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Preparation method for D-type rare ketohexose

A rare ketohexose, D-type technology, applied in the field of preparation of D-type rare ketohexose, can solve the problems of low conversion rate, difficult separation and purification of products, etc., and achieve the effect of low production cost, low price and cost reduction

Active Publication Date: 2020-05-19
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the thermodynamic equilibrium involved, the conversion rate is low, and the product is difficult to separate and purify

Method used

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  • Preparation method for D-type rare ketohexose
  • Preparation method for D-type rare ketohexose
  • Preparation method for D-type rare ketohexose

Examples

Experimental program
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preparation example Construction

[0034] The preparation method of D-psicose and D-sorbose of the present invention comprises the following steps: (1) using glycerol and pyrophosphate as substrates, adding L-rhamnosugar-1-phosphate aldolase (L-rhamnosin) -rhamnulose-1-phosphate aldolase, EC4.1.2.19), glycerol phosphate oxidase (L-α-glycerophosphate oxidase, EC: 1.1.3.21) catalase (Catalase, EC: 1.11.1.6), acid phosphatase (acid phosphatase, EC: 3.1.3.2) multi-enzyme catalyst of sugar alcohol oxidase (alditol oxidase, EC: 1.1.3.41) Establish a multi-enzyme reaction system for enzymatic reactions. (2) Separating and purifying the product of the enzyme-catalyzed reaction to obtain the product.

[0035] Wherein, the glycerol concentration in step (1) is 300-800mM; preferably, the glycerol concentration is 500-800mM, most preferably 700mM; the pyrophosphate concentration is 20-100mM; most preferably 40mM; wherein The pyrophosphoric acid is pyrophosphate, preferably disodium dihydrogen pyrophosphate: tetrasodium py...

experiment example 1

[0045] Experimental example 1: In vitro multi-enzyme catalyzed conversion of glycerol to D-psicose and D-sorbose

[0046] Glycerol was converted into D-psicose and D-sorbose ( figure 1 ). These key enzymes include: (1) acid phosphatase (PhoN-Sf, EC: 3.1.3.2), which phosphorylates glycerol to generate DL-3-phosphate glycerol. At the same time, sugar phosphate is dephosphorylated to generate rare ketose; (2) glycerol phosphate oxidase (GPO, EC: 1.1.3.21), which catalyzes DL-3-glycerol phosphate to dihydroxyacetone phosphate (DHAP); (3) sugar alcohol Oxidase (AldO, EC: 1.1.3.41), converts glycerol to D-glyceraldehyde; (4) L-rhamnosan-1-phosphate aldolase (RhaD, EC 4.1.2.19), converts DHAP and D-glyceraldehyde is sugar phosphate; (5) catalase (Catalase, EC: 1.11.1.6), which decomposes hydrogen peroxide into water and oxygen.

[0047] In the present invention, acid phosphatase is derived from Shigella flexneri (Shigelaflexneri), and its gene sequence is numbered CP0190 on KEGG, ...

experiment example 2

[0061] Experimental Example 2: Using L-fucose-1-phosphate aldolase to convert glycerol into D-psicose and D-sorbose in vitro with multi-enzyme catalysis

[0062] L-fuculose-1-phosphate aldolase (L-fuculose-1-phosphate aldolase, EC 4.1.2.17) and L-rhamnosin-1 derived from Thermus thermophilus HB8 -Phosphate aldolase (RhaD, EC4.1.2.19) has the same catalytic activity, so the present invention adds L-fucose-1-phosphate aldolase to the multi-enzyme catalytic system.

[0063] In the present invention, glycerol is converted into D-psicose and D-sorbose ( Figure 5 ). L-fucose-1-phosphate aldolase from Thermus thermophilus (Thermus thermophilus) HB8, its gene sequence number on NCBI is 2827875, codon optimization was carried out according to the codon preference of Escherichia coli ; The corresponding expression vector pET28a-fucA was obtained by molecular cloning. This plasmid was transformed into Escherichia coli expression bacteria BL21 (DE3), and protein expression and purific...

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Abstract

The invention discloses a preparation method for D-type rare ketohexose. The preparation method comprises the steps that glycerin and pyrophosphoric acid are taken as substrates, and a multi-enzyme catalyst containing L-rhamnulose-1-phosphate aldolase, L-alpha-glycerophosphate oxidase, catalase, acid phosphatase and alditol oxidase is added to establish a multi-enzyme reaction system to perform anenzyme catalysis reaction, and products of the enzyme catalysis reaction are separated and purified. According to the preparation method, the glycerin and the pyrophosphoric acid are taken as raw materials instead of expensive D-glyceraldehyde or D-fructose, and therefore, the D-type rare ketohexose is low in production cost, and is suitable for large-scale production. According to the preparation method disclosed by the invention, glycerol kinase and adenosine triphosphate (ATP) are not needed in performing substrate phosphorylation to produce glycerol-3-phosphate without, thus greatly reducing the cost of producing the D-type rare ketohexose.

Description

technical field [0001] The invention belongs to the technical field of enzyme-catalyzed preparation of D-type rare ketohexose, and in particular relates to a preparation method of D-type rare ketohexose. Background technique [0002] Rare ketoses are a class of monosaccharides and their derivatives that exist in nature but at very low levels and have important physiological functions. The D-configuration includes D-sorbose and D-psicose, etc. It has the advantages of low calorie and natural sweetness, and can be used as a functional sweetener. It also has anti-cancer, anti-obesity, neuroprotection, and scavenging free radicals It has broad application prospects in life medicine, cosmetics, diet and other fields. Therefore, it is of great significance to improve the rare sugar synthesis system. [0003] There are two methods for producing D-type rare ketose, chemical synthesis and biotransformation. The traditional chemical synthesis method has cumbersome steps, many side ...

Claims

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Application Information

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IPC IPC(8): C12P19/02
CPCC12P19/02
Inventor 李子杰高晓冬李芬
Owner JIANGNAN UNIV
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