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1.
Shiyuan Hu Jun Chen Zhongyi Yang Lijun Shao Hua Bai Jiali Luo Weihong Jiang Yunliu Yang 《Applied microbiology and biotechnology》2010,85(5):1383-1391
N-Acetyl-d-neuraminic acid (Neu5Ac) can be produced from N-acetyl-d-glucosamine (GlcNAc) and pyruvate by a chemoenzymatic process in which an alkaline-catalyzed epimerization transforms GlcNAc to N-acetyl-d-manosamine (ManNAc). ManNAc is then condensed biocatalytically with pyruvate in the presence of N-acetyl-d-neuraminic acid lyase (NAL) or by a two-step, fully enzymatic process involving bioconversions of GlcNAc to ManNAc and ManNAc to Neu5Ac using N-acetyl-d-glucosamine 2-epimerase (AGE) and NAL. There are some drawbacks to this technique, such as lengthy reaction time, and the low conversion rate when the soluble forms of the enzymes are used in the two-step enzymatic process. In this study, the Escherichia coli-expressed AGE and NAL in the supernatant were purified by FP-based affinity chromatography and then immobilized on Amberzyme oxirane resin. These two immobilized enzymes, with a specific activity of 78.18 U/g for AGE and 69.30 U/g for NAL, were coupled to convert GlcNAc to Neu5Ac directly in one reactor. The conversion rate of the two-step reactions from GlcNAc to Neu5Ac was ~73% within 24 h. Furthermore, the immobilized AGE and NAL could both be used up to five reaction cycles without loss of activity or significant decrease of the conversion rate. 相似文献
2.
The purification and characterisation of viral, bacterial and mammalian sialidases (EC 3.2.1.18, neuraminidases, neuraminosylglycohydrolases) prompted a search for a colorimetric technique to localize the enzymes on electropherograms. The 5-bromo substituted indol-3-yl -ketoside of 5-N-acetyl-d-neuraminic acid (BIN), the synthesis of which is described here, seemed to be the appropriate substrate, because of its relative ease of enzymatic hydrolysis to 5-N-acetyl-d-neuraminic acid and 5-bromoindoxyl. The latter is readily transformed to the insoluble, intensely coloured 5,5-dibromo-indigo. This precipitates and can be seen readily at the sites of enzymatic activity. The new substrate is of definitive advantage as it provides a simple and direct method for the demonstration of sialidases without the need of a coupling reaction.Abbreviations Neu5Ac
5-N-acetyl-d-neuraminic acid
- BIN
5-bromo-indol-3-yl -ketoside ofN-acetyl-d-neuraminic acid 相似文献
3.
Bai-Xue Lin Zi-Juan Zhang Wei-Feng Liu Zhi-Yang Dong Yong Tao 《Applied microbiology and biotechnology》2013,97(11):4775-4784
N-Acetyl-d-neuraminic acid (Neu5Ac) has attracted considerable interest due to its promising potential applications in medicine. Significant efforts have been made in whole-cell biocatalyst for Neu5Ac production, but the processes often result in suboptimal performance due to poor expression of enzymes, imbalances of pathway components, disturbance of competing pathways, and barriers of mass transport. In this study, we engineered Escherichia coli strains capable of producing Neu5Ac by assembling a two-step heterologous pathway consisting of N-acetyl-d-glucosamine 2-epimerase (AGE) and Neu5Ac aldolase (NanA). Multiple approaches were used to improve the efficiency of the engineered pathway and process for enhanced Neu5Ac production. Firstly, we identified that NanA was the rate-controlling enzyme in this pathway. With increased expression of NanA, a ninefold increase in Neu5Ac production (65 mM) was observed. Secondly, knocking out nanTEK genes blocked Neu5Ac uptake and the competing pathway, which kept the reactions to the synthetic direction as the final product went outside of the cells and enhanced the Neu5Ac production by threefold, resulting in 173.8 mM of Neu5Ac. Thirdly, we improved the performance of the system by promoting substrate transport and optimizing concentrations of substrates. An overall whole-cell biocatalytic process was developed and a maximum titer of 240 mM Neu5Ac (74.2 g/L) was achieved, with productivity of 6.2 g Neu5Ac/L/h and conversion yield of 40 % from GlcNAc. The engineered strain could be reused for at least five cycles with a productivity of >6 g/L/h. It is a cost-effective process for Neu5Ac production with potential applications in large-scale industrial production. 相似文献
4.
