Team:HK SSC/Poster

Poster: HK_SSC



Production of PdRp-CBD complex for recovery of palladium from palladium(II) ions

Presented by team HK_SSC 2020

Student Leaders:

Young Hong Yui,

Poon Curtis Long Yiu

Student Members:

Wong Hoi Ling,

Angela Leung,

Chan Cheuk Hin,

Choi Justin Yuet Hei,

Huang Helene Heling,

Mok Yu Yiu,

Zhang Hiu Lo Constance,

Chloe Leung Tsz Ching,

Hsu Ting Ruei

Abstract

Palladium is widely adapted in industrial processes such as catalytic converters or ceramic capacitors. It also holds biomedical applications such as acting as a drug carrier and is utilised in cancer treatment. However, palladium is an environmental pollutant and difficult to be recycled. In this project, twenty-five palladium(II) reducing peptides (PdRp) are modified from a selection of known palladium binding peptides. The PdRp are fused to a cellulose binding domain 3 (CBD 3) derived from Clostridium thermocellum which acts as an affinity tag. The PdRp-CBD complex is expressed in Escherichia coli BL21(DE3) in pETBlue-2 vector and purified using regenerated amorphous cellulose (RAC). Peptide-Palladium interactions are determined by computational methods of Molecular Dynamics. Downstream analysis and further experiments will be performed to determine the efficiency and performance of the PdRp with potassium hexachloropalladate.

Introduction

  • Palladium is a transitional metal that is used in pharmaceutical industries, the jewellery business, catalytic converters and many more productions.
  • The current supply of palladium is not able to fulfil our demands, a shortage of palladium would likely occur in the future. Therefore, in order to meet as many demands as possible, palladium is being recycled so that more of this rare metal could be reused.
  • If palladium is recycled, not only can the metal be reused for other purposes, it can also contribute in saving the environment as palladium is an environmental pollutant which is found to be highly toxic to aquatic lives and can cause cardiac malformation in zebrafish embryos or harm terrestrial plants as palladium can alter their cell structure.
  • Many technologies for recycling of palladium like pyrometallurgy processes and smelting have been developed and implemented, but they are mostly unfavourable as they either use harmful chemicals, cause hazardous gas and waste emission, or recycle palladium into forms that are not commonly used.
  • These current recycling methods can’t provide us with the elemental palladium we need without further reduction that are costly and not fully developed yet. So if we could reduce palladium(II) ions through a peptide with high binding and reducing efficiency, more palladium could be reused.

Design

Palladium Reducing Peptides (PdRp)

  • Designed from palladium binding peptides reported in the past
Sequence (AA)​
QQSWPIS​​1
SVTQNKY2
TSNAVHPTLRHL​3
TSNAVAPTLRCL​4
​

Library peptides used in this project

The binding residues are serine, threonine, histidine and cysteine

Our modified palladium reducing peptide (PdRp)

  • Binding residues are identified
  • Single tryptophan or double tryptophan mutations are implemented at different positions
  • Tryptophan can reduce palladium(II) ions1
  • Efficiency of reduction by single tryptophan and double tryptophan can be compared
  • Efficiency of reduction at different positions can be compared

Cellulose Binding Domain (CBD)

  • From Clostridium thermocellum CipA5
  • Binds to cellulose
  • Protein tag and stabiliser
  • Can be purified using regenerated amorphous cellulose column (RAC)

PdRp-CBD

PdRp is fused with CBD and forms the complex

References

[1] Chiu, C., Li, Y., & Huang, Y. (2010, May 12). Size-controlled synthesis of Pd nanocrystals using a specific multifunctional peptide. Retrieved November 09, 2020, from https://pubs.rsc.org/en/content/articlelanding/2010/NR/c0nr00194e

[2] Sarikaya, M., Tamerler, C., Jen, A., Schulten, K., & Baneyx, F. (2002). Molecular biomimetics: Nanotechnology through biology. Retrieved November 09, 2020, from https://www.nature.com/articles/nmat964

