Method for preparing glucaric acid through whole-cell transformation

A technology for whole-cell transformation and glucaric acid, which is applied in the field of whole-cell transformation to prepare glucaric acid, can solve the problems of expensive catalyst, high nitric acid oxidation process cost, low yield, etc. Single ingredient and mild production process

Inactive Publication Date: 2020-08-28
UNIV OF JINAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the nitric acid oxidation process has high cost, harsh conditions, high energy consumption, high cost, low yield and poor safety, so it cannot be used in food, medicine and other fields.
Kochkar H and Lassalle L (Kochkar H, Lassalle L, Morawietz M, et al.Regioselective oxidation ofhydroxyl groups of sugar and its derivatives using silver catalysts mediated by TEMPO and peroxodisulfate in water.J Catal,2000,194(2):343–351 .) reported the use of 2,2,6,6-tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated electrochemical oxidation of glucose to synthesize glucaric acid, although the TEMPO oxidation method is relatively mild, but the catalyst It is expensive and difficult to carry out large-scale production. Therefore, the preparation of glucaric acid by microbial fermentation is more and mor

Method used

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  • Method for preparing glucaric acid through whole-cell transformation
  • Method for preparing glucaric acid through whole-cell transformation
  • Method for preparing glucaric acid through whole-cell transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the construction of genetically engineered bacteria

[0031] The Udh genomes derived from Pseudomonas synxantha strain KENGFT3 (Pseudomonas synxantha strain KENGFT3) and Pseudomonas fragilis P121 (Pseudomonas fragi strain P121) were respectively used as templates for PCR amplification reactions, and the amplified fragments were used with SalI, NotI and NdeI, XholI endonuclease was connected to the expression vector pETDuet-1 to construct the plasmid pETDuet-2×Udh, primers F1, R1, F2, R2, the primers are as follows

[0032] F1: 5'-ATGCGTCCGGCGTAGA-3'

[0033] R1: 5'-GATTATGCGGCCGTGTACAA-3'

[0034] F2: 5'-TTGTACACGGCCGCATAATC-3'

[0035] R2: 5'GCTAGTTATTGCTCAGCGG3'

[0036] ①Transform the expression vector into the host strain E.coli BL21(DE3), smear / streak the recombinant strain on a plate containing ampicillin resistance, select positive clones, and verify by extracting the plasmid for enzyme digestion and electrophoresis , to obtain the recombinant Esc...

Embodiment 2

[0039] Example 2: High-density fermentation culture of recombinant Escherichia coli pETDuet-2×Udh

[0040] Seed medium (LB): yeast extract powder 5g / L, tryptone 10g / L, sodium chloride 10g / L.

[0041] Fermentation medium: glucose 25g / L, tryptone 7g / L, ammonium sulfate 3.5g / L, magnesium sulfate 0.55g / L, potassium dihydrogen phosphate 3.5g / L, dipotassium hydrogen phosphate 3.5g / L.

[0042] In Example 1, single colonies were picked from the LB plate containing ampicillin antibiotic resistance, inoculated into a 25mL test tube containing 5mL of medium, 37°C, 220rpm, and cultivated for 12h to obtain the seed solution of recombinant Escherichia coli; by 1% The amount of inoculum inoculated the seed liquid into a 1L shake flask with 250mL of fermentation medium, the concentration of ampicillin finally reached 100μg / mL, adjusted the pH to 7, and continued to cultivate at 37°C and 220rpm until OD 600 When the value reaches 0.6, add IPTG to make the final concentration reach 0.4mM, cult...

Embodiment 3

[0043] Embodiment 3: the method for preparing glucaric acid by transformation of whole cells

[0044] The recombinant Escherichia coli fermentation broth obtained in Example 2 was centrifuged at 6000 rpm for 15 minutes to recover the Escherichia coli sludge I, resuspended the bacteria sludge I with the washing liquid and centrifuged again to obtain the bacteria sludge II; take 5-10 g of the bacteria sludge II and add 1000 mL The reaction solution (in the reaction solution, MOPS is 20.9g, and the initial amount of glucuronic acid is 2g) is placed in a 2.5L shake flask, and the reaction is stirred at a low speed at 37°C for 24 hours. During the reaction, the decrease of the substrate glucuronic acid is detected. Amount, after the reaction finishes, detect the content of glucaric acid in the conversion solution by MS and HPLC, the result is: the conversion solution containing glucaric acid is 1.5g / L.

[0045] The HPLC method for detecting glucaric acid in the conversion solution ...

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PUM

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Abstract

The invention discloses a method for preparing glucaric acid through whole-cell transformation, and belongs to the technical field of biology. Escherichia coli is used as a production strain to heterologously express glucuronic acid dehydrogenase from different sources, escherichia coli cells with protein overexpression are obtained through high-density fermentation culture, and glucuronic acid isused as a substrate to produce glucaric acid. A conversion liquid has the advantages of single component, short production period, simple operation, low production cost, no potential safety hazard and the like.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing glucaric acid by transforming whole cells. Background technique [0002] Glucaric acid (Glucaric acid, referred to as GA) is a non-toxic glucose derivative, which naturally exists in apples, citrus and other fruits and broccoli, cabbage and other cruciferous vegetables, and is also secreted in a small number of mammals and humans ( Qiu Yuying, Fang Fang, Du Guocheng, etc. Research progress of glucaric acid[J]. Chinese Journal of Bioengineering, 2015,31(4):481-490). Glucaric acid was rated as "the most valuable biorefinery product" by the U.S. Department of Energy and can be used as a raw material to produce a variety of polymer intermediates and bio-new energy (Yuan Haibo, Li Jianghua, Liu Long, etc. Based on systems biology and synthesis Research progress in biomanufacturing of important platform chemicals in biology [J]. Acta Chemical Society, 2016, 67(21): ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12N15/70C12P7/58C12R1/19
CPCC12N9/0006C12N15/70C12P7/58C12Y101/01203
Inventor 叶春江牛林林董启圣赵晓畅
Owner UNIV OF JINAN
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