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A method for synthesizing lacto-n-disaccharides

A galactose and galactokinase technology, applied in biochemical equipment and methods, glycosylase, glycosyltransferase, etc., to achieve important economic value and social benefits, improve lacto-N-disaccharide synthesis and substrates Effective utilization and low cost

Active Publication Date: 2020-03-13
SHANDONG HENGLU BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the removal of E. coli endotoxin is a major challenge in large-scale industrial production

Method used

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  • A method for synthesizing lacto-n-disaccharides
  • A method for synthesizing lacto-n-disaccharides
  • A method for synthesizing lacto-n-disaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Embodiment 1: take galactose as substrate synthetic milk- N - disaccharide

[0054] Add 10 mM galactose, 10 mM ATP, 10 mM GlcNAc, 3 mM MgCl to 1 mL reaction system 2 , 100mM Tris-HCl, 5 U Galk, 168 U milk- N - Disaccharide phospholases. As shown in Table 1, wherein Control is no milk- N - Disaccharide phospholyase (LNBP) control group, after reacting at 37°C for 12 hours, the reaction system was boiled for 5 minutes, centrifuged to take the supernatant, after filtering the membrane, Biorad-HPX column, RID detection of corresponding substrates and products, reaction detection Spectrum (HPLC) comparison results verify that milk- N - Disaccharides.

[0055] Gal mM ATP mM GalK mg MgCl 2 mM

Embodiment 2

[0056] Example 2: Introduction of ATP regeneration cycle synthetic milk - N - disaccharide

[0057] Add 10 mM galactose, 7.5 mM ATP, 2.5 mM acetyl phosphate, 10 mM GlcNAc, 3 mM MgCl to 1 mL reaction system 2 , 100 mM Tris-HCl, 5 U Galk, 3 U ACK, 168 U LNBP. As shown in Table 2, the initial concentration of ATP in the Control control was 10 mM, and the ATP cycle reaction was not introduced into the reaction system. After reacting at 37°C for 12 h, the reaction system was boiled for 5 min, and the supernatant was collected by centrifugation. After filtering the membrane, Biorad-HPX Column, RID detects the corresponding substrate and product. Such as figure 2 As shown, under the ATP-1 reaction condition, the synthesis of LNB was increased by 1.60 times compared with the control group (without adding ATP).

Embodiment 3

[0058] Example 3: Add 10 mM galactose, 5 mM ATP, 5 mM acetyl phosphate, 10 mM GlcNAc, 3 mM MgCl to 1 mL reaction system 2 , 100 mM Tris-HCl, 5 U Galk, 3 U ACK, 168 U LNBP. As shown in Table 2, the initial concentration of ATP in the Control control was 10 mM, and the ATP cycle reaction was not introduced into the reaction system. After reacting at 37°C for 12 h, the reaction system was boiled for 5 min, and the supernatant was collected by centrifugation. After filtering the membrane, Biorad-HPX Column, RID detects the corresponding substrate and product. Such as image 3 As shown, under the ATP-2 reaction condition, the synthesis of LNB was increased by 1.49 times compared with the control group (without adding ATP).

[0059] Table 2 The reaction table of the two-step catalyzed production of lacto-N-disaccharide with galactose substrate after the introduction of ATP regeneration cycle

[0060]

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Abstract

The present invention provides a kind of synthetic milk- N A method for disaccharides belongs to the technical field of bioengineering and oligosaccharide synthesis. The present invention utilizes the multi-enzyme catalytic system with good biological safety and wide application, and improves milk- N ‑Disaccharide synthesis and substrate utilization. The present invention provides a new milk- N ‑disaccharide synthesis pathway for milk‑ N The large-scale industrial production of disaccharides has laid the foundation and has important economic value and social benefits. Simultaneously, the synthesis method of the present invention is efficient, mild, simple and easy to implement, low in cost, suitable for industrialized production, and has good practical application value.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and oligosaccharide synthesis, in particular to a synthetic milk- N - The method of disaccharides. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Breast milk, as the only material and energy link with the mother after the newborn is born, is considered the gold standard of infant nutrition. In addition to providing nutrients such as protein, fat and carbohydrates necessary for infant growth, breast milk also provides active components such as enzymes, antibodies, growth factors and oligosaccharides that can promote infant health and development. Further research found that compared wi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/26
CPCC12P19/26C12P19/12C12N9/1051C12N9/1205C12N9/1217C12Y204/01211C12Y207/01006C12Y207/02001C12N9/2402C12Y302/0114
Inventor 方诩杜志强
Owner SHANDONG HENGLU BIOTECH CO LTD
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