[3] Pacardo, D., Sethi, M., Jones, S., Naik, R., & Knecht, M. (2009). Biomimetic synthesis of Pd nanocatalysts for the Stille coupling reaction. Retrieved November 09, 2020, from https://pubmed.ncbi.nlm.nih.gov/19422199/

[4] Coppage, R., Slocik, J., Heinz, H., Ramezani-Dakhel, H., Naik, R., Knecht, M., & Bedford, N. (2013). Exploiting localized surface binding effects to enhance the catalytic reactivity of peptide-capped nanoparticles. Retrieved November 09, 2020, from https://pubmed.ncbi.nlm.nih.gov/23865951/

[5] Yunus, I., & Tsai, S. (2015, February 12). Designed biomolecule–cellulose complexes for palladium recovery and detoxification. Retrieved November 09, 2020, from https://pubs.rsc.org/en/content/articlelanding/2015/ra/c4ra16200e

Proposed Implementation

Proposed end users of our implementation are the electronics industry, metal recycling industry and the automobile industry.

Step 1:

  • Engineered baceria are grown in large scale fermenter
  • PdRp-CBD are synthesized
  • Cells are lysed using homogenizer

Step 2:

  • Supernatant poured into a regenerated amorphous cellulose column1
  • CBD in PdRp-CBD will absorb to the column
  • Impurities can be washed away
  • PdRp-CBD can be eluted

Step 3:

  • PdRp-CBD and Pd(II) ions are put into a pH resistant tank
  • Pd(II) ions will be reduced to elemental palladium

Step 4:

  • The reacted solution will be put into a decanting centrifuge
  • The palladium in solid form will be ejected
  • Palladium will be purified after a few centrifugations

Safety Concerns

Leakage of palladium or engineered bacteria

Future Improvement:

Provide methods to utilize palladium metal alloy as a raw material for our implementation

References

[1] Wang, D., & Hong, J. (2014). Purification of a recombinant protein with cellulose-binding module 3 as the affinity tag. Retrieved November 09, 2020, from https://pubmed.ncbi.nlm.nih.gov/24943312/

Modelling

Molecular Dynamics

Our team performed 21 in silico molecular dynamic simulation to gain preliminary insights on the functionality of the designed peptides.

MD preparation:

  • Structure of peptide was prepared by PEPFOLD 3.51,2,3
  • Forcefield of the simulation was CHARMM274.
  • Parameterization of the force field was preformed to add the molecular topology of Pd (II) compound.
  • Equilibration of the system is performed.

Results

Results will be discussed in the next session.

ODE Model

  • Our ODE model is based on a tryptophan reduction pathway in Pd (II) compounds suggested in literature.
  • We aimed to optimize the reaction conditions and maximize the production of palladium nanoparticles.
  • Tryptophan may remain its structure, or form tryptophan or kynurenine in the pathway.
  • Tryptophan is the main product of reduction.
  • The tryptophan reduction process is irreversible.
  • An overall equation and 11 equations rate equations based on the overall equation are made based on different products formed in the reduction process.

ODE graph

  • A ODE graph of our model that roughly estimates the amount of palladium nanoparticles formed.
  • The rates in the graph are based on experiments done in literature.
  • The rate constants that are not mentioned in literature are assumed to be the same.
  • Experimental data is required to find out the rate constants in the equations and curve fitting.
  • The simulation started with 50M of Pd (II) compounds and tryptophan.
  • The theoretical yield of palladium nanoparticles produced in reduction is about 11M.

References

[1] Lamiable A, Thévenet P, Rey J, Vavrusa M, Derreumaux P, Tufféry P. PEP-FOLD3: faster de novo structure prediction for linear peptides in solution and in complex. Nucleic Acids Res. 2016 Jul 8;44(W1):W449-54.

[2]Shen Y, Maupetit J, Derreumaux P, Tufféry P. Improved PEP-FOLD approach for peptide and miniprotein structure prediction J. Chem. Theor. Comput. 2014; 10:4745-4758

[3]Thévenet P, Shen Y, Maupetit J, Guyon F, Derreumaux P, Tufféry P. PEP-FOLD: an updated de novo structure prediction server for both linear and disulfide bonded cyclic peptides.Nucleic Acids Res. 2012. 40, W288-293.

