A kind of biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid

A technology for the biosynthesis of uridine diphosphate glucose and uridine diphosphate glucuronic acid, which is applied in the field of biosynthesis of uridine diphosphate glucose and uridine diphosphate glucuronic acid, can solve the problems of complicated preparation steps and high cost of raw materials, and achieve simplification of operation steps and saving Time and Cost Effects

Active Publication Date: 2019-09-06
安徽禾庚生物技术有限公司
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

This preparation method uses maltodextrin as one of the raw materials, the raw material cost is high, and the crude enzyme solution of uridine diphosphate glucose pyrophosphorylase and maltodextrin phosphorylase needs to be extracted, and the preparation steps are relatively complicated

Method used

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  • A kind of biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid
  • A kind of biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid
  • A kind of biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0096] Example 1. Induced expression of recombinant Escherichia coli high temperature α-glucan phosphorylase (TmαGP) and high temperature sugar-1-phosphate nucleotidylase (StUSP)

[0097] Inoculate the recombinant Escherichia coli BL21 (DE3) containing the target gene TmαGP or StUSP synthesized by GenScript into 30 mL of LB medium (containing 100 μg / mL of ampicillin), and activate at 225 rpm for 12 to 14 hours at 37°C; then , the activated recombinant Escherichia coli BL21 (DE3) was inoculated in 250 mL of LB medium (containing 100 μg / mL of ampicillin) for expansion culture, and the initial OD 600 The value is 0.05, cultured to OD at 37°C 225rpm 600 When the value reaches 0.6-0.8, add IPTG with a final concentration of 0.2mM to induce, the induction condition is 22°C 225rpm, 18-20h, measure the OD of the recombinant E. coli liquid 600 Value, wherein, the OD after induced expression of recombinant Escherichia coli containing the target gene TmαGP 600 The value is 2.1-2.3; the...

Embodiment 2

[0099] Example 2. High-temperature whole-cell catalytic reaction of high-temperature α-glucan phosphorylase (TmαGP)

[0100] 1) High-temperature whole-cell catalytic reaction of TmαGP

[0101] First, centrifuge the recombinant Escherichia coli bacterium containing induced expression TmαGP prepared in Example 1 to collect the bacterial pellet, freeze the obtained bacterial pellet at -20°C for about one day, take it out and melt it on ice during the reaction, and use 60 mL of bacteria The bacterial pellet obtained by liquid centrifugation was resuspended in 2 mL triple distilled water. The reaction system is shown in Table 4, wherein the KP solution is KH 2 PO 4 and K 2 HPO 4 Prepared by dissolving in water, KH in KP solution 2 PO 4 and K 2 HPO 4 Concentrations of 0.38M and 0.62M, pH 7, PO 4 3- The total concentration of TmαGP cells is 1M, and the amount of TmαGP cells added is 6.6g cell dry weight / L reaction solution; in addition, a control group is set up, respective...

Embodiment 3

[0114] Example 3. High-temperature whole-cell catalytic reaction of high-temperature sugar-1-phosphate nucleotidylase (StUSP)

[0115] 1) High-temperature whole-cell catalytic reaction of StUSP

[0116] First, centrifuge the recombinant Escherichia coli liquid containing induced StUSP prepared in Example 1 to collect the bacterial precipitate, freeze the obtained bacterial precipitate at -20°C for about one day, take it out and melt it on ice during the reaction, and use 60mL bacterial The bacterial pellet obtained by liquid centrifugation was resuspended in 2 mL triple distilled water. The reaction system is shown in Table 5, wherein, Mg 2+ Produced by the hydrolysis of magnesium chloride, the component of sodium dihydrogen phosphate buffer is NaH 2 PO 4 0.2M, NaOH to adjust the pH to 7.5; in addition, set up a control group, respectively, no StUSP cells in the reaction solution, and uridine diphosphate glucose (UDP-Glc) in the reaction solution, each reaction solution wa...

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Abstract

The invention discloses a biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid. The method includes adopting soluble starch as a main initial raw material, conducting recombinant expression on high-temperature alpha-glucan phosphorylase and high-temperature sugar-1-nucloside phosphorylase in escherichia coli respectively, and utilizing high-temperature whole cell catalysis of expressed bacteria to synthesize uridine diphosphate glucose; on this basis, conducting recombinant expression on high temperature uridine diphosphate glucose dehydrogenase in the escherichia coli, coupling a synthesis system of the uridine diphosphate glucose to conduct high temperature whole cell catalysis to synthesize the uridine diphosphate glucuronic acid, and introducing awhole cell catalysis system of high temperature NADH oxidase into the synthesis system of the uridine diphosphate glucuronic acid to form a high temperature NAD+ / NADH circulating system to reduce theuse of coenzyme NAD+. The high temperature whole cell catalysis method is utilized to successfully avoid the interference of various metabolic pathways of the bacteria in the synthesis process and reduce the purification difficulty.

Description

technical field [0001] The invention relates to a biosynthesis method of uridine diphosphate glucose and uridine diphosphate glucuronic acid, belonging to the technical field of biosynthesis. Background technique [0002] In the study of carbohydrates, oligosaccharides and glycoconjugated complexes with uniform structure are important information molecules in organisms and play an important role in life activities. Nowadays, they are more and more used as probes for biological research or lead compounds for drug and vaccine discovery, and the research on their synthesis has attracted more and more attention. As a research hotspot in recent years, chemoenzyme method has high efficiency and high specificity, and has great application potential in the synthesis of structurally uniform oligosaccharides and glycoconjugated complexes. In this synthesis process, high-energy nucleotide sugars are indispensable as monosaccharide donor substrates. [0003] As a high-energy form of m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/30C12R1/19
Inventor 生举正徐以泓孟丹华
Owner 安徽禾庚生物技术有限公司
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