Gérard Strecker Jean-Michel Wieruszeski Claude Martel Jean Montreuil 《Glycoconjugate journal》1987,4(4):329-337
Alkaline borohydride reductive cleavage of hen ovomucin resulted in the release of a series of neutral and acidic oligosaccharide-alditols.1H-NMR spectroscopy in combination with fast ion bombardment-mass spectrometry in negative ion mode were used for investigation of the structures of three oligosaccharide-alditols. The following structures were established:
Abbreviations NeuAc
N-acetyl-d-neuraminic acid
- Gal
d-galactose
- GlcNAc
N-acetyl-d-glucosamine
- Gal-NAc-ol
N-acetyl-d-galactosaminitol
- NMR
nuclear magnetic resonance
- FAB-MS
fast atom bombardmentmass spectrometry 相似文献
5.
Zimmermann V Hennemann HG Daussmann T Kragl U 《Applied microbiology and biotechnology》2007,76(3):597-605
In this work, a model describing the complete enzyme catalysed synthesis of N-acetylneuraminic acid (Neu5Ac) from N-acetyl-d-glucosamine (GlcNAc) is presented. It includes the combined reaction steps of epimerisation from GlcNAc to N-acetyl-d-mannosamine (ManNAc) and the aldol condensation of ManNAc with sodium pyruvate yielding Neu5Ac. The model is expedient to
predict the reaction course for various initial and feed concentrations and therefore to calculate reaction times and yields.
The equilibrium constants calculated from the kinetic constants via the Haldane relationship correspond with experimental
values very well (0.26 calculated and 0.24 experimental value for the epimerisation, 27.4 l mol−1 calculated and 28.7 l mol−1 experimental for the aldol condensation). The actual relevance of the model is shown by a scale-up. Using the model, an optimisation
of reaction conditions in consideration of different targets is possible. Exemplarily, it is presented how the optimal ratio
of the two enzymes in the reaction can be determined and how the composition of the reaction solution in a fed-batch reactor
can be designed to meet downstream processing needs. 相似文献
6.
The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu2+ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac
N-acetylneuraminic acid
- Neu5Ac2en
2-deoxy-2,3-dehydro-N-acetylneuraminic acid
- 4MU-Neu5Ac
4-methylumbelliferyl--N-acetyl-d-neuraminic acid 相似文献
7.
Xu X Gao C Zhang X Che B Ma C Qiu J Tao F Xu P 《Applied and environmental microbiology》2011,77(10):3197-3201
Production of N-acetyl-d-neuraminic acid (Neu5Ac) via biocatalysis is traditionally conducted using isolated enzymes or whole cells. The use of isolated enzymes is restricted by the time-consuming purification process, whereas the application of whole cells is limited by the permeability barrier presented by the microbial cell membrane. In this study, a novel type of biocatalyst, Neu5Ac aldolase presented on the surface of Bacillus subtilis spores, was used for the production of Neu5Ac. Under optimal conditions, Neu5Ac at a high concentration (54.7 g liter−1) and a high yield (90.2%) was obtained under a 5-fold excess of pyruvate over N-acetyl-d-mannosamine. The novel biocatalyst system, which is able to express and immobilize the target enzyme simultaneously on the surface of B. subtilis spores, represents a suitable alternative for value-added chemical production. 相似文献
8.
N-Acetyl-d-neuraminic acid (Neu5Ac) and its derivates are a very important group of biomolecules because these sugars occupy the terminal
positions in numerous macromolecules, such as the glycans of glycoproteins, and are involved in many biological and pathological
phenomena. The synthesis and applications of Neu5Ac are attracting much interest due to the potential applications of this
compound in the pharmaceutical industry, such as in the synthesis of the anti-flu drug zanamivir. In this review article,
we discuss existing knowledge on the biotechnological production and applications of Neu5Ac and also propose some guidelines
for future studies. 相似文献
9.
The dominant glycosylation mutants of MDAY-D2 mouse lymphoma cells, designated class 2 (D33W25 and D34W25) were selected for their resistance to the toxic effects of wheat germ agglutinin (WGA) and shown to express elevated levels of Neu5Gc. In accordance with this, the activity of CMP-Neu5Ac hydroxylase was found to be substantially higher in the mutant cells. The hydroxylase in the D33W25 mutant cells exhibited kinetic properties identical to those of the same enzyme from mouse liver. Growth rate experimentsin vivo andin vitro, where the mutant cells grew more slowly at low cell densities in serum-free medium and also formed slower growing tumours in syngeneic mice, indicate that CMP-Neu5Ac hydroxylase expression may be associated with altered growth of the mutant cells.Abbreviations WGA
wheat germ agglutinin
- Neu5Ac
N-acetyl--d-neuraminic acid
- Neu5Gc
N-glycology--d-neuraminic acid
- CMP-Neu5Ac
cytidine-5-monophospho-N-acetylneuraminic acid
- CMP-Neu5Gc
cytidine-5-monophospho-N-glycoloylneuraminic acid
- FACS
fluorescence-activated cell sorting
- buffer A
triethylamine hydrogen carbonate, pH 7.6 (concentration given at appropriate points in the text)
- SFM
serum free medium
- IMDM
Iscove's modified Dulbecco's medium
- CMP-Neu5Ac hydroxylase
CMP-N-acetylneuraminate: NAD(P)H oxido-reductase (N-acetyl hydroxylating) (EC 1.14.99.18); CMP-sialate hydrolase (EC 3.1.4.40); sialic acid-pyruvate lyase (EC 4.1.3.3) 相似文献
10.