[4]Vanommeslaeghe K., et. al. CHARMM general force field: A force field for drug‐like molecules compatible with the CHARMM all‐atom additive biological force fields. J. Comput. Chem., 2010. 31: 671-90.

Results and Conclusion

  • Root mean square deviation (RMSD) and Radius of gyration (Rg) is used to evaluate peptide’s structural stability.

RMSD results:

TSNAVAWWLRCL:

TSNAVAWTLRCL:

SVWWNKY:

WWTQNKY:

Peptide Average(nm) Standard deviation(nm)
TSNAVAWWLRCL 0.161120326 0.080106553
TSNAVAWTLRCL 0.097607266 0.052898493
SVWWNKY 0.209204939 0.024462841
WWTQNKY 0.083749003 0.056350154

Rg results:

TSNAVAWWLRCL:

TSNAVAWTLRCL:

SVWWNKY:

WWTQNKY:

Peptide Average(nm) Standard deviation(nm)
TSNAVAWWLRCL 0.676006502 0.029810114
TSNAVAWTLRCL 0.6603765 0.021057266
SVWWNKY 0.533118367 0.016722055
WWTQNKY 0.556363889 0.020679286
  • Distance between tryptophan indole ring’s nitrogen and the Pd (II) is used to evaluate the binding ability of the peptide.

TSNAVAWWLRCL:

TSNAVAWTLRCL:

SVWWNKY:

WWTQNKY:

Peptide Average(nm) Standard deviation(nm)
TSNAVAWWLRCL 1.240814273 0.31742214
SVWWNKY 0.8264017 0.19382964
WWTQNKY 0.705289089 1.0422625
  • Total energy is used to validate the system follows the law of conservation of energy.

Total energy of the system:

TSNAVAWWLRCL:

TSNAVAWTLRCL:

SVWWNKY:

WWTQNKY:

Peptide Average (kJmol-1) Standard deviation (kJmol-1)
TSNAVAWWLRCL -178422.7609 617.08549
TSNAVAWTLRCL -387309.6473 882.61857
SVWWNKY -106262.0135 472.53544
WWTQNKY -314810.0785 803.85032

Conclusion:

The small standard deviation in RMSD and Rg shows the four peptide structure is stable.

The small and consistent distance of tryptophan indole ring’s N and the Pd (II) suggests the peptide can sequester the Pd(II).

The small standard deviation of total energy shows the system is physically valid.

Collaboration

First, we joined a virtual symposium by Hong Kong iGEM teams. We had a chance to present our projects and were able to see our shortcomings through the advice. We were able to see other teams’ presentation, which were extremely beneficial to us. Besides, we were able to learn new knowledge from keynote speakers’ lectures.

Our team collaborated with team HK-CPU-WFN-WYY and hosted a sharing section with their team. We presented on how molecular dynamics was done as their team wanted to know about single peptide. Their team shared about signal peptide.

Last but not least, our team collaborated with team PuiChing Macau and had a few meetings to discuss the binding ability of cellulose binding domain. Since we do not have access to laboratory this year, their team helped us evaluate the CBD.

Engineering Success

To create a project for IGEM, we have decided to approach current issues and tackle them through biological methods, and we will be focusing on environmental issues. Moreover, we have noticed that rivers are polluted and fish are dead, and we discovered that contamination by Palladium (II) ion is the cause of water pollution. In order to solve the issue, we have designed palladium-specific reducing peptides to reduce palladium (II) ions to Pd nanoparticles.

After literature review, we decided to incorporate tryptophan in our peptide sequences. As double tryptophan-based peptides rare suggested to be more effective in palladium (II) ion reduction haven’t been investigated, we decided to incorporate it into our design and then evaluate its binding and reducing properties through modelling and experiments. Also, we used a BFM to simulate the peptide structure and interaction matrix provided by a journal we read to simulate the interaction of peptide and the palladium.

