Application of polyphenol oxidase and method for synthesizing flavone

A polyphenol oxidase and compound technology, applied in the biological field, can solve problems such as lack and achieve the effect of high thermal stability

Active Publication Date: 2021-07-27
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the research on the flavonoid synthesis pathway in tomato and even Solanaceae still lacks an important link, that is, the key enzyme responsible for the synthesis of flavonoids - flavone synthase (flavone synthase, FNS) has not been reported so far.

Method used

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  • Application of polyphenol oxidase and method for synthesizing flavone
  • Application of polyphenol oxidase and method for synthesizing flavone
  • Application of polyphenol oxidase and method for synthesizing flavone

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment 1, the cloning of tomato SlPPOs gene

[0030] (1) Extraction of tomato total RNA

[0031] The total RNA of tomato was extracted using the RNA kit of Chengdu Befit Technology Co., Ltd. The specific steps are as follows: transfer 40-100 mg of tomato fibrous roots into a 2 mL grinding tube, add steel balls with a diameter of 5 mm, close the lid and freeze in liquid nitrogen for 5 min, then Quickly transfer to a grinder for crushing; then immediately add solution A, vortex and mix well, briefly centrifuge for 5-10s, then add 300μL solution B and 200μL chloroform, vortex for 30s to fully emulsify the solution, room temperature, 12000g, centrifuge for 5min, Transfer 800 μL of supernatant to a new 2 mL centrifuge tube, add an equal volume of solution C, and immediately vortex to mix. Then transfer the above mixture into the RNA adsorption column twice, centrifuge at 12000g at room temperature for 1min, discard the waste liquid in the collection tube; add 700μL solu...

Embodiment 2

[0053] Embodiment 2, construct the Escherichia coli recombinant expression vector and engineering bacteria containing SlPPOs gene

[0054] The amplified SlPPOs gene (DNA sequence shown in SEQ ID NO.1-4) was connected to the vector pDONR207 through the BP reaction in the Gateway cloning technique, and the intermediate vectors obtained were named pDONR207-SlPPO A and pDONR207-SlPPO B respectively , pDONR207-SlPPO D, pDONR207-SlPPO F. Then Sangon Bioengineering (Shanghai) Co., Ltd. sequenced to confirm the correctness of the gene.

[0055] Then the intermediate vectors pDONR207-SlPPO A, pDONR207-SlPPO B, pDONR207-SlPPO D, pDONR207-SlPPO F were respectively connected with the expression vector pDEST17 using the LR reaction in Gateway cloning technology and transformed into Escherichia coli Trans10, positive clones were screened, and then the plasmid was extracted PCR detection was performed to obtain recombinant expression vectors containing four genes of SlPPO A, SlPPOB, SlPPPO ...

Embodiment 3

[0057] Embodiment 3, SlPPOs recombinant protein shows polyphenol oxidase and lyase activity

[0058] Induced expression of SlPPOs recombinant protein: the screened engineered bacteria BL21-pDEST17-SlPPO A / B / D / F were respectively cultured in 5 mL of LB liquid medium containing 50 mg / L Carb (carbenicillin) at 37 ° C and 200 rpm overnight, Then transfer it to 50 mL of the same LB liquid medium and shake it to OD 600 = 0.6-0.9. Then add IPTG (isopropylthio-β-D-galactoside) and CuSO to the bacterial solution 4 Make the final concentration 0.5mM; 20°C, 180rpm induction for 48h, room temperature, 5000g, 10min to collect the bacterial cells, which can be extracted immediately or frozen in -80°C refrigerator for later use.

[0059] Extraction and purification of SlPPOs recombinant protein: add 5mL lysis buffer (50mMNaH 2 PO 4 , 300mM NaCl, 10mM imidazole, pH 8.0), sonicate the bacterial cells (work 3s, interval 6s, 15min). After the bacterial cells were crushed, the supernatant wa...

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Abstract

The invention discloses an application of polyphenol oxidase and a flavone synthesis method, and further limits the application of polyphenol oxidase in catalyzing oxidation of a compound with a structure as shown in a formula 1 to generate a compound with a structure as shown in a formula II or a formula III, and the amino acid sequence of the polyphenol oxidase is as shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8. It is verified that the polyphenol oxidase can be used for converting eriodictyol into luteolin and 5,7-dihydroxychromone, and converting butin into 3',4',7-trihydroxyflavone and 7-hydroxy-4-benzopyrone; and the method is of great significance to research on a tomato flavone biosynthesis mechanism. The invention further verifies that the SlPPO F protein has very high thermal stability, and luteolin is synthesized by using escherichia coli engineering bacteria of the gene, thereby providing a new tool for developing microbial engineering and enzyme engineering.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of polyphenol oxidase, and also to a method for synthesizing flavonoids. Background technique [0002] Flavonoids (flavonoids) are a large class of compounds that exist widely in plants, with various types and complex functions. Flavonoids are divided into six main subtypes: flavones, flavonols, flavanones, isoflavones, chalcones and anthocyanins. Among them, flavonoids are an important subfamily of flavonoids, and their compound structure is characterized by the presence of double bonds between C2-C3. They are widely present in many food-derived plants such as fruits, vegetables, beans and tea in the form of bound (flavone glycosides) or free (flavone aglycones), and can be used in beverages, wines, cakes, baked goods, etc. production. At the same time, it can also be widely used in the medical industry as a medicine, and its economic application value is very hig...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P17/06
CPCC12N9/0059C12Y110/03001C12P17/06Y02A50/30
Inventor 张阳魏硕付饶
Owner SICHUAN UNIV
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