We demonstrate that 9-amino-NeuAc transferred to asialo-1-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC
high performance liquid chromatography
- BSA
bovine serum albumin
- NeuAc
N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid
- 9-Amino-NeuAc
9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid
- CMP-NeuAc
cytidine-5-monophospho-N-acetyl-d-neuraminic acid
- CMP-9-amino-NeuAc
cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid
- 9-azido-NeuAc
5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid.
Enzymes EC 3.2.1.18
sialidase, acylneuraminylhydrolase
- EC 2.4.99.1
Galß1-4GlcNAc a(2-6)-sialytransferase 相似文献
11.
CMP-N-acetylneuraminate hydroxylase was isolated from mouse liver high speed supernatant with a yield of 0.4% and an apparent 1000-fold purification. The enzyme is a monomeric protein with a molecular weight of 66 kDa, as determined by gel filtration and SDS-PAGE. The hydroxylase system was reconstituted with Triton X-100-solubilized mouse liver microsomes and purified soluble or microsomal forms of cytochrome b5 reductase and cytochrome b5. The systems were characterized in detail and kinetic parameters for each system were determined.Abbreviations Neu5Ac
N-acetyl--d-neuraminic acid
- Neu5Gc
N-glycoloyl--d-neuraminic acid
- CMP-Neu5Ac
cytidine-5-monophospho-N-acetylneuraminic acid
- CMP-Neu5Gc
cytidine-5-monophospho-N-glycoloylneuraminic acid
- TCA
trichloroacetic acid
- Chaps
3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate
- SOD
superoxide dismutase
Enzymes: CMP-N-acetylneuraminate: NADH oxidoreductase (N-acetyl hydroxylating) (E.C. 1.14.13.45), CMP-Neu5Ac hydroxylase; NADH: cytochrome b5 oxidoreductase (E.C. 1.6.2.2), cytochrome b5 reductase; hydrogen peroxide: hydrogen peroxide oxidoreductase, catalase (E.C. 1.11.1.6); superoxide:superoxide oxidoreductase (E.C. 1.15.1.1), superoxide dismutase.This paper is dedicated to Professor Harry Schachter on the occasion of his 60th birthday. 相似文献
12.
A novel integrated optical technique is used to monitor the kinetics of incorporation of glycophorin A (GPA) from solution into a planar dimyristoylphosphatidylcholine-cholesterol bilayer membrane, and the subsequent binding of wheat germ agglutinin (WGA) to the membrane-incorporated GPA. The technique significantly improves the attainable accuracy of kinetic measurements. The number of bound molecules can be determined to a precision of ca ± 80 mol µm–2. Our results show that GPA incorporates spontaneously into the bilayer. Binding of WGA to GPA is optimal in the presence of human serum albumin, and can be reversed byN-acetyl-d-glucosamine. The kinetics of the binding are consistent with the presence of two classes of kinetically distinguishable binding sites with association rates of 2.0×104 and 9.6×102 M–1 s–1, and dissociation rates of 2.7×10–3 s–1 and <10–5 s–1, respectively. A stoichiometry of 4 WGA monomers per GPA monomer was determined as characteristic of the overall binding interaction.Abbreviations DMPC
dimyristoylphosphatidylcholine
- GlcNAc
N-acetyl-d-glucosamine
- GPA
glycophorin A
- HSA
human serum albumin
- NeuNAc
N-acetyl-d-neuraminic acid
- TE
transverse electric
- TM
transverse magnetic
- WGA
wheat germ agglutinin 相似文献
13.
