Revision as of 17:19, 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）. 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.