Method for biocatalytically synthesizing D-(+)-glucuronic acid and application of method

A technology of glucuronic acid and biocatalysis, applied in the field of biocatalytic synthesis of D-glucuronic acid, can solve the problems of difficult separation and purification, long production cycle of microbial fermentation and the like

Active Publication Date: 2020-11-10
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are relatively few studies on the production of glucuronic acid by biological fermentation, and microbial fermentation has the disadvantages of long production cycle and difficult separation and purification; enzymatic synthesis has short production cycle, simple separation and purification, less environmental pollution, and high specificity. Advantages, the use of enzymatic methods to produce glucuronic acid has great development prospects

Method used

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  • Method for biocatalytically synthesizing D-(+)-glucuronic acid and application of method
  • Method for biocatalytically synthesizing D-(+)-glucuronic acid and application of method
  • Method for biocatalytically synthesizing D-(+)-glucuronic acid and application of method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The preparation of embodiment 1 enzyme

[0067] 1. Preparation of sucrose phosphorylase, glucose phosphate mutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase:

[0068]In the present invention, sucrose phosphorylase (Sucrose phosphorylase, SP, EC: 2.4.1.7) is derived from E.coli BL21 (DE3), and its NCBI accession number is NC_012892.2; Phosphoglucomutase (Phosphoglucomutase, PGM, EC :5.4.2.2) derived from B.methnoillus PB1 (Bacillusmethanolicus PB1), its NCBI accession number is NZ_AFEU01000003.1:850496-851473; inositol-1-phosphate synthase (Inositol-1-phosphate synthase, MIPS, EC:5.5 .1.4) derived from S.cerevisiae (Saccharomyces cerevisiae S288C, Saccharomyces cerevisiae S288C), its NCBI accession number is Chromosome: X; NC_001142.9 (134332..135933); inositol monophosphatase (Inositol monophosphatase, IMP, EC :3.1.3.25) derived from B.methnoillus PB1, its NCBI accession number is NZ_AFEU01000003.1:122318-123091; inositol oxidase (myo...

Embodiment 2

[0085] Example 2 In vitro multi-enzyme catalysis converts sucrose into D-(+)-glucuronic acid

[0086] The present invention converts sucrose into D-(+)-glucuronic acid ( figure 1 ). These key enzymes include:

[0087] 1) Sucrose phosphorylase (SP), which catalyzes the decomposition of sucrose into glucose-1-phosphate and fructose;

[0088] 2) Phosphoglucomutase (PGM), which catalyzes glucose-1-phosphate to glucose-6-phosphate;

[0089] 3) Inositol-1-phosphate synthase (MIPS), which catalyzes glucose-6-phosphate to inositol-1-phosphate;

[0090] 4) Inositol monophosphatase (IMP), which catalyzes the dephosphorylation of inositol-1-phosphate to inositol;

[0091] 5) Inositol oxidase (MIOX), which catalyzes inositol to D-(+)-glucuronic acid.

[0092] Among them, the first step and the second step reaction are reversible, and the subsequent reactions are all irreversible reactions, so the enzyme catalytic system can obtain a high conversion rate.

[0093] The specific experi...

Embodiment 3

[0097] Example 3 In vitro multi-enzyme catalysis converts sucrose into glucuronic acid

[0098] (1) The preparation of sucrose phosphorylase, glucose phosphomutase, inositol-1-phosphate synthase, inositol monophosphatase and inositol oxidase is the same as in Example 1.

[0099] (2) Contain 150mM Tris-HCl buffer solution, 20mM NaH in a 2.0ml reaction system 2 PO 4 , 5mmol / L MgCl 2 , 2mmol / L L-cysteine, 1U / mL SP, 1U / mL PGM, 2U / mL MIPS, 1U / mL IMP and 5U / mL MIOX, 50mmol / L sucrose, the pH of the catalytic reaction system is 7.5, in The catalytic reaction was carried out at 35°C for 60 hours.

[0100] (3) After the reaction, the final concentration of D-(+)-glucuronic acid was 19.5 mM (the quantitative method was the same as in Example 2), and the conversion rate of sucrose atoms reached 39%.

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Abstract

The invention discloses a method for biocatalytically synthesizing D-(+)-glucuronic acid and an application of the method. The method comprises the following steps: by taking cane sugar as a substrate, adding enzyme to perform a catalytic reaction to obtain D-(+)-glucuronic acid, wherein the enzyme comprises sucrose phosphorylase, phosphoglucose mutase, inositol-1-phosphate synthase, inositol monophosphate esterase and inositol oxidase. The enzyme catalysis system can obtain a very high conversion rate. In addition, phosphate, phosphofructokinase and phosphoglucose isomerase are added into thereaction system, the conversion rate of the D-(+)-glucuronic acid can be further improved. The method is high in raw material utilization rate, high in D-(+)-glucuronic acid conversion rate, simple in step, low in production cost, small in pollution, small in environmental influence and capable of achieving large-scale production of D-(+)-glucuronic acid.

Description

technical field [0001] The invention belongs to the field of enzyme-catalyzed production of D-(+)-glucuronic acid, in particular to a method for biocatalytically synthesizing D-(+)-glucuronic acid and its application. Background technique [0002] D-(+)-glucuronic acid (C 6 h 10 o 7 , D-glucuronic acid), is a compound formed by oxidation of the primary alcohol hydroxyl group of glucose to form a carboxyl group. Its aqueous solution is unstable and easily converted to 3,6 glucuronic acid lactone. The two are in an interconverting equilibrium state in aqueous solution. When glucuronic acid is heated and there is a strong acid, it is easy to decarboxylate and generate CO 2 , Furan and other products. [0003] Glucuronic acid widely exists in animals and plants and has important biological functions. It can combine toxic substances containing groups such as hydroxyl, amino, carboxyl, and mercapto groups, and strengthen the aqueous solution of toxic substances, so that they ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12P19/18C12P19/24
CPCC12P19/02C12P19/18C12P19/24
Inventor 娄文勇苏慧慧区晓阳杨继国曾英杰彭飞倪子富徐培
Owner SOUTH CHINA UNIV OF TECH
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