Summary Peroxidase-labelled lectins specific for various carbohydrate residues were used as histochemical reagents in the investigation of Hurler's syndrome. Peanut lectin was used to detect terminald-galactose, wheatgerm lectin forN-acetyl-d-glucosamine, soybean lectin forN-acetyl-d-galactosamine,Tetragonolobus lotus lectin for -l-fucose andBandeiraea S. lectin for -d-galactose. It was found that Kupffer cells in the liver and splenic reticulo-endothelial cells contain acid mucopolysaccharides which bind lectins in paraffin sections after appropriate fixation. The pattern of lectin binding suggests that such cells contain significant amounts ofd-galactose,l-fucose,N-acetyl-d-galactosamine andN-acetyl-d-glucosamine. It is likely that the last named carbohydrate is present as a polymer. Neurones contain a different carbohydrate, rich in galactose and fucose but poor inN-acetyl-d-glucosamine. This compound is resistant to lipid extraction. Hepatocytes, as a rule, do not react with lectins, most likely because of loss of the more soluble mucopolysaccharides during fixation. The results are consistent with the biochemical data of Hurler's syndrome and indicate that lectins can be a useful tool for the investigation of the cytochemistry of storage disorders. 相似文献
14.
Deqiang Zhu Xiaobei Zhan Jianrong Wu Minjie Gao Zhongsheng Zhao 《Biotechnology letters》2017,39(1):55-63
Objective
To develop a strategy for producing N-acetyl-d-neuraminic acid (Neu5Ac), which is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and pyruvate, but without using pyruvate.Result
An efficient three-module whole-cell biocatalyst strategy for Neu5Ac production by utilizing intracellular phosphoenolpyruvate was established. In module I, the synthetic pathway was constructed by coexpressing GlcNAc 2-epimerase from Anabaena sp. CH1 and Neu5Ac synthase from Campylobacter jejuni in Escherichia coli. In module II, the Neu5Ac degradation pathway of E. coli was knocked out, resulting in 2.6 ± 0.06 g Neu5Ac l?1 after 72 h in Erlenmeyer flasks. In module III, the transmembrane mode of GlcNAc was modified by disruption of GlcNAc-specific phosphotransferase system and Neu5Ac now reached 3.7 ± 0.04 g l?1. In scale-up catalysis with a 1 l fermenter, the final Neu5Ac yield was 7.2 ± 0.08 g l?1.Conclusion
A three-module whole-cell biocatalyst strategy by manipulating synthetic, degradation and transmembrane pathways in E. coli was an economical method for Neu5Ac production.15.
A strictly anaerobic, mesophilic and chitinolytic bacterial strain was isolated from human feces. Based on morphological and
physiological properties and 16S rRNA sequence analysis the strain was identified asClostridium paraputrificum. The strain utilized chitin andN-acetyl-d-glucosamine, grew on glucose and hydrolyzed starch. Cultivation of the strain with colloidal chitin as the growth substrate
resulted in the production of gas (hydrogen and carbon dioxide) and formation of acetate and lactate (21.6 and 18.9 mmol/L,
respectively) and only small quantities of propionate and butyrate (1.7 and 2.6 mmol/L, respectively). In the course of a
10-d cultivation with chitin, the endochitinase activity was detected after 1 d and gradually increased, reaching maximum
after 3 d (251 nkat/LN-acetyl-d-glucosamine). The β-N-acetyl-glucosaminidase activity appeared just at the beginning of the cultivation, increased to day 2 and then remained nearly
constant. More than 90% of chitin added was degraded within 2 d of cultivation. On the zymogram of the extracellular chitinolytic
complex were visible at least 6 isoenzymes with molar mass 43.5–65.0 kDa. The temperature optimum of endochitinase and β-N-acetylglucosaminidase activities was 50°C; the optimum activity of both enzymes was found at pH 4–6. 相似文献
16.
Sgambati E Marini M Vichi D Zappoli Thyrion GD Parretti E Mello G Gheri G 《Histochemistry and cell biology》2007,128(3):263-273
The aim of this study was to investigate the distribution of the oligosaccharides of the glycoconjugates in placentas from
pregnancies complicated by different degree of altered glycaemia. Placentas from women with physiological pregnancies (group
1), with pregnancies complicated by minor degree of glucose intolerance (group 2) and with pregnancies complicated by gestational
diabetes mellitus (GDM) treated with insulin (group 3) were collected. Ten lectins were used (ConA, WGA, PNA, SBA, DBA, LTA,
UEA I, GSL II, MAL II and SNA) in combination with chemical and enzymatic treatments. The data showed a decrease of sialic
acid linked α(2–6) to galactose/N-acetyl-d-galactosamine and an increase of N-acetyl-d-glucosamine in the placentas of the pathological groups, in particular the group 3, comparing to the group 1. A decrease
of l-fucose (LTA) and d-galactose-(β1–3)-N-acetyl-d-galactosamine, and an increase and/or appearance of l-fucose (UEA I) and N-acetyl-d-galactosamine were observed in both the pathological groups, particularly in the group 2, with respect to the group 1. In
GDM, and even in pregnancies with a simple alteration of maternal glycaemia, the changes in the distribution of oligosaccharides
could be related to alteration of the structure and functionality of the placenta. 相似文献
17.
