In order to verify the properties of the reaction between bromelain and substrate, BAEE was selected as the substrate for molecular docking, and the RMSD value and RMSF value of the enzyme combined with the substrate were calculated. The results show that the reactivity of bromelain and BAEE is very good, and it can react stably with BAEE under the condition of simulated PH=7.
We tested the activity and stability of the directed evolved enzyme to validate our concept.
In the experiment of formulating the working curve, we configured several standard L-tyrosine with gradient concentrations of 0, 10, 20, 30 and 40 respectively μg/ml; After a thorough reaction with Folin reagent for 20min, microplate reader was applied to measure the absorbance of the system after reaction at the wavelenth680nm. The A values obtained are 0.001, 0.436, 0.801, 1.096 and 1.293 respectively. From this series of data, the following working curve can be obtained:
The formula we applied to is: A=0.03246c+0.076, and the reaction is carried out at 40℃ then the absorbance of the system is measured:
| 40℃ | A1 | A2 | A3 |
|---|---|---|---|
| Wild type | 0.556 | 0.549 | 0.550 |
| p.(S16G) | 0.772 | 0.794 | 0.689 |
| p.(S16G,W67L) | 0.802 | 0.789 | 0.796 |
Taking the wild stem bromelain for an example. The average value of the data is 0.552, and the concentration of L-tyrosine can be calculated as 14.66μg/ml according to the function. The amount of substance consumed in the reaction time is 0.08091μmol per minute. According to the formula provided in GB/T23527-2009:
Where X is the enzyme activity of the sample, A_1 is the enzyme activity of the reaction diluent obtained from the working curve (0.08091μmol here), V_1 is the volume of reaction vessel (15ml here), V_0 is the volume of the reaction system (10ml here), n is the dilution multiple of the sample (2 here), m_0 is the mass of the sample taken (0.5g here), and T is the time consumed (10min here). It can be calculated that the activity of our p.(S16G), p.(S16G,W67L) and wild type are respectively 6900U, 7344U, 4854U per gram of coarse cell extracts in the buffer.
The experimental process is basically consistent with the contents above. The reaction temperature is raised to 50, 55 and 60℃, and three parallel groups are still set at each temperature. After reacting with Folin reagent, the absorbance of the system is measured at 680nm:
| A1 | A2 | A3 | |
|---|---|---|---|
| 50℃ | 0.548 | 0.518 | 0.466 |
| 55℃ | 0.214 | 0.307 | 0.279 |
| 60℃ | 0.085 | 0.079 | 0.096 |
The calculation process will not be repeated. Therefore, the average absorbance values at three temperatures are 0.510, 0.266 and 0.087, and the enzyme activities at these three temperatures are 4412U, 1915U and 1541U per gram of coarse cell extracts in the buffer respectively. The enzyme activity-temperature diagram can be plotted from the above data:
In order to verify the decomposition effect of bromelain on protein, and improve the decomposition ability of bromelain by our improvement, we designed polyacrylamide gel electrophoresis experiment to extract the soluble protein in the treated soybean meal, observe the bands, and analyze the distribution of protein sub components with molecular weight less than 30 kDa (analyze the hydrolysis ability of bromelain on the sensitive protein in soybean meal). Because the lab was closed in the middle of the experiment due to the epidemic, we did not get the final results. You can check the detailed plan in Design Page.
