3 with N-propionylneuraminic acid (GM3(NeuPr)) was synthesized from GM3(NeuAc) by N-deacylation followed by N-propionylation. Folch partition was used to decrease lipophilic materials included in the sample, and the sialic acids released were recovered from the upper phase. The present method has a satisfactory sensitivity in the simultaneous quantification of NeuAc and NeuGc in purified gangliosides as well as in crude lipid fractions containing a variety of gangliosides.     

Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli.

Escherichia coli K-12 and K-12 hybrid strains constructed to express a polysialic acid capsule, the K1 antigen, were able to efficiently use sialic acid as a sole carbon source. This ability was dependent on induction of at least two activities: a sialic acid-specific transport activity, and an aldolase activity specific for cleaving sialic acids. Induction over basal levels required sialic acid as the apparent inducer, and induction of both activities was repressed by glucose. Induction also required the intracellular accumulation of sialic acid, which could be either added exogenously to the medium or accumulated intracellularly through biosynthesis. Exogenous sialic acid appeared to be transported by an active mechanism that did not involve covalent modification of the sugar. Mutations affecting either the transport or degradation of sialic acid prevented its use as a carbon source and have been designated nanT and nanA, respectively. These mutations were located by transduction near min 69 on the E. coli K-12 genetic map, between argG and glnF. In addition to being unable to use sialic acid as a carbon source, aldolase-negative mutants were growth-inhibited by this sugar. Therefore, the intracellularly accumulated sialic acid was toxic in aldolase-deficient E. coli strains. The dual role of aldolase in dissimilating and detoxifying sialic acids is consistent with the apparent multiple controls on expression of this enzyme.     

Diversity of sialic acids revealed using gas chromatography/mass spectrometry of heptafluorobutyrate derivatives

The fine structural motifs of sialic acids, a frequent terminal monosaccharide of glycans, seem to contain essential biological properties. To identify such subtle structural differences, a reliable method was developed for the qualitative and quantitative identification of sialic acids present in different tissues and fluids. This method involved, after liberation of sialic acids by mild acid hydrolysis, their methyl esterification using diazomethane in the presence of methanol and the formation of volatile derivatives using heptafluorobutyric anhydride. The derivatives were analyzed by gas chromatography coupled to mass spectrometry in the electron impact mode. This technique allowed the separation and identification of a large variety of sialic acids, including different O-acylated forms of N-acetyl and N-glycolyl neuraminic acids and of 3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn). This method allowed also identifying 8-O-methylated and 8-O-sulfated derivatives, de-N-acetylated neuraminic acid, and 1,7-sialic acid lactones. Compounds present in very complex mixtures could be identified through their fragmentation patterns. Because of the stability of the heptafluorobutyrate derivatives, this method presents important improvements compared to the previous techniques, because it can be frequently applied on very small amounts of crude samples. This methodology will support progress in the field of the biology of sialic acids.     

A sialylated glycan microarray reveals novel interactions of modified sialic acids with proteins and viruses

Many glycan-binding proteins in animals and pathogens recognize sialic acid or its modified forms, but their molecular recognition is poorly understood. Here we describe studies on sialic acid recognition using a novel sialylated glycan microarray containing modified sialic acids presented on different glycan backbones. Glycans terminating in β-linked galactose at the non-reducing end and with an alkylamine-containing fluorophore at the reducing end were sialylated by a one-pot three-enzyme system to generate α2-3- and α2-6-linked sialyl glycans with 16 modified sialic acids. The resulting 77 sialyl glycans were purified and quantified, characterized by mass spectrometry, covalently printed on activated slides, and interrogated with a number of key sialic acid-binding proteins and viruses. Sialic acid recognition by the sialic acid-binding lectins Sambucus nigra agglutinin and Maackia amurensis lectin-I, which are routinely used for detecting α2-6- and α2-3-linked sialic acids, are affected by sialic acid modifications, and both lectins bind glycans terminating with 2-keto-3-deoxy-D-glycero-D-galactonononic acid (Kdn) and Kdn derivatives stronger than the derivatives of more common N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Three human parainfluenza viruses bind to glycans terminating with Neu5Ac or Neu5Gc and some of their derivatives but not to Kdn and its derivatives. Influenza A virus also does not bind glycans terminating in Kdn or Kdn derivatives. An especially novel aspect of human influenza A virus binding is its ability to equivalently recognize glycans terminated with either α2-6-linked Neu5Ac9Lt or α2-6-linked Neu5Ac. Our results demonstrate the utility of this sialylated glycan microarray to investigate the biological importance of modified sialic acids in protein-glycan interactions.     

