Difference between revisions of "Part:BBa K4228000"
 
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<span class='h3bb'><h1>Brief introduction</h1></span>
 
<span class='h3bb'><h1>Brief introduction</h1></span>
 
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.  
 
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.
 
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.
  
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<h1>Characterization</h1>
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<b>Molecular Dynamics Simulation Analysis</b>
  
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<h1>Methods</h1>
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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).
  
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<b>Selection of target segments
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[[File:BBa K4228000-picture9.png|center|thumb|600px|Figure 1: the result of temperature balance]]
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</b>
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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）.
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Finally, we chose the stem bromelain sequence containing 212 amino acids as target segment as it was reviewed and thus, more reliable.
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[[File:BBa K4228000-picture10.png|center|thumb|600px|Figure 2: the result of pressure balance]]
  
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<b>Construction of point mutation model
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After the whole system was balanced, the molecular simulation started to run, which took 10h (1ns) in total.
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</b>
+
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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.
+
  
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<b>Synthesis of the target fragment
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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.
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</b>
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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.  
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[[File:BBa K4228000-picture11.png|center|thumb|600px|Figure 3: the RMSD of Stem Bromelain(212) and BAEE complex]]
  
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<b>Molecular Docking Analysis
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[[File:BBa K4228000-picture12.png|center|thumb|600px|Figure 4: the comparing RMSD of Stem Bromelain(212) and BAEE complex]]
  
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</b>
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[[File:BBa K4228000-picture13.png|center|thumb|600px|Figure 5: the RMSF of Stem Bromelain(212) and BAEE complex]]
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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.
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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.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

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.

Brief introduction

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.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 312
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 312
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 312
    Illegal BamHI site found at 1
    Illegal XhoI site found at 646
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 312
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 312
  • 1000
    COMPATIBLE WITH RFC[1000]