Team:Xiamen city/Result
Result
Overview
We purified the superoxide dismutase SOD and catalase CAT proteins we selected, and successfully established the reaction system to detect the enzyme activity in vitro, and characterized their activity. In the same time, we successfully constructed two plasmids: 1) co-expressing SOD and CAT and 2) superficially displaying SOD and CAT and successfully transforming them into Escherichia coli Nissle 1917.
1. Characterization of the biochemical characteristics of SOD.
SOD was expressed in Escherichia coli, bacterial cells were collected and broken, and SOD solution was obtained through isolation and purification, and further confirmed by the SDS-Page method, protein bands of corresponding size were found (Fig.1).
Fig.1 SDS-Page assay the expression of SOD protein
M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole.
We used the classic nitroblue tetrazolium (NBT) color development method. Superoxide anion (O2-.) was produced by Xanthine and Xanthine Oxidase (XO) reaction system to reduce NBT to blue formazan, which had strong absorption at 560nm.While SOD can remove superoxide anions, so dirty formation is inhibited. The bluer the reaction solution is, the lower the activity of superoxide dismutase is, and vice versa. The activity level of superoxide dismutase can be calculated by colorimetric analysis. The detection principle is shown in Fig.2, and the detected absorbance is shown in Table.1.
Fig.2
Table1
The data is substituted into the formula for calculation:
Inhibition percentage=[(Ablank1-Ablank2) - (Asample-Ablank3)]/(Ablank1-Ablank2) * 100%
=69.543%
Enzyme activity of sample=inhibition percentage / (1-inhibition percentage) (units)
=2.283 U
Specific activity of SOD= enzyme activity of sample / amount of protein (units/mg)
=1936.12 U/mg.
The results showed that SOD protein became dissolved in this E. coli expressing condition, and the target protein is very pure. And SOD had excellent catalytic properties, which could successfully degrade ROS into H2O2
2. Characterize the biochemical characteristics of CAT.
CAT was expressed in E. coli, bacterial cells were collected and broken, and the CAT enzyme solution was isolated and purified, and further confirmed by the SDS-Page Method (Fig3).
Fig. 3 SDS-Page assay the expression of CAT protein
M: Protein Ladder; FT: Flow-through sample; W: Washing sample; 50: Elution sample with 50mM imidazole; 100: Elution sample with 100mM imidazole; 250: Elution sample with 250mM imidazole; 500: Elution sample with 500mM imidazole.
When hydrogen peroxide is relatively abundant, catalase can catalyze hydrogen peroxide to produce water and oxygen. The residual hydrogen peroxide can oxidize the color substrate under the catalysis of Peroxidase to produce the red product (N-(4-antipyryl) -3-chloro-5-sulfonate-Pbenzoquinonemonoimine), and the maximum absorption wavelength is 520nm.We used the hydrogen peroxide standard to make the standard curve (Fig4), so that the amount of residual hydrogen peroxide in the sample could be measured, and the catalase catalyzed the conversion of hydrogen peroxide to water and oxygen in unit volume of unit time could be calculated, thus the enzyme activity of catalase in the sample could be calculated.
Fig.4
We added different amounts of enzymes to obtain the maximum rate of CAT catalytic substrates, and we found that the catalytic efficiency of enzymes gradually decreased with the increase of enzyme concentration (Fig5).This indicated that CAT could be well expressed in Escherichia coli, and the purified enzyme also had high catalytic efficiency, which could rapidly transform a large amount of hydrogen peroxide into water and oxygen in a short time.
Future approch
Due to time constraints, we did not fully achieve the final goal of our experiment.
In the following work, we have the following goals:
1) We further determined the engineered Escherichia coli Nissle 1917 for the co-expression of two ROS degrading enzymes, and measured the degradation efficiency of the substrates of the synthetic strains.
2) In intestinal environment simulation, different antioxidants were added to compare with engineered strains to determine the scavenging efficiency of reactive oxygen species by different methods.
3) Further improve the ROS removal efficiency of ROS degradation engineering bacteria through bacterial surface display technology.
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