Diversity of microbial sialic acid metabolism.

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.     

The Golgi CMP-sialic acid transporter: A new CHO mutant provides functional insights

A CHO mutant line, MAR-11, was isolated using a cytotoxic lectin, Maackia amurensis agglutinin (MAA). This mutant has decreased levels of cell surface sialic acid relative to both wild-type CHO-K1 and Lec2 mutant CHO cells. The CMP-sialic acid transporter (CMP-SAT) gene in the MAR-11 mutant cell has a C-T mutation that results in a premature stop codon. As a result, MAR-11 cells express a truncated version of CMP-SAT which contains only 100 amino acids rather than the normal CMP-SAT which contains 336 amino acids. Biochemical analyses indicate that recombinant interferon-gamma (IFN-gamma) produced by the mutant cells lack sialic acid. Using MAR-11 as host cells, an EPO/IEF assay for the structure-function study of CMP-SAT was developed. This assay seems more sensitive than previous assays that were used to analyze sialylation in Lec2 cells. Cotransfection of constructs that express CMP-SAT into MAR-11 cells completely converted the recombinant EPO to a sialylation pattern that is similar to the EPO produced by the wild-type CHO cells. Using this assay, we showed that CMP-SAT lacking C-terminal 18 amino acids from the cytosolic tail was able to allow high levels of EPO sialylation. Substitution of the Gly residues with Ile in three different transmembrane domains of CMP-SAT resulted in dramatic decreases in transporter's activity. The CMP-SAT only lost partial activity if the same Gly residues were substituted with Ala, suggesting that the lack of side chain in Gly residues in the transmembrane domains is essential for transport activity.     

Factors influencing the potential use of Aliquat 336 for the in situ extraction of carboxylic acids from cultures of Pseudomonas putida

The use of extraction techniques to alleviate product inhibition in bioprocesses is one of a number of potential separation methods. However, the intimate contact of an organic phase with the broth implies that the organic components of this phase may be present in the aqueous phase at saturation levels. The quaternary amine Aliquat 336 (trioctyl/decylmethylammonium entity), dissolved in octan-1-ol showed no inhibition on the growth of Pseudomonas putida, at least with respect to molecular toxicity. Nevertheless, it is important to point out two main effects of Aliquat 336 associated with its ion exchange properties. It is able (1) to complex hydroxyl ions and therefore drastically lower the pH of the broth and (2) release its counter ion through these exchanges. Therefore, a strict control of the pH of the cultivation must be conducted, with the constraint that Ps. putida has an optimal pH growth of 7.4-7.5. The pH range tolerated by this strain is, however, between 5.0 and 9.0. In addition, the counter ion of Aliquat 336 needs to be carefully chosen and HSO4- should be preferred to Cl-.     

Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are alpha2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes.     

Sequential GC/MS analysis of sialic acids,monosaccharides, and amino acids of glycoproteins on a single sample as heptafluorobutyrate derivatives

A GC/MS procedure was developed for the analysis of all major constituents of glycoproteins. The rationale for this approach is that by using GC/MS analysis of the constituents as heptafluorobutyrate derivatives, it was possible to quantitatively determine the sialic acid, monosaccharide, fatty acids (when present), and the amino acid composition with the sample remaining in the same reaction vessel during the entire procedure. A mild acid hydrolysis was used to liberate sialic acids and was followed by formation of methyl-esters of heptafluorobutyrate (HFB) derivatives. After GC/MS analysis of sialic acids, the remaining material was submitted to acid-catalyzed methanolysis followed by the formation of HFB derivatives. After GC/MS analysis of the monosaccharides, the sample was supplemented with norleucine (as internal standard) and hydrolyzed with 6 M HCl followed by the formation of isoamyl-esters of HFB derivatives and GC/MS analysis. His and Trp residues were modified during the step of acid-catalyzed methanolysis, but the resulting derivatives were stable during acid hydrolysis and quantitatively recovered by GC/MS analysis. As a result, all constituents of glycoproteins (sialic acids, monosaccharides (or di- and trisaccharides) and amino acids) are identified in the electron impact mode of ionization and quantified using three GC/MS analysis in the same chromatographic conditions and using a limited number of reagents, a considerable advantage over previous techniques. This method is very sensitive, all data (qualitative and quantitative) being obtained at the sub-nanomolar level of initial material.     