After the preliminary modelling, we learnt that the BFM has its shortcomings. For example, cysteine bond isn’t defined in the interaction matrix. The energetics involving the thiol side chain of cysteine residue is not included in the BFM. So, we chose to build a molecular dynamic model of our peptide with the Pd (II) compound, GROMACS 2020.3 was used for the software package for the simulation, and CHARMM27 were chosen after some literature review.

Lastly, we succeeded in using GROMACS to test the peptide sequences designed. In the future, peptide structures with a protonation state in an acidic or basic environment will be prepared or further analysis.

Human Practice

From interviews we learnt that,

  • Frequently used disposal methods of e-waste are not very effective and can cause huge damage to the environment
  • Cost is a major concern to recycling organizations

Thus, we aimed to design a cost-effective recycling palladium by peptide reduction which is environmentally friendly. Electronic suppliers and the automobile industry can simply adapt to our solution to recycle palladium in the form of palladium nanoparticles.

Survey:

  • Answered by 113 respondents from the general public.
  • Informed us of the general public’s current recycling methods and attitude towards e-waste, and their opinions towards current government support.
  • The general public's awareness of e-waste recycling and enthusiasm for centralized e-waste recycling should be enhanced to establish a complete system for e-waste recycling.

Contribution

Our team carried out an in silico simulation to predict the functionality and stability of thea peptide BBa_K925005 with Pd binding abilities designed by iGEM team 2012 St_Andrews that had not been characterized before.

Methods:

  • The structure of the peptide was predicted using I-TASSER.
  • The peptide was solvated in water model spc216.
  • The Molecular Dynamics simulation was performed using GROMACS 2020.3

MD Results

The interaction between the peptide and Pd was analyzed through:

  • RMSD of peptide backbone atoms

Average (avg): 0.2429242nm; Standard deviation (SD): 0.036509137nm

  • Radius of gryation (Rg)

avg: 1.867476428nm; SD: 0.015650297nm

  • Total energy of the system

avg: -808423.6426 kJ/mol; SD: 1443.8496 KJ/mol

  • The distance of the O in hydroxyl group between threonine and Pd

avg: 2.286150981nm; SD: 0.45291467nm

Conclusion

The distance of the O in hydroxyl group between threonine and Pd was larger than expected, which may be because the GST tag may have stronger molecular forces with Pd(II) ions, weakening intermolecular forces between the Pd peptide and Pd(II) ions. The distance of the O in hydroxyl group between threonine and Pd was larger than expected which may be because the GST tag may have stronger molecular forces with Pd(II) ions, weakening intermolecular forces between the Pd peptide and Pd(II) ions. Further in vitro analysis is required to prove the binding ability of this part.

Science Communication

In order promote synthetic biology to younger generations, we hosted a synthetic biology workshop in September and we have planned to host another 15-week workshop with two streams, biology and chemistry.

The Synthetic biology workshop included:

  1. Brief introduction on Synthetic Biology
  2. What is iGEM and why is it important for the promotion of synthetic biology
  3. Applications of Synthetic Biology

The synthetic biology workshop achieved:

  1. Increased understanding of synthetic biology
  2. Raised peers’ interest towards iGEM and synthetic biology

What the 15-week workshop will include:

  • Biology Stream (Achieved mainly through lectures, group discussions and laboratory sessions)
    • Biotechnology techniques and principles
      • Pipetting
      • Gel electrophoresis
      • PCR
      • Cloning
    • Basic Molecular Biology
      • DNA, RNA, Proteins
      • Translation and Transcription processes
  • Chemical Stream (Achieved through lectures, projects and laboratory sessions)
    • Basic Chemistry
    • Green Chemistry

Attributions and Sponsors

Special thanks to:

Prof. Chi-Wai Chan for his support and advice.

Prof. Shen Long Tsai for providing our sequence of the cellulose binding domain.

Dr. Ho Chi Wang John

Mr. Sze-Ho Ng

Mrs. Cheung (Ng) Bik Wan, Josephine

Mr. Yeung Ho Lam, Jeremy

Mr. Wong Chung Hin

Mr. Chung Tsun Ho Anson

Ms. Lee Hong Kiu

Integrated DNA Technologies

Matlab by MathWorks

Snapgene

St. Stephen’s College

Twist Bioscience