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Biological synthesis method of xylosic acid

A technology of xylonic acid and xylose, applied in the field of recombinant engineering acceptor bacteria, can solve problems such as restricting application, restricting commercial application, environmental pollution, etc., and achieves the effects of reducing production cost, reducing oxidation, and avoiding reaction conditions

Inactive Publication Date: 2014-06-18
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Traditional xylonic acid is mainly catalyzed by chemical methods to oxidize xylose, and the common ways mainly include iodine catalysis and palladium catalysis: (1) in a strong alkaline environment, using methanol as a solvent and iodine to oxidize xylose can To protect the hydroxyl group from being oxidized, only the aldehyde group is oxidized to the carboxyl group to obtain xylose salt, and concentrated sulfuric acid is added dropwise to the potassium xylate salt dissolved in methanol to obtain xylose crystals; this method requires a large amount of use Iodine and concentrated sulfuric acid are likely to cause serious environmental pollution
(2) Using 5% palladium loaded on activated carbon as a catalyst, under the conditions of temperature 35-50°C and pH=10.0, 4g of catalyst can catalyze xylose acid from an aqueous solution containing 20g of xylose (Chun et al., 2006 Applied Biochemistry and Biotechnology, 6: 654-658); this method can be carried out at room temperature, and the selectivity is good, but it needs noble metal palladium as a catalyst, which limits its commercial application
Liu et al. have made preliminary explorations in the production of xylonic acid using genetically engineered Escherichia coli (2012, Bioresource Technology, 115: 244-248), but the product obtained in this study is xylonolactone instead of xylonic acid, which is restricted. downstream application

Method used

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  • Biological synthesis method of xylosic acid
  • Biological synthesis method of xylosic acid
  • Biological synthesis method of xylosic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] The construction of the co-expression vector of xylose dehydrogenase gene (xylB) and xylonolactonase gene (xylC) is as follows:

[0059] Using oligonucleotides 5'-CAT GCC ATG GGC ATG TCC TCA GCC ATC TATCCC AG-3' and 5'-CGC GGA TCC TCA ACG CCA GCC GGC GTC GAT C-3' as primers, Genomic DNA of Caulobacter crescentus As a template, the xylose dehydrogenase gene (xylB gene) (SEQ ID No: 1) was amplified by the polymerase chain reaction (PCR) method, and NcoI and BamHI restriction enzymes were introduced at the 5' end and 3' end respectively. site, and then clone the gene into the pACYCduet-1 (purchased from Novagen) vector with the above-mentioned restriction site to obtain the recombinant plasmid pA-xylB;

[0060] Use oligonucleotides 5'-GGG AAT TCC ATA TGA CCG CTC AGG TTA CATGCG-3' and 5'-CCG CTC GAG TTAAAC CAG ACG AAC TTC GTG C-3' as primers, and Genomic DNA of Caulobacter crescentus as template , the xylonolactonase gene (xylC gene) (SEQ ID No: 3) was amplified by the pol...

Embodiment 2

[0062] Preparation of recombinant engineered Escherichia coli strains for synthesizing xylonic acid, and using the strain to ferment and convert D-xylose to generate xylonic acid, the specific process is as follows:

[0063] The recombinant plasmid pA-xylBC constructed in Example 1 was extracted by alkaline lysis, and 10 μl of recombinant plasmid pA-xylBC was transformed into Escherichia coli BL21 (DE3) competent cells by the heat shock transformation method, and then 50 μl of the transformed bacterial solution was applied to Cloth contains 34μg·mL -1 Positive clones were screened on chloramphenicol LB plates, and the grown colonies were recombinant E. coli strains co-expressing xylose dehydrogenase and xylonolactonase. The recombinant E. coli strain containing the recombinant plasmid pA-xylBC can be further verified by sequencing.

[0064] Pick a single colony of recombinant Escherichia coli that has been constructed and inoculate it to a concentration of 34 μg·mL -1 Chlora...

Embodiment 3

[0067] Preparation of recombinant engineered Escherichia coli strains for synthesizing xylonic acid, and using the strain to ferment and convert D-xylose to generate xylonic acid, the specific process is as follows:

[0068] The recombinant plasmid pA-xylBC constructed in Example 1 was extracted by alkaline lysis, and 1 μl of recombinant plasmid pA-xylBC was transformed into Escherichia coli BL21 (DE3) competent cells by the heat shock transformation method, and then 100 μl of transformed bacterial liquid was applied to Cloth contains 34μg·mL -1 Positive clones were screened on chloramphenicol LB plates, and the grown colonies were recombinant E. coli strains co-expressing xylose dehydrogenase and xylonolactonase. The recombinant E. coli strain containing the recombinant plasmid pA-xylBC can be further verified by sequencing.

[0069] Pick a single colony of recombinant Escherichia coli that has been constructed and inoculate it to a concentration of 34 μg·mL -1 Chlorampheni...

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Abstract

The invention discloses a method for synthesis of xylosic acid by biological catalysis of D-xylose. The method comprises the following steps: A) constructing a recombinant engineering receptor bacteria strain which is capable of converting the D-xylose to the xylosic acid and contains a xylose dehydrogenase gene xylB gene fragment and a xylose acid lactonase gene xylC gene fragment; and B), using the engineering receptor bacteria in the A for fermentation cultivation in a D-xylose-containing culture medium, using an appropriate inducer for introduction, and separating and purifying to obtain the xylose acid.

Description

technical field [0001] The invention relates to a recombinant engineering acceptor bacterium for synthesizing xylonic acid, preferably a recombinant engineering Escherichia coli for synthesizing xylonic acid. [0002] The present invention also relates to a method for synthesizing xylonic acid by using the above-mentioned recombinant engineered recipient bacteria (preferably, recombinant engineered Escherichia coli). Background technique [0003] Xylose acid is an important chemical intermediate, which can not only be used to synthesize various chemical products, but also be used as a high-efficiency cement binder, and its bonding efficiency is more than twice that of existing binders. In the US Department of Energy report, xylonic acid was listed as one of the 30 most promising biorefinery products. [0004] Traditional xylonic acid is mainly catalyzed by chemical methods to oxidize xylose, and the common ways mainly include iodine catalysis and palladium catalysis: (1) in...

Claims

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Application Information

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IPC IPC(8): C12P7/58C12N15/70C12N1/21C12R1/19C12R1/01
Inventor 咸漠曹玉锦刘炜
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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