Bernard Priem Julien Solokwan Jean-Michel Wieruszeski Gérard Strecker Hassan Nazih Henri Morvan 《Glycoconjugate journal》1990,7(2):121-132
The oligosaccharides Man5GlcNAc and Man3(Xyl)GlcNAc(Fuc)GlcNAc presumed to originate fromN-glycosyl proteins have been purified from an extracellular medium (concentration: 2–5 mg/l of 14 day cultures) of white campion (Silene alba) suspension culture. Their primary structures have been determined by1H-400-MHz NMR spectroscopy and FAB-MS spectrometry. They are probably the result of an autophagic process including protein catabolism due to sucrose starvation. Additional identification of digalactosylglycerol (galactolipid breakdown) argues for this hypothesis.Abbreviations Fuc
l-fucose
- Man
d-mannose
- Xyl
d-xylose
- GlcNAc
N-acetyl-d-glucosamine
- Gal
d-galactose
- Glc
d-glucose
- FAB-MS
fast atom bombardment mass spectrometry
- NMR
nuclear magnetic resonance 相似文献
18.
Maria Montserrat Sala Jesús M. Arrieta Julia A. Boras Carlos M. Duarte Dolors Vaqué 《Polar Biology》2010,33(12):1683-1694
Global warming and the associated ice melt are leading to an increase in the organic carbon in the Arctic Ocean. We evaluated
the effects of ice melt on bacterioplankton at 21 stations in the Greenland Sea and Arctic Ocean in the summer of 2007, when
a historical minimum of Arctic ice coverage was measured. Polar Surface Waters, which have a low temperature and low salinity
and originate mainly from melted ice, contained a very low abundance of bacteria (7.01 × 105 ± 2.20 × 105 cells ml−1); however, these bacteria had high specific bacterial production (2.40 ± 1.61 fmol C bac−1 d−1) compared to those in Atlantic Waters. Specifically, bacterioplankton in Polar Surface Waters showed a preference for utilizing
carbohydrates and had significantly higher specific activities of the glycosidases assayed, i.e. β-glucosidase, xylosidase,
arabinosidase and cellobiosidase. Furthermore, bacterioplankton in Polar Sea Waters showed preferential growth on some of
the carbohydrates in the Biolog Ecoplate, such as d-cellobiose and N-acetyl-d-glucosamine. Our results suggest that climate change and the associated melting of Arctic ice might induce changes in bacterioplankton
functional diversity by enhancing the turnover of carbohydrates. Since organic aggregates are largely composed of polysaccharides,
higher solubilization of aggregates might modify the carbon cycle, weaken the biological pump and have biogeochemical and
ecological implications for the future Arctic Ocean. 相似文献
19.
The sialic acid analogue,N-acetyl-4-deoxy-neuraminic acid, is readily activated by CMP-sialic acid synthase from bovine brain. We also show that sialyl-transfer from CMP-N-acetyl-4-deoxy-neuraminic acid to asialo-
1-acid glycoprotein is achieved at a high rate using Gal1-4GlcNAc (2.6)-sialyltransferase from rat liver.In contrast toVibrio cholerae sialidase, fowl plague virus sialidase liberates boundN-acetyl-4-deoxy-neuraminic acid from the glycoprotein. Thus, as opposed to the general view, the action of neither synthase nor transferase depends on the presence of the hydroxy group at C-4 ofN-acetylneuraminic acid.Abbrevations BSA
bovine serum albumin
- DTE
dithioerythritol
- HPLC
high performance liquid chromatography
- NeuAc
N-acetyl-d-neuraminic acid
- 4-deoxy-NeuAc
N-acetyl-4-deoxy-d-neuraminic acid
- 4-epi-NeuAc
4-acetamido-3,5-dideoxy-d-glycero-d-talononulosonic acid
- CMP-NeuAc
Cytidine-5-monophospho-N-acetylneuraminic acid
- CMP-4-deoxy-NeuAc
Cytidine-5-monophospho-N-acetyl-4-deoxy-neuraminic acid
- FPV-sialidase
Fowl plague virus sialidase
- VCN
Vibrio cholerae neuraminidase 相似文献
20.
Deqiang Zhu Jianrong Wu Xiaobei Zhan Li Zhu Zhiyong Zheng Minjie Gao 《Biotechnology letters》2017,39(2):227-234