Latest revision as of 09:11, 12 October 2022

Bromelain from pineapple

Bromelain is a mixture of enzymes derived from pineapple. Its effects are mainly a product of its proteolytic activity, which stimulates fibrinolysis by increasing plasmin.

Bromelain is a group of thiol hydrolytic proteases extracted from the tropical plant pineapple and mainly exists in the fruit, bud, leaf and stem of pineapple, with a molecular weight of 33000. It belongs to the papain family of cysteine proteases, and the enzymatic activity is dependent on the thiol group of a cysteine residue within its active site. Bromelain has a variety of properties, including anti-cancer activity, anti-inflammatory effect, antimicrobial effect, antibiotic potentiation, skin protection, postsurgery recovery and so on. Therefore, it has a wide range of applications in the medical and food fields. We choose fodder processing as the application scenario of the study. Bromelain is a plant protease, which can convert protein in feed into peptides and small peptides easily absorbed by animals, improve the conversion rate of feed, so as to reduce the pollution of breeding industry to the environment. It also has a certain therapeutic effect on diarrhea caused by pathogenic bacteria, avoiding the negative effects of antibiotics, improving the growth performance of animals, and resisting the damage of parasites to animals.

In order to get better bromelain with higher enzyme activity, we carried out directional evolution on the selected target fragments. The screening tool for point mutations was Hotspot Wizard, the 3D structure of the protease was demonstrated by Pymol, and the protease stability after mutation was calculated by RELAX section in R2, an online platform. After Hotspot Wizard is used to screen out the possible mutation sites, pymol is used to mutate them to the amino acids suggested by Hotspot Wizard, and then the mutation with the lowest energy is selected as the target variant. Second mutation is carried out on this variant to obtain our optimal enzyme mutant. Next, we used Molecular Dynamics Simulations to verify the stability of bromelain reaction with BAEE.

Characterization

Molecular Dynamics Simulation Analysis

A molecular dynamics simulation was conducted to analyze the binding stability of Stem Bromelain(212aa) and BAEE complexes, where multiple descriptors were analyzed to understand the flexible and stable nature of the complexes. The system has been balanced in advance (the result of temperature and pressure balance is shown in below(Figure 9,10).

Figure 1: the result of temperature balance

Figure 2: the result of pressure balance

After the whole system was balanced, the molecular simulation started to run, which took 10h (1ns) in total.

RMSD and RMSF analysis were performed on the simulated results, and the results were shown in the figure below. It can be seen that Stem bromelain(212) has good reactivity with BAEE in neutral environment. The RMSD value was less than 0.4, and the RMSD value structure did not change much before and after simulation, indicating that the reaction between the complex was very stable. At the same time, the RMSF value changed greatly, which reflected that the atomic motion of Stem bromelain (212) was relatively free when it reacted with BAEE.

Figure 3: the RMSD of Stem Bromelain(212) and BAEE complex

Figure 4: the comparing RMSD of Stem Bromelain(212) and BAEE complex

Figure 5: the RMSF of Stem Bromelain(212) and BAEE complex

As for the mutant, since it can also dock with BAEE and its binding energy is better than that of Stem Bromelain (212), we predict that its RMSD map fluctuation should be smaller, and the data will be made up in the later molecular simulation.