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Carbohybrate purification using ultrafiltration, reverse osmosis and nanofiltration

A technology of reverse osmosis membrane and sugar, applied in the direction of purification by adsorbent, extraction of sucrose by physical method, application, etc., can solve the problem of hindering application, expensive chromatography purification method, and chromatography purification method is not suitable for large-scale purification, etc. question

Inactive Publication Date: 2000-01-26
NEOSE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, chromatographic purification methods are not suitable for large-scale purification, thus hindering their application in the commercial production of sugars
Moreover, chromatographic purification methods are expensive

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example

[0036] The invention provides a rapid and effective purification method for purifying specific sugars and oligosaccharide structures to high purity by using semipermeable membranes (such as reverse osmosis membranes and / or nanofiltration membranes). The method is particularly suitable for separating the desired oligosaccharide compound from the reactants and other contaminants remaining in the reaction mixture after the synthesis or degradation of the oligosaccharide. For example, the present invention provides a reaction mixture used to synthesize or enzymatically degrade oligosaccharides, nucleotide sugars, glucolipids, glycolipids, nucleotides, nucleosides and other sugar-containing compounds from a reaction mixture Methods of separating enzymes and / or other components. In addition, it also provides methods for removing salts, sugars and other components from the raw material liquid by ultrafiltration, nanofiltration or reverse osmosis. By adopting these technologies, sugars (s...

Embodiment 1

[0096] Synthesis and purification of sialic acid

[0097] This example illustrates the method of synthesizing sialic acid with the relatively inexpensive substrate GlcNAc (instead of using the more expensive ManNAc to synthesize or directly using sialic acid). A procedure similar to that described by Simon et al., J. Am. Chem. Soc. 110: 7159 (1988) was used to convert GlcNAc to ManNAc. Briefly, GlcNAc (1000 g, 4.52 mol) was dissolved in 500 ml of water. The pH was adjusted to 12.0 with 50% sodium hydroxide (approximately 115 mL). The solution was stirred under argon for 7.5 hours, then cooled in an ice bath, and the pH was adjusted to 7.7 with concentrated hydrochloric acid (about 200 ml). Then sialic acid is formed by the aldol condensation of ManNAc. ÷

[0098]In order to obtain sialic acid, the ManNAc produced in the previous step is subjected to aldol condensation mediated by N-acetylneuraminic acid (Neu5Ac) aldolase and pyruvate. To a 1.5 liter aqueous sol...

Embodiment 2

[0100] Cyclic synthesis of sialyllactose with sialyltransferase

[0101] Add lactose monohydrate (79.2 g, 0.22 mol), 0.7 g bovine serum albumin, and phosphoenolpyruvate monopotassium salt (37 g, 0.22 mol) to the sialic acid prepared in Example 1, and adjust the pH to 7.5 . CMP (2.48 g, 0.0088 mol) and ATP (0.54 g, 0.0009 mol) were added to adjust the pH to 7.5. Add sodium azide (0.35 g), and the following enzymes: pyruvate kinase (19800U), myokinase (13200U), CMP sialic acid synthase (440U) and sialyltransferase (165U). Add 66 ml MnCl 2 , Adjust the final volume to 2.2 liters with water. The reaction was allowed to proceed at room temperature.

[0102] The reaction is monitored daily by thin layer chromatography (TLC) and measured by ion chromatography [Mn 2+ ]. Add / adjust as shown in Table 1:

[0103] Table 1

[0104] Day 2 Add 44ml 1M MnCl 2

[0105] Day 4 Add 43ml 1M MnCl 2

[0106] Day 6 Add 34.3ml 1M MnCl 2 , 37 grams of PEP; readjust pH 7.5; pyruvate kinase...

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Abstract

The invention provides methods for purifying carbohydrates, including oligosaccharides, nucleotide sugars, and related compounds, by use of ultrafiltration, nanofiltration and / or reverse osmosis. The carbohydrates are purified away from undesired contaminants such as compounds present in reaction mixtures following enzymatic synthesis or degradation of oligosaccharides.

Description

[0001] Cross-documentation of related applications [0002] This application is a partial renewal application of U.S. Provisional Application No. 60 / 028,226 (filed on October 10, 1996), and the content of this application is incorporated herein for reference. Invention field [0003] The present invention relates to the synthesis of oligosaccharides. Specifically, the present invention relates to an improved method of purifying oligosaccharides using ultrafiltration, nanofiltration and / or reverse osmosis. Background of the invention [0004] As the understanding of the role of carbohydrates as recognition elements on the cell surface continues to increase, so does the interest in producing carbohydrate molecules with defined structures. For example, compounds containing oligosaccharide moieties sialyl lactose have become interesting neutralizers against bacterial enterotoxins such as Vibrio cholerae, Escheri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01D61/00B01D61/24C07H1/06C08B30/00C13B10/08C13B20/12C13B30/08C13B35/08
Inventor S・德弗里斯
Owner NEOSE TECH
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