Preparation method of uridine diphosphate glucuronic acid

A technology of uridine diphosphate glucose and aldehyde acid, which is applied in the field of enzyme catalysis, can solve the problems of low synthesis efficiency, high cost, and high cost of raw materials, and achieve the effects of efficient synthesis, cost saving, and controllable reaction system

Pending Publication Date: 2021-04-06
JIANGNAN UNIV
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Problems solved by technology

Chemical methods often have long synthesis time, low synthesis efficiency, high cost, and poor stereoselectivity, which are not conducive to industrial development, while enzymatic methods have mild reaction conditions, strong selectivity, low energy consumption, and friendly process environment, which are unmatched by chemical synthesis.
Qichen carried out recombinant expression and optimization of UGD derived from Escherichia coli, adding UGPase and UGD to the system, two-step coupling catalytic reaction to synthesize UDP-glucuronic acid, UGPase enzyme catalyzed the synthesis of UDP-glucose with UTP as substrate, and UGD with UDP -Glucose was used as a substrate to catalyze the synthesis of UDP-glucuronic acid, and the conversion rate of the substrate was found to be unsatisfactory
Ji Xiaohu used fresh bovine liver as raw material, prepared UGD crude product by step-by-step salting-out-thermal denaturation method, and used commercially available rabbit muscle lactate dehydrogenase to synthesize UDP-glucuronic acid by one-pot enzymatic method. The product shows better advantages, but the use of commercially available rabbit muscle lactate dehydrogenase has high raw material costs, and the UGD crude enzyme solution needs to be extracted from fresh bovine liver, and the preparation process is relatively complicated.

Method used

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  • Preparation method of uridine diphosphate glucuronic acid
  • Preparation method of uridine diphosphate glucuronic acid
  • Preparation method of uridine diphosphate glucuronic acid

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1: the preparation of UDP-glucose

[0034] Weigh 50g of anion exchange resin into a 250mL beaker, add 50mL of 10% isopropanol to soak for 20min, filter with suction, rinse with 10% isopropanol, rinse with deionized water, filter with suction, rinse with 0.5M HCl, filter with suction, Resuspend in 45mL 0.5M HCl, soak for 2h, filter with suction, rinse with deionized water, and stir in 45mL CoCl2 aqueous solution overnight. Wash with 50mM pH7.5 phosphate buffer for later use. Prepare 20mM Tris buffer, adjust the pH to 7.0, mix the volume of enzyme solution and buffer solution at a ratio of 1:4, take 194 mg of purified protein GlmU enzyme and 3 mg of PPA enzyme and place them in a triangular flask, and add 21.5 mg of the treated resin respectively. g, 0.15g, put into a shaker, the rotation speed is 120rpm, the temperature is 25°C, and the time is 1h. Suction filter, rinse with 50mM pH7.5 phosphate buffer, and store at 4°C for later use.

[0035] Weigh 2.16g o...

Embodiment 2

[0037] Example 2: Single enzyme catalyzes the oxidation of UDP-glucose to generate UDP-glucuronic acid

[0038] Mix 2.0mL 10mM UDPG, 2.5mL 10mM NAD, 100μL 1M Tris, 10μL β-mercaptoethanol, 20μL 1M MgCl2, 3.37mL deionized water, then add 2.5mL 1.0mg / mL UGD enzyme solution, at the optimum temperature React overnight at 25°C; take 100 μL of the reaction solution to pass through an aqueous filter membrane with a pore size of 0.22 μm, and perform HPLC analysis (such as Figure 6 shown), and the reaction substrates UDP-glucose, NAD + and product NADH, UDP-glucuronic acid standards for HPLC analysis (such as figure 2 , 3 , 4, 5 shown), Figure 6 It is shown that there is a preliminary conclusion that there are new substances NADH and UDP-glucuronic acid formed, and the calculated amount of UDP-glucuronic acid is about 0.51mg / mL.

Embodiment 3

[0039] Example 3: Dual-enzyme coupling catalyzes the oxidation of UDP-glucose to generate UDP-glucuronic acid

[0040] Mix 2.4mL 10mM UDPG, 1.5mL 10mM NAD, 100μL 1M Tris, 10μL β-mercaptoethanol, 20μL 1M MgCl2, 1mL 1M sodium pyruvate, 1.97mL deionized water, and then add 2.5mL 1.0mg / mL UGD enzyme solution, 0.5mL 2.7mg / mL LDH enzyme solution, and react overnight at the optimum temperature of 25°C; take 100μL of the reaction solution to pass through an aqueous filter membrane with a pore size of 0.22μm for HPLC analysis, and UDP-glucosaldehyde can be detected acid generation, the yield was about 1.01mg / mL, and the yield was nearly doubled (such as Figure 7 shown).

[0041] The UDP-glucuronic acid standard product and the reaction product UDP-glucuronic acid were detected by mass spectrometry, and the LC-MS analysis was carried out by WATERSMALDI SYNAPT MS, as shown in Figure 8 , 9 As shown, the test result of the sample is consistent with the test result of the UDP-glucuronic ...

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Abstract

The invention relates to a preparation method of uridine diphosphate glucuronic acid. Uridine diphosphate glucose is oxidized by uridine diphosphate glucose dehydrogenase to generate uridine diphosphate glucuronic acid, and lactic dehydrogenase is added for double-enzyme coupling reaction, so that cyclic regeneration of NAD<+> is realized, the addition of NAD<+> is reduced, the cost is saved, and at the same time, the circulation of NAD<+> / NADH reduces the feedback inhibition effect of the byproduct NADH on the product uridine diphosphate glucuronic acid, and the generation amount of the target product can reach 1.01 mg / mL. The nucleotide sequence of lactic dehydrogenase is optimized and modified, so that the target protein can be better expressed in escherichia coli, and efficient synthesis of the target product uridine diphosphate glucuronic acid is facilitated. In-vitro reaction is adopted, a reaction system is more controllable, and compared with intracellular reaction, in-vitro reaction can be more beneficial to reaction under limited conditions.

Description

technical field [0001] The invention relates to the technical field of enzyme catalysis, in particular to a preparation method of uridine diphosphate glucuronic acid. Background technique [0002] Nucleotide sugars are high-energy forms of monosaccharide activation in nature, and the common ones are uridine diphosphate sugar (UDP-sugar), thymidine diphosphate sugar (TDP-sugar), guanosine diphosphate sugar (GTP-sugar) Wait. Especially uridine diphosphorylated sugars are the most widely used, among which UDP-glucose is the initial substrate for the formation of other UDP-monosaccharides, and UDP-glucuronic acid is the UDP-monosaccharide that changes from six-carbon sugar to five-carbon sugar key substrates. [0003] UDP-glucuronic acid, a derivative derived from the dehydrogenation of UDP-glucose, is an important sugar donor in cells and participates in multiple metabolic pathways of organisms. In microbial cells, UDP-glucuronic acid is the key synthetic substrate of capsul...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30C12N9/04C12N15/53C12N15/70
CPCC12P19/305C12N9/0006C12N15/70C12Y101/01022C12N2800/22
Inventor 何丽丽窦文芳吕海超
Owner JIANGNAN UNIV
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