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Method for enzymic synthesis of kaempferol

A technology of fusion protein and enzyme cleavage site, applied in chemical instruments and methods, biochemical equipment and methods, enzymes, etc., can solve the large metabolic burden of host bacteria, inhibit the growth of host bacteria, and coordinate expression without rational regulation methods, etc. problems, to achieve the effect of low cost, short cycle and easy operation

Active Publication Date: 2018-04-06
YANGZHOU UNIV
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Problems solved by technology

However, there are still many shortcomings in the production of flavonoids by microbial fermentation
First of all, the key enzymes involved in the synthetic pathway and the intermediate products produced during the synthetic process will inhibit the growth of the host bacteria
Secondly, the expression of multiple genes is involved in the early stage of bacterial growth, and the coordinated expression between genes lacks a rational regulation method, which brings a large metabolic burden to the host bacteria

Method used

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  • Method for enzymic synthesis of kaempferol
  • Method for enzymic synthesis of kaempferol
  • Method for enzymic synthesis of kaempferol

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Embodiment

[0047] 1. Cloning, induced expression and purification of key enzyme genes in the biosynthetic pathway of kaempferol

[0048]1.1 Cloning of flavanone-3-hydroxylase (F3H) gene f3h: design PCR amplification primers, the forward primer is The base in italics indicates the enzyme cutting site BamHI, and the reverse primer is Bases in italics indicate the enzyme cutting site EcoRI. The f3h gene (gene accession number: NM_001203121.1) was cloned from Arabidopsis thaliana into the prokaryotic expression vector pET-32a, and the recombinant plasmid pET-32a-f3h was constructed.

[0049] 1.2 Cloning of flavanone synthase (FLS) gene fls1: design PCR amplification primers, the forward primer is The base in italics indicates the enzyme cutting site BamHI, and the reverse primer is Bases in italics indicate the enzyme cutting site EcoRI. The fls1 gene (gene accession number: NM_120951.3) was cloned from Arabidopsis thaliana into the prokaryotic expression vector pET-32a, and the ...

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Abstract

The invention discloses a method for enzymic synthesis of kaempferol. Preparation and detection of enzyme activity of two kinds of fusion protein, TrxA-His-F3H and TrxA-His-FLS1 are involved, and a buffer solution system is designed, wherein the reaction buffer solution is 0.1 M of Tris-HCl with a pH value of 7.2, and every 100 muL of a reaction system contains 0.4% of ascorbic acid, 10% of glycerol, 8.2 mM of alpha-ketoglutaric acid, 0.01 mM of ferrous sulfate, 0.5 mM of naringenin, 3.3 umg of TrxA-His-F3H and 1.8 umg of TrxA-His-FLS1. The method for efficient one-step synthesis of kaempferolis established in vitro by optimizing the pH value, reaction time and temperature of the reaction system, and a CCK-8 method is applied to the detection of the inhibiting effect of the synthesized kaempferol on the growth of tumor cells.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for enzymatically synthesizing kaempferol. Background technique [0002] Kaempferol is a secondary metabolite widely found in fruits, vegetables and Chinese herbal medicines—flavonols, which has various biological functions such as anti-inflammation, anti-oxidation, anti-tumor, and antibacterial, and is safe and non-toxic. The field of food and medicine has a good application prospect (Zhang Yawen et al. Research progress on the biological function of kaempferol. Life Science, 2017; 29(4):400-5). [0003] At present, the common preparation method of kaempferol is the organic solvent extraction method, but the method steps are cumbersome and complicated, the cycle is long, the yield is low, the cost is high, a large amount of organic solvents are needed, and the source of raw materials is easily restricted by seasons, so Not conducive to industrialized pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N9/02C12N9/10C12N15/70C12P17/06
CPCC07K2319/21C07K2319/35C12N9/0071C12N9/1029C12N15/62C12N15/70C12P17/06C12Y114/11009C12Y203/01041
Inventor 张新跃张智萍何妍之黄岳陈磊丁笠刘雅娴
Owner YANGZHOU UNIV
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