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Method for Escherichia coli whole cell catalyzed production of phloretin

A technology of Escherichia coli and phloretin, applied in the field of phloretin biosynthesis, can solve problems such as serious pollution and serious chemical pollution, and achieve the effects of good purity, high content and easy production cost

Inactive Publication Date: 2018-03-16
嘉兴欣贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is discharge of chemical waste liquid in this method, and the chemical pollution is serious
In addition, apple peel is used as raw material, and ethanol solution is used as extractant, acid hydrolysis after concentration and then extraction and drying to obtain phloretin. This method adopts acid hydrolysis and serious pollution

Method used

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  • Method for Escherichia coli whole cell catalyzed production of phloretin
  • Method for Escherichia coli whole cell catalyzed production of phloretin
  • Method for Escherichia coli whole cell catalyzed production of phloretin

Examples

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Effect test

Embodiment 1

[0036] Example 1 Plasmid construction method.

[0037] According to the amino acid sequence of the corresponding expressed gene and the codon preference of Escherichia coli, this patent optimizes the nucleic acid sequence of the gene, and sends it to a commercial gene synthesis company to synthesize the expressed gene, and integrates two or more genes into the expression vector pET-28a or pET On -22b, specifically, the expression genes of 4-coumaric acid coenzyme A ligase and chalcone synthase are constructed on the vector pET-28a, as shown in Figure 1(a), phenylalanine ammonia lyase, cinnamon The expression genes of acid-4-hydroxylase and double bond reductase are constructed on the vector pET-22b, as shown in Figure 1(b), the expression genes on the same vector share a T7 promoter, at the end of the expression gene, before the terminator Modify the N-terminal primer, add 6×his gene sequence (CAT CATCAT CAT CAT CAT) to the primer, see Figure 1(c), and connect the expressed ge...

Embodiment 2

[0038] Embodiment 2 Whole cell catalyzes the synthesis of phloretin

[0039] A method for catalyzed production of phloretin by Escherichia coli whole cells:

[0040] 1) 37°C, 220rpm, in LB medium supplemented with corresponding resistance, culture Escherichia coli overnight;

[0041] 2) Transfer to 20mL, 37℃, 220rpm, LB medium, culture to OD 600 = about 1;

[0042] 3) Transfer to 800mL large bottle of 2YT medium, culture at 37°C, 220rpm until OD 600 = about 0.8;

[0043] 4) 16°C, 220rpm, IPTG induction, 16h-18h, harvest bacteria;

[0044] 5) Suspend with phosphate buffer and concentrate 20 times;

[0045] 6) Add the substrate, react at 37° C., 220 rpm for 24 hours, and collect the produced phloretin.

[0046] Note: LB (Luria-Bertani) medium:

[0047] Liquid medium: tryptone: 1%, yeast extract: 0.5%, NaCl: 1%, pH7.5

[0048] Solid medium: add 1.5% agar powder to LB liquid medium

[0049] 2YT medium: tryptone: 1.6%, yeast extract: 1%, NaCl: 0.5%.

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Abstract

The invention relates to a method for Escherichia coli whole cell catalyzed production of phloretin. The synthesis method is characterized in that the production of the phloretin is completed by adopting phenylalanine as a raw material and an Escherichia coli engineering bacterium as a host bacterium through catalysis of multiple enzymes in the host bacterium; and the host bacterium at least can express phenylalnine ammonialyase, cinnamic acid-4-hydroxylase, 4-coumaric acid coenzyme A ligase, double-bond reductase and chalcone synthetase. The method has the following advantages: 1, the phenylalanine is used as the raw material, so the raw material cost of the method is low; 2, catalytic synthesis of biological enzymes in microorganisms is carried out based on microorganism modification, solarge-scale extraction, separation and purification processes are avoided, and the production cost and the environmental protection are easy to control; 3, the size and the quantity of production devices used in the method are less than those of other methods, so the method can be easily industrially converted; and 4, only the final step in the production of the method contains separation and purification processes, so the product purifying process is simple, and the product has better quality and higher content than the product obtained through general methods.

Description

technical field [0001] The invention relates to a biosynthesis method of phloretin, in particular a method for catalyzing the production of phloretin by whole cells of Escherichia coli. Background technique [0002] Phloretin molecular formula: C 15 h 14 o 5 ; English name: Phloretin; CAS: 60-82-2; Molecular weight: 274.28; Physical properties: easily soluble in methanol, ethanol and acetone, slightly soluble in chloroform, insoluble in water, petroleum ether and benzene, square prism or tablet crystals (ethanol or methanol). [0003] Phloretin is a new type of natural skin whitening agent newly researched and developed abroad. It is another natural beauty product following arbutin. It is mainly distributed in the peels and root bark branches and leaves of juicy fruits such as apples and pears. It is mainly used as a natural skin whitening agent. It can inhibit the activity of tyrosinase and melanin, and has a lightening effect on various skin spots. According to the la...

Claims

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Application Information

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IPC IPC(8): C12P7/26C12N1/21C12R1/19
CPCC12N9/0004C12N9/0073C12N9/1037C12N9/88C12N9/93C12P7/26C12Y114/13011C12Y203/01074C12Y403/01005C12Y602/01012
Inventor 陈贤情王筱王文江会锋刘晓楠杨月
Owner 嘉兴欣贝莱生物科技有限公司
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