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Preparation method of sedoheptulose and aldose in immobilized enzyme cascade reaction

A technology of sedum heptulose and sedum aldoheptulose, applied in the direction of immobilized enzymes, biochemical equipment and methods, enzymes, etc., can solve the problems of expensive raw materials, low product purity, and low reaction efficiency

Active Publication Date: 2018-07-13
SHENZHEN READLINE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The raw materials of the method adopted in the prior art are expensive, the reaction efficiency is low, and the product purity is not high

Method used

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  • Preparation method of sedoheptulose and aldose in immobilized enzyme cascade reaction
  • Preparation method of sedoheptulose and aldose in immobilized enzyme cascade reaction
  • Preparation method of sedoheptulose and aldose in immobilized enzyme cascade reaction

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1 fermentation production enzyme

[0079] Fermentative production of erythrulose activating enzyme (EK), erythrulose phosphate isomerase TRI, erythrulose phosphate isomerase RPI, aldolase (FSA), sedoheptulose-7-phosphate iso Constructase (SPI), Sedoheptose-7-phosphate activating enzyme (SPK), Sedoheptose-1,7-bisphosphate hydrolase (SPH), Inorganic phosphohydrolase (IPH) and ATP regeneration enzyme (PPK).

[0080] EK, TRI, and RPI were amplified by PCR using the extracted DNA of Escherichia coli BL21 strain (purchased from General Biotech) and the chromosomes of Saccharomyces cerevisiae and Bacillus bronchiseptica purchased from ATCC as templates. , FSA, SPI, SPK, SPH and IPH gene fragments, wherein the amplification primers used are as follows:

[0081] Gene sequence amplification primers:

[0082] EK forward primer: 5'-GAAGGACGGACATATGACGTACCTCCTGAAC-3'SEQ ID No.1

[0083] EK reverse primer: 5'-GTCGGTCGGGCCTCGAGCCGGGTGAGGTGC-3'SEQ ID No.2

[0084] TRI f...

Embodiment 2

[0099] Example 2 Enzyme immobilization on the carrier

[0100] Isomerase (TRI, erythrulose phosphate isomerase RPI, SPI), activating enzyme (EK, SPK), phosphohydrolase (SPH, IPH) and aldolase (FSA) cell crushing supernatant were added gradually 65% saturated ammonium sulfate was precipitated, and then the solid was slowly dissolved in 25mM Tris buffer solution with pH 8.0, desalted through a G25 size exclusion column (purchased from Sigma) and then used DEAE Seplite FF (Xi’an Lanxiao Company) for anion exchange Column separation to obtain primary purified EK, TRI, RPI, FSA, SPI, SPK, SPH, IPH enzymes, all enzymes are immobilized individually or mixed with LX-1000EP epoxy resin (Xi’an Lanxiao Company) according to the following method: 1000U purified enzymes Dissolve in 1L 100mM potassium phosphate solution with pH 8.0, then add 60mM phenoxyacetic acid and 200g LX-1000EP epoxy resin to the buffer, stir at room temperature for 24 hours, filter out the immobilized enzyme, and fin...

Embodiment 3

[0101] Example 3 Preparation of D-Sedum heptulose-7-phosphate with immobilized enzyme

[0102] Using immobilized isomerase TRI, erythrulose phosphate isomerase RPI, immobilized activating enzyme (EK), immobilized transketolase (FSA), immobilized ATP regeneration enzyme (PPK) and starting material L- Preparation of D-sedoheptulose-7-phosphate by one-pot method of erythrulose and 1,3-dihydroxyacetone; separation and purification of D-sedoheptulose-7-phosphate;

[0103] Add 9.0 g of L-erythrulose (75 mM), 7.2 g of 1,3-dihydroxyacetone (80 mM), and 551 mg of adenosine triphosphate to 1 L of 25 mM pH 7.0 tris-hydrochloride (Tris.HCl) solution Disodium salt ATP (1mM), 7.6 grams of polyphosphoric acid (Sigma, 25 poly, 74mM phosphoric acid), 2.8 grams of magnesium chloride (30mM), 1.5 grams of potassium chloride (20mM); after the pH value was adjusted to 7.0, 500U was immobilized Activating enzyme (EK), 800U immobilized isomerase (TRI, RPI), 1200U immobilized aldolase (FSA) and 1500U...

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Abstract

The invention relates to a preparation method of sedoheptulose and aldose in an immobilized enzyme cascade reaction, and specifically discloses a method for preparing sedoheptulose and aldose in the immobilized enzyme cascade reaction. The method comprises the following steps: taking L-erythrulose and 1,3-dioxyacetone as starting materials, and preparing D-sedoheptulose-7-phosphoric acid by usinga one-pot method of immobilized kinase EK, immobilized isomerase TRI, erythrulose phosphoric acid isomerase RPI, immobilized aldolase FSA and immobilized ATP regenerated enzyme PPK; and further generating sedoheptulose and aldose. The method is high in preparation efficiency and cheap in raw materials, and is applicable to large-scale industrial production, so that the preparation cost is greatlyreduced.

Description

technical field [0001] The invention relates to a preparation method of sedoheptulose and aldose, in particular to a method for preparing sedoheptulose and aldose by using immobilized enzyme cascade reaction. Background technique [0002] Sedoheptulose jing-tian geng ketone sugar / aldose is a monosaccharide with molecular formula C 7 h 14 o 7 , The molecular weight is 210. Sedoheptulose was discovered by LaForge and Hudson in Sedum spectabile, a plant of Sedum spectabile. It is one of the few seven-carbon sugars in nature. It is an important primary and secondary metabolite in the synthesis of shikimic acid and aromatic ammonia. Although a large number of documents have reported the important physiological functions of sedoheptulose / aldose in organisms, the high price has seriously restricted the further application and promotion of this seven-carbon sugar; It becomes very important to reduce the application cost of heptulose / aldose method. [0003] The conventional pre...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12P19/24C12N11/00
CPCC12N11/00C12P19/02C12P19/24
Inventor 黄华何遂平张新帅王俊梅刘建
Owner SHENZHEN READLINE BIOTECH CO LTD
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