Preparation method for L-type rare ketohexose

A kind of rare ketohexose, L-type technology, applied in the field of preparation of L-type rare ketohexose, can solve the problems of low conversion rate, difficult separation and purification of products, etc., achieve low production cost, reduce the use of NAD+, and low price Effect

Active Publication Date: 2020-05-01
JIANGNAN UNIV
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  • Abstract
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Problems solved by technology

However, due to the thermodynamic equilibrium involved, the convers

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

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preparation example Construction

[0046]The preparation method of L-fructose of the present invention comprises the following steps: (1) using glycerol and pyrophosphate as substrates, adding L-rhamnulose-1-phosphate aldolase (L-rhamnulose-1-phosphate aldolase, EC 4.1.2.19), 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 catalysts of horse liver alcohol dehydrogenase (horse livercohol dehydrogenase, EC: 1.1.1.2), NADH oxidase (NADH oxidase, EC: 1.6.3.4) to establish a multi-enzyme reaction system for enzymatic reactions; (2 ) to separate and purify the enzyme-catalyzed reaction product.

[0047] Wherein, the glycerol concentration in step (1) is 300-900mM; preferably, the glycerol concentration is 500-800mM, most preferably 800mM; the pyrophosphate concentration is 5-125mM, preferably, the pyrophosphate The concentration is 20-100mM; the most preferred is 40mM; wherein the pyrophosphate ...

experiment example 1

[0050] Experimental example 1: In vitro multi-enzyme catalyzed conversion of glycerol to L-fructose

[0051] Horse liver alcohol dehydrogenase can specifically oxidize glycerol to generate L-glyceraldehyde, without producing other enantiomers, and can be used for L-glyceraldehyde production. However, NAD is required for its production + , the production cost is higher. NADH oxidase can transfer electrons from NADH to oxygen to generate H2O. This study is the first to combine horse liver alcohol dehydrogenase and NADH oxidase at low concentrations of NAD + L-GA is produced under the action.

[0052] The 0.5mL reaction system contains 50mM Tris-Hcl (pH7.5), the dosage of horse liver alcohol dehydrogenase is 16U / mL, the dosage of NADH oxidase is 32U / mL, glycerol 100mM and NAD + 1 mM; another reaction without NADH oxidase served as a control. The amount of D-glyceraldehyde in the reaction solution was measured by HPLC at 6h of reaction. The result is as figure 1 As shown, t...

experiment example 2

[0057] Experimental example 2: In vitro multi-enzyme catalyzed conversion of glycerol into L-fructose and L-tagatose

[0058] Conversion of glycerol to L-fructose and L-tagatose ( Figure 5 ). 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 ketohexose; (2) glycerol phosphate oxidase (GPO, EC: 1.1.3.21), which catalyzes DL-3-glycerol phosphate to dihydroxyacetone phosphate (DHAP); (3) liver Alcohol dehydrogenase (horse liver alcohol dehydrogenase, EC: 1.1.1.2), converts glycerol into L-glyceraldehyde; (4) L-fucose-1-phosphate aldolase (FucA, EC 4.1.2.17) , will convert DHAP and L-glyceraldehyde into sugar phosphate; (5) catalase (Catalase, EC: 1.11.1.6), decompose hydrogen peroxide into water and oxygen; (6) NADH oxidase (NADH oxidase, EC:1.6.3.4), convert NADH to NAD + .

[0059] L-fucose-1-phosphate aldolase der...

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Abstract

The invention discloses a preparation method for L-type rare ketohexose. The preparation method comprises the following steps: with glycerol and pyrophosphoric acid as substrates, adding aldolase containing L-rhamnose-1-phosphate, glycerophosphate oxidase, catalase, acid phosphatase, equine dehydrogenase and NADH oxidase into the glycerol and pyrophosphoric acid so as to establish a multi-enzyme reaction system, and carrying out enzyme catalytic reaction; and separating an enzyme catalytic reaction product, and carrying out purifying. According to the invention, raw materials are low in price;glycerol triphosphate is produced without glycerol kinase and ATP for substrate phosphorylation; the cost of producing the L-type rare ketohexose is greatly reduced; equine dehydrogenase and NADH oxidase are combined to produce L-glyceraldehyde; and use of NAD+ is reduced.

Description

technical field [0001] The invention belongs to the technical field of enzyme-catalyzed preparation of L-type rare ketohexose, and in particular relates to a preparation method of L-type rare ketohexose. Background technique [0002] Rare ketohexoses are a class of monosaccharides and their derivatives that exist in nature but at very low levels and have important physiological functions. The L-configuration includes L-sorbose, L-fructose and L-tagatose, 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, Physiological activities such as scavenging free radicals have broad application prospects in the fields of life medicine, cosmetics, and diet. Therefore, it is of great significance to improve the rare sugar synthesis system. [0003] There are two methods for producing L-type rare ketohexose, chemical synthesis and biotransformation. The traditional chemical ...

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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