Recombinant escherichia coli bacteria producing equol derivative and synthesis method of equol derivative using same
A technology of recombinant Escherichia coli and synthetic method, which can be applied in biochemical equipment and methods, recombinant DNA technology, and the introduction of foreign genetic material by using a carrier, which can solve the problem of not elucidating the physiological activity of 5-hydroxy-equol, and achieve high levels. Added value, high research value, and the effect of reducing production costs
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
preparation example Construction
[0110] Preparation method of recombinant escherichia coli
[0111] After amplifying the DNA sequences corresponding to daidzein reductase, dihydrodaidzein racemase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase by polymerase chain reaction, put them into pRSFDuet or pCDFDuet vector, and expressed in E. coli with His tag (6 histidine).
[0112] Specifically, after transforming the plasmid (plasmid) inserted with the base sequence of the above enzyme into BL21 competent cells, they were cultured in LB solid medium containing antibiotics corresponding to the plasmid in an incubator at 37°C for 12 Hour. One colony was inoculated in 3 mL of LB liquid medium containing antibiotics, and cultured in an incubator at 37° C. at a speed of 200 rpm for 12 hours. 2v% (1 mL) of this culture solution was inoculated into a new LB liquid medium containing 50 mL of antibiotics. If the O.D. (600nm) of the culture solution reaches 0.6-0.8, add 0.1mM isopropylthiogalactopyranoside ...
Embodiment 1
[0117] Example 1: Synthesis of (S)-equol from daidzein using recombinant E. coli expressing all four enzymes involved in equol conversion
[0118] For 0.2 to 5 mM daidzein, the reaction was carried out in the same manner as in Reaction Method 1 using recombinant Escherichia coli expressing the four above-mentioned enzymes of O.D.10. Substrates, intermediates, and products were extracted with more than 4 times of ethyl acetate (hereinafter referred to as "EA"), and after the solvent was removed by a centrifugal decompressor, they were redissolved in a specified amount of methanol and passed through a high-efficiency liquid phase Chromatography confirms the reactivity and concentration of the substance. Such as figure 1 The yield of (S)-equol shown in can be confirmed to be 95% or more at 0.2mM, 80% or more at 0.4mM and 0.6mM, 50% or more at 2mM, and 50% at 5mM. More than 20%.
Embodiment 2
[0119] Example 2: Synthesis of 5-hydroxy-equol or 5-hydroxy-dehydroequol from genistein using recombinant E. coli expressing all four enzymes involved in equol conversion
[0120] For 500 μM genistein ( figure 2 ), the reaction was carried out in the same manner as in Reaction Method 1 using recombinant Escherichia coli expressing the four above-mentioned enzymes of O.D.10. Substrates, intermediates, and products were extracted with more than 4 times of ethyl acetate (hereinafter referred to as "EA"), and after the solvent was removed by a centrifugal decompressor, they were redissolved in a specified amount of methanol and passed through a high-efficiency liquid phase Chromatography confirms the reactivity and concentration of the substance. After 6 hours of reaction, it was confirmed that 500 μM genistein was converted by 99% or more, and 97 mg / L of 5-hydroxy-equol and 22 mg / L of 5-hydroxy-dehydroequol were produced.
[0121] With the composition as described above, 1 mM ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More