Background
We are concerned about the digestion and absorption of flavonoid, which is a healthy compound beneficial in blood circulation and anti-inflammation, in human’s body, thereafter, we have been making every effort to find out possible solutions to enhance the degradation efficiency of flavonoid in human’s body.
Design
According to Yang, G., et al. (2021), flavone reductase (FLR) discovered from Flavonifractor plautii ATCC 49531 (originally assigned as Clostridium orbiscindens DSM 6740) plays a key step in catalyzing flavonoid. Thus, we plan to over-express FLR in Eco. li (BL21) and test its function in degrading flavonoid.
This is the general degradation process of flavonoid, in which FLR serves as a catalyst in the initial step.
This is the plasmid profile we designed to over-express FLR.
The composite part is transcripted by the T7 promoter and stopped by the T7 terminator. Meanwhile, a His protein tag is inserted for future protein purification.
Build
We carried out molecular biology experiments and successfully constructed the composite part above in the vector of Eco. Li (BL21). Also, we obtained the purified protein accordingly.
Test
After obtaining the purified protein containing FLR enzyme, we tested its effectiveness in degrading flavonoid.
We conducted enzyme activity tests by using 4 kinds of flavonoid samples: apigenin, chrysin, luteolin, diosmetin with the initial concentration 10mg/L and the concentration of FLR enzyme was 1mM/L. Each sample was guaranteed three replicates of the enzyme activity test in order to to ensure the credibility of the test results. The test results are listed as follows:
Figure 1. Bar graph of the enzyme activity test results
Learn
According to our test results, the concentration of these 4 flavonoids remarkably decreases after 2 hours which were degraded by the enzyme, even there were only 0.3-5% left after 6 hours. All these results indicate that the FLR enzyme has high activity and is capable of degrading multiple flavonoid with a certain universality. It means that the composite part pET28a-FLR is able to realize the expected function in our design.