Revision as of 18:04, 11 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.

Methods

Selection of target segments

The AlphaFold2 platform was used to search for bromelain. Two different stem bromelain sequences were found, one with 212 amino acids and the other with 291 amino acids. So we must choose which one to use as the target sequence, and this selection process requires comparing the two sequences. The global protein sequence alignments of these two sequences were performed by Clustal W. The 291-amino acid stem bromelain sequence had extra 122 amino acids at the beginning and the 212-amino acid stem bromelain sequence had extra amino acids at the end. However, there was a length of constant amino acids in the middle of both sequences that are highly similar. Clustal W showed that the amino acids in this region are extremely conserved. At the same time, 291 and 212 structure comparison analysis was conducted. The RMSD value was 0.42. It could be seen that they were highly similar in structure, which also indicated that both of them were stem bromelain. Their structure was shown below（Figure 1）.

Figure1

Finally, we chose the stem bromelain sequence containing 212 amino acids as target segment as it was reviewed and thus, more reliable.

Construction of point mutation model

In order to enhance bromelain 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 2.4, 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 2.4 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.

Synthesis of the target fragment

Our target segment was synthesized by our company (Engines, Nanjing). The company also performed two point mutations (serine at position 16 and leucine at position 67) and codon optimization for Bacillus coli.

Molecular Docking Analysis

The simulation of molecular docking was performed on the Hdock protein-protein docking server. The docking results were visualized in the Hdock to confirm the binding position of bromelain and the BAEE.

Molecular Dynamics (MD) Simulation

The dynamic behavior of Stem Bromelain (212aa) and BAEE complexes were studied by MD simulation performed on Gromacs.2020-fosscuda-2019b using the CHARMM36 all atom force field. The solvation of the system was done by using the TIP3P water model with a margin of 1Å. The system was neutralized by the addition of ions(NA and CL). Thenceforward, a consecutive minimization step was performed before the long MD simulation. 50000 steps were submitted and atom coordinates for the whole system were restrained to their initial coordinates with a force constant of 1000 KJ/mol Å−2. Before starting the molecular simulation, the temperature and pressure balance of the whole system was necessary. (Each balancing process took 50,000 steps.) The pdb2pqr module of Gromacs was used for protein-protein complex preparation and the gmx programs was used for counter ion addition, solvation, and preparation of topology files. Gromacs.2020-fosscuda-2019b on Beikun Cloud supercomputing platform was used for MD simulation production and trajectory processing.

Results

Search for mutable sites to obtain superior variants

Table 1 and 2 show the point mutation sites provided by Hotspot Wizard. In the search for point mutations, reference was made to the original analysis of residue 291. All conserved residues in enzymes are composed of polar, nonpolar, acidic and basic groups. Most of the conserved residues, such as Tyr, Asn, Gly, Thr, Gln and Cys, belong to polar groups, followed by non-polar groups (Phe, Trp, Ala, Val and Pro), acidic groups (Glu and Asp) and basic groups (Arg and His). So we evaluated some functional residues as well as other residues.

表1

表2

Rosseta's Relax module was used to score these point mutation models. The initial model energy was calculated as -607.691. The energy of No. 18 Arg mutation was -332.407 and the structure was disorganized（Figure 2）, so the mutation scheme has been abandoned. The possible reason was that site 18 was mutated into Arg, and the surrounding area was positively charged, while Arg was negatively charged in the past, and the energy was too high, so the structure appeared to fall apart.

Figure2

The energy of the Gln mutation 18 was -605.077. Although its structure did not fall apart, its energy increased and thus it was abandoned. The structure of Arg 20 was disintegrated after mutation, so it was abandoned too. (Figure 3)

Figure3

However, the energy of Gly 16 was -610.561 after mutation. Meanwhile, compared with the structure before mutation, its RMSD value was 0.683, so it was selected for further mutation.

Figure4

Finally, we screened the model with Leu mutation at site 67, and its energy was -614.826. Compared with the original model, the RMSD value was 0.97. Its reduced energy and relatively stable structure suggested that it might be a better variant of bromelain, as the experiments would prove.

Exploration of upper relationship between residues of stem bromelain

The definition of upper relationship means that some single mutations of A are unfavorable, but the addition of B mutation will have better performance than single B mutation, which means that B has upper relationship with A. After calculating by Relax, we found that the Gly single mutation at site 16 had an upper relationship with the Leu single mutation at site 67: when Leu single mutation was detected, the structure was disordered, RM value was 1.4, and energy was -348. But with the addition of Gly, the structure stabilizes and the energy drops to -614.82. At the same time, the single mutation of Gly at site 16 has an upper relationship with the single mutation of Val at site 64: the energy of single mutation of Val is -599, while the energy of mutation of both Gly and Val decreases to -608.

Substrate docking of stem bromelain and its variant

After literature review and network search, BAEE was finally determined as the reaction substrate of bromelain, and its docking and energy calculation were performed with bromelain and mutated variants. The calculations were done online by Hdock.

Finally, the binding energy of bromelain in wild-type was -125.09, while that in mutant was -126.26, which decreased. This also proves that the stem bromelain variant we developed is more easily bound to the substrate and its reactivity is enhanced（Figure 5, 6, 7 and 8）

Figure5

Figure6

Figure7

Figure8

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 9: the result of temperature balance

Figure 10: 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 11: the RMSD of Stem Bromelain(212) and BAEE complex

Figure 12: the comparing RMSD of Stem Bromelain(212) and BAEE complex

Figure 13: 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.