Biological catalysis system for oxidizing phenolic compound and application thereof

A technology of phenolic compounds and catalytic systems, applied in the field of biocatalytic systems, can solve problems such as insufficient mild conditions, pollution, and poor stereoselectivity, and achieve the effects of safe reaction production, simple preparation, and mature technology

Inactive Publication Date: 2017-07-18
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The methods for synthesizing compounds mainly include traditional chemical synthesis and biocatalysis / transformation. Chemical synthesis has the disadvantages of many reaction steps, poor stereoselectivity, not mild enough conditions, easy to produce by-products and serious environmental pollution.

Method used

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  • Biological catalysis system for oxidizing phenolic compound and application thereof
  • Biological catalysis system for oxidizing phenolic compound and application thereof
  • Biological catalysis system for oxidizing phenolic compound and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Construction of CreH, CreI, CreJ, CreE, CreF, CreD, CreG and CreC enzyme expression strains.

[0056] First, according to the corresponding coding genes creH (Ncgl0528), creI (Ncgl0529), creJ (Ncgl0530), creE (Ncgl0525), creF (Ncgl0526), The sequence design primers of creD (Ncgl0524), creG (Ncgl0527) and creC (Ncgl0523) are as follows:

[0057] creH-F: GCGCCATATGGCTAATAAATCTTTTCCCCAAGCCC

[0058] creH-R: ACGCGGATCCGTGGACTAGGCATGTGTATC

[0059] creI-F: TCGCCATATGGGAGACACCATGACCAACAGT

[0060] creI-R: GCCCAAGCTTAACGAGGTAGTACGGGTACA

[0061] creJ-F: CGTGCCATGGGCACAATGACTTCCCAGACT

[0062] creJ-R: CGGGAAGCTTAGCGTTCCAAGTCACGGGAA

[0063] creE-F: GACACATATGAATACTTCAGCTGAAACTGGA

[0064] creE-R: GGAGCTCTCCCAAGCGGGTAAAT

[0065] creF-F: GCGCCATATGAAGATCATGTCTACTATTCATT

[0066]creF-R: TCATAAGCTTTCACACTTGCGTTTCTGGCG

[0067] creD-F: GACACATATGACTCGCAGTAATTTACCCGC

[0068] creD-R: CGGAATTCGAGAAGCACGCCTGGTTG

[0069] creG-F: GACACATATGCCTAGTCCACGCACTGTTC

[0070] creG-...

Embodiment 2

[0075] Protein expression and purification of CreH, CreI, CreJ, CreE, CreF, CreD, CreG and CreC.

[0076] The expression strains obtained above were respectively inoculated in LB containing 50 ug / mL kanamycin at 37° C., 220 rpm, and cultured overnight. Inoculate fresh LB medium (containing 4% glycerol, 50 μg / mL kanamycin) at 1:100 (v / v), culture at 37°C, 220 rpm for 2-4 hours, until OD 600 = 0.4-0.6. Add IPTG with a final concentration of 0.2mM, and culture at 20°C and 220rpm for 18 hours. 4°C, centrifuge at 5000rpm for 5min to collect the cells, and use 50mL of lysis buffer (NaH 2 PO 4 50mM, NaCl 300mM, glycerol 10%, imidazole 10mM) dissolved and mixed with a vortex shaker. The bacteria were ultrasonically lysed with an ultrasonic instrument, the lysate was collected separately, centrifuged at 12000g for 30min at 4°C, and the supernatant was taken. Add 1mL Ni-NTA resin to every 50mL supernatant, and incubate at 4°C for 20min. Then add it to the protein purification colu...

Embodiment 3

[0079] Phosphorylation of p-cresol (4-methylphenol).

[0080] Establish an enzyme reaction system:

[0081] p-cresol 1mM,

[0082] Mg 2+ 20mM,

[0083] mn 2+ 2mM,

[0084] ATP 1mM,

[0085] CreH protein 10 μM,

[0086] CreI protein 10 μM.

[0087] After incubation at 30°C for 30 minutes, p-cresol can be converted into p-cresol phosphorylation product, and the conversion rate is 100%. The reaction formula is as follows:

[0088]

[0089] High-resolution mass spectrometry of p-cresol phosphorylation products see figure 1 , NMR spectrum see figure 2 .

[0090] Among them, CreH and CreI proteins can be replaced by their homologous proteins or functional equivalents, and p-cresol can also be converted into p-cresol phosphorylation products.

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PUM

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Abstract

The invention belongs to the technical field of bioengineering and specifically relates to a biological catalysis system for oxidizing a phenolic compound and an application thereof. The biological catalysis system for oxidizing the phenolic compound at varying degrees is CreH, CreI, CreJ, CreE, CreF, CreD or a functional equivalent of the enzymes. Meanwhile, the added CreC and CreG can enhance the oxidizing process of the phenolic compound. The biological catalysis system provided by the invention can widely and efficiently oxidize various phenolic compounds and has ultrahigh application potential and economic value in the fields of biochemical engineering and so on.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, in particular to a biocatalytic system for oxidizing phenolic compounds and its application. Background technique [0002] Phenolic compounds have a wide range of physiological activities, such as anti-oxidation, scavenging free radicals, anti-ultraviolet radiation, antibacterial effect and anti-viral effect, etc. They also have a wide range of uses in the production of medicines, pesticides, cosmetic raw materials and food additives. People's life and industrial production are closely related (Cuvelier et al., 2014). [0003] As an important production raw material and organic intermediate, phenolic compounds are widely used in various fields, so they have considerable market demand and application value. p-Hydroxybenzaldehyde is a very important fine chemical product, which is widely used in the fields of medicine, spices, pesticides, petrochemicals, electroplating and liquid crystals, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12P7/02C12P7/24C12P7/26C12P7/40
CPCC12N9/00C12P7/02C12P7/24C12P7/26C12P7/40
Inventor 李盛英杜磊刘双江
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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