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Method for preparing 4-ethylphenol from glycerol

A technology of ethyl phenol and glycerol, applied in the field of bioengineering, can solve problems such as unfavorable environment, no discovery of 4-ethyl phenol metabolic pathway, complicated process, etc., and achieves less environmental pollution, important economic value and social benefit, and mild conditions. Effect

Active Publication Date: 2020-06-12
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current industrial production of 4-ethylphenol only relies on the petrochemical industry and mainly includes three steps, namely sulfonation of ethylbenzene to produce ethylbenzenesulfonic acid mixture, separation of p-ethylbenzenesulfonic acid and finally fusion with alkali to obtain 4-ethylbenzene based phenols, but the process is complex and environmentally unfriendly
Therefore, biotechnological approaches would be an alternative for the sustainable production of 4-ethylphenol, but no metabolic pathways for microbial biosynthesis of 4-ethylphenol from simple carbon sources have been identified so far.

Method used

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  • Method for preparing 4-ethylphenol from glycerol
  • Method for preparing 4-ethylphenol from glycerol
  • Method for preparing 4-ethylphenol from glycerol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Construction of Escherichia coli recombinant plasmid pETDuet-tal-pad-vpr

[0036] The primer sequences are shown in Table 1, where the underlined part is the restriction site.

[0037] Table 1 The primers used in this embodiment

[0038]

[0039]

[0040] The recombinant plasmid pETDuet-tal-pad-vpr contains three genes, namely setal, bapad and lpvpr, each gene is controlled by an independent T7 promoter and sugar body binding site RBS, the main construction steps are as follows:

[0041] 1) Using pET21b-setal as a template, using setal-F and setal-R as primers, perform PCR amplification on the sequence of setal. The setal-PCR fragment was cloned into the vector pETDuet-1, and the restriction sites were SacI and NotI to generate pETDuet-setal.

[0042] 2) Using pET28a-bapad as a template and using bapad-F and bapad-R as primers to perform PCR amplification on the sequence of bapad. The bapad-PCR fragment was digested with NdeI and XhoI and cloned into ...

Embodiment 2

[0045] Expression and purification analysis of three kinds of enzymes in the recombinant bacterial strain of embodiment 2

[0046] The constructed recombinant plasmid pETDuet-tal-pad-vpr was transformed into Escherichia coli competent E.coli TransB(DE3) to obtain the recombinant strain E.coli TransB-TPV. Recombinant bacteria were cultured overnight at 37°C in 4 mL of LB medium, then inoculated into 50 mL of LB medium at a 2% inoculum size, and osmotic agents (1M sorbitol and 2.5mM betaine) were added to the medium to increase enzyme activity. Soluble expression. Cultured at 37℃ to OD 600 When the temperature was 0.8, IPTG with a final concentration of 0.2mM was added, and the cells were collected after induction at 20°C for 16h. The collected cells were centrifuged at 8,000 rpm for 10 min at 4°C, the supernatant was removed, and 5 mL of Lysis buffer (50 mM NaH 2 PO 4 , 300mM NaCl, pH8.0), and vortexed to mix evenly. Then, the cells were disrupted with an ultrasonic disrup...

Embodiment 3

[0048] Shake flask fermentation culture of embodiment 3 recombinant strains

[0049] (1) Effect of host bacteria and penetrant on the production of 4-ethylphenol

[0050] The recombinant plasmid pETDuet-tal-pad-vpr was transformed into Escherichia coli E.coli BL21(DE3) and E.coliTransB(DE3) respectively to obtain recombinant bacteria E.coli BL21-TPV and E.coli TransB-TPV. The recombinant bacteria were cultured overnight at 37°C in 4 mL of LB medium, and then inserted into 100 mL of TB medium at an inoculum size of 2%. The TB medium was divided into two parts, one with osmotic agent added (1 M sorbitol and 2.5 mM betaine) and the other without osmotic agent added. Cultured at 37℃ to OD 600 When it is 0.8, add IPTG with a final concentration of 0.2mM, and culture at 20°C for 72h. The concentration of product 4-ethylphenol was measured by sampling every 12h.

[0051] Such as figure 2 As shown, after shaking the flask for 72 hours, the 4-ethylphenol production of the recombi...

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Abstract

The invention discloses a method for preparing 4-ethylphenol from glycerol, and belongs to the technical field of bioengineering. According to the method, a biosynthetic route of 4-ethylphenol is constructed in escherichia coli; the method comprises the following steps: taking the glycerol as a substrate, synthesizing tyrosine in the escherichia coli through a glycolysis way and a shikimic acid way, then converting the tyrosine into p-coumaric acid under the catalytic action of tyrosine ammonia-lyase, and then generating 4-vinylphenol from the p-coumaric acid under the catalytic action of phenolic acid decarboxylase; and finally, under the catalysis of phenol reductase, converting the 4-vinylphenol into the 4-ethylphenol. The invention provides a new 4-ethylphenol production way, and the method has the advantages of low production cost, low production energy consumption, small environmental pollution and the like, lays a foundation for large-scale industrial application in the future,and has important economic value and social benefit.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a method for preparing 4-ethylphenol from glycerin. Background technique [0002] Due to the depletion of fossil fuel resources and concern for the sustainable development of the traditional petrochemical industry, biorenewable fuels and chemicals have received increasing attention. With the development of metabolic engineering and the discovery of new metabolic pathways, compounds that were previously difficult to obtain from nature can be obtained through microbial biosynthesis. Compared with the traditional chemical synthesis method, the biosynthesis method has the advantages of mild reaction conditions, good substrate affinity, and no impact on the environment, and the products synthesized by the biosynthesis method can be regarded as natural products, which can be directly used in food and medicine industry. In recent years, many researchers have established artific...

Claims

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Application Information

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IPC IPC(8): C12P7/22C12N15/60C12N15/53C12N15/70C12R1/19
CPCC12P7/22C12N9/88C12N9/001C12Y403/01023C12N15/70
Inventor 丁少军张莹
Owner NANJING FORESTRY UNIV
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