Enzymatic synthesis of sialic acid derivative by immobilized lipase from Candida antarctica

The effects of important reaction parameters on the enhancement of sialic acid derivative lipophilic properties through the lipase-catalyzed esterification of N-acetyl neuraminic acid methyl ester are investigated in this study. It is found that the lipase Novozym 435 from Candida antarctica is particularly useful in the preparation of sialic acid methyl ester monononanoate (SAMEMN). The optimum temperature for the SAMEMN synthesis reaction using Novozym 435 is 60 °C, and nonanoic anhydride is found to be the best substrate among all acyl donors. The Novozym 435-catalyzed esterification of N-acetyl neuraminic acid methyl ester gave a maximum yield of 87.7% after 6 h in acetonitrile at 60 °C. Because the novel method developed is simple, yet effective, it could potentially be used industrially for the production of sialic acid derivatives.     

Diversity of Microbial Sialic Acid Metabolism

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.     

“Just a spoonful of sugar...”: import of sialic acid across bacterial cell membranes

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.     

One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities

Chemoenzymatic synthesis, which combines the flexibility of chemical synthesis and the high selectivity of enzymatic synthesis, is a powerful approach to obtain complex carbohydrates. It is a preferred method for synthesizing sialic acid-containing structures, including those with diverse naturally occurring and non-natural sialic acid forms, different sialyl linkages and different glycans that link to the sialic acid. Starting from N-acetylmannosamine, mannose or their chemically or enzymatically modified derivatives, sialic acid aldolase-catalyzed condensation reaction leads to the formation of sialic acids and their derivatives. These compounds are subsequently activated by a CMP-sialic acid synthetase and transferred to a wide range of suitable acceptors by a suitable sialyltransferase for the formation of sialosides containing natural and non-natural functionalities. The three-enzyme coupled synthesis of sialosides can be carried out in one pot without the isolation of intermediates. The time for synthesis is 4-18 h. Purification and characterization of the product can be completed within 2-3 d.     

A facile preparation of uronates via selective oxidation with TEMPO/KBr/Ca(OCl)(2) under aqueous conditions

Addition of solid Ca(OCl)(2) as the terminal oxidant in the TEMPO-mediated selective oxidation has the benefit of easier operation. A variety of partially protected saccharide derivatives (1a-l) have been successfully converted into the corresponding uronate derivatives, including disaccharide building blocks for GAG fragments and precursors to saponins. The beneficial effect of Aliquat 336 was also disclosed in the oxidation of certain substrates.     

Sialic Acid Derivatives and their Distribution in Rat Sublingual Gland Acini during Pre- and Post-natal Development

Sialoglycoconjugates in rat sublingual gland acinar cells, at different stages of pre- and post-natal development, were investigated in situ with specific lectins and by the selective removal of terminal sialic acids. Cleavage of acetyl substituents sited in the pyranose ring and/or polyhydroxyl side chain was used as an additional means of characterising the glycoconjugates. The first expression of terminal sialic acid linked to -galactose was found at gestational day 17 and progressive different derivatives were observed. The terminal disaccharide sialic acid-N-acetylgalactosamine was constantly visualized in the sublingual gland from gestational day 18. In both terminal disaccharides, sialic acids were characterized by variable degrees of acetylation and were found to be highly packaged and responsible for the hydration coat. The complex data obtained indicated that the sublingual gland is characterized by a marked fluctuation of complex sialoglycoconjugates that differ from those in the submandibular gland of the same species.