Design

The project e-PHAESTUS revolves around the optimized production of the tripeptide, Glutathione (GSH) which is used as a bioleaching agent in e-waste recycling while it forms glutathione coated metal nanoparticles. But...how did we end up here?

When our team made the choice to focus on bioleaching, we began searching for scientific literature in order to gain a deeper understanding of the technology used for metal recovery, in terms of recycling of WEEE (Waste Electrical and Electronic Equipment), otherwise referred to as e-waste.

Metal Selection

Initially, we explored the various metals present in e-waste materials. According to sources, gold (Au) is known to be the metal with the highest value per gram of device. In contrast, lithium (Li) was perceived as a hazardous metal with distinctive chemical identities. ^{[1] [2] [3]}

To make an informed decision about which metals to pursue, we needed to consult the experts. We first spoke with specialists such as Dr. Methenitis, who teaches students about bioleaching and metal recycling technologies. From this discussion, we compiled an initial list of metals including Au, Ag, Cu, Pt, Pd, Fe, Cd, Li, In, Si, Ge, Ni, Ti, and Co. Given the diversity of metals with varying properties in this list, we turned to industry professionals for further insight, which was also a big part of our integrated human practices.

• Verde Tec: At this expo, we had the opportunity to speak with over 40 companies engaged in recycling and the green economy. Companies involved in solar panel production emphasized the value of recovering metals and metalloids found in panels. However, our team believed that recovering these metals would involve significant challenges and limited applications.
• MetalLogic: During our meeting, they highlighted the significance of copper and gold, metals that dominate in e-waste. Consequently, we began to concentrate more on these metals.

Combining the knowledge we gained from our literature review and expert consultations, we decided to focus on Au, Cu, and Ag. These 3 metals have similar properties.

Gene Selection

In order to select the bioleaching concept and its genes, that occurred simultaneously with metal selection, we considered factors such as novelty and feasibility. In our initial idea, we wanted to harness the potential of the microorganism Acidithiobacillus ferrooxidans, an acidophilic bacterium that has interested the scientists for its applications in bioleaching. Specifically, the following reactions facilitate the solubilization of metals at low pH levels. The first reaction is part of its respiratory chain, where Fe³⁺ can act as a reductant to oxidize metals when paired with an appropriate reductant: ^{[4] [5]}

However, this technology is limited to metals with suitable oxidation potentials, such as Ni and Cu. We decided to ultimately discard this idea, even though we spent a lot of time analyzing mechanisms and industrial applications. Our initial thought was to incorporate responsible genes into industry-friendly strains like E.coli, as using Acidithiobacillus ferrooxidans proved economically unsustainable. Furthermore, it became clear that the mechanism was not well-studied and involved many genes. Therefore, the implementation would be really difficult.^[6]

For these reasons, our attention shifted from Acidithiobacillus ferrooxidans to various pseudomonas strains, such as Pseudomonas putida and Pseudomonas fluorescens. More specifically, the HCNabc operon enables the production of hydrogen cyanide (HCN) from simple, inexpensive amino acids like glycine. HCN is crucial because CN^- ions are the most traditional leachate in gold dissolution, yielding very high recovery rates: ^{[7] [8] [9]}

Interaction of Au and cyanide ions, Gaussian software

Further literature review indicated that glycine is the optimal amino acid and that a pH level of 9 would minimize the emission of the highly toxic HCN, while enhancing CN^- activity.¹⁰

To recover the metals, we consulted professors from the Chemistry Department specializing in Inorganic and Polymer Chemistry. Dr. Efthimiadou, Dr. Methenitis, and Dr.Pitsikalis guided us in synthesizing an amphiphilic copolymer. The first part is made from polystyrene (PS) ensures that the polymer is insoluble, while the second hydrophilic part from polyacrylic acid (PAA) is responsible for binding metallic ions. This membrane can interact with and bind metals depending on the solution's pH. Specifically, at pH < 3, the hydrophilic group exists in a molecular form (OH), whereas at pH > 9, the ionized group interacts as shown below.

Interaction of the polymer and gold, GaussView software

Overall, the PCBs (printed circuit boards) would be placed in a culture of transformed bacteria during their exponential phase. The pH of the growth medium would be regulated at 9, and the metals would be left for one week. Afterward, the solution would be filtered, retaining the metal residues and bacteria in the original container. The filtrate would contain soluble Au-CN complexes. To recover the metals, the filtrate would pass into a vessel containing the aforementioned copolymer. Subsequently, the copolymer would be acidified in a new vessel, releasing the desired metallic ions. Since we wanted to broaden the concept and expand it, we explored the idea of using microorganisms that are able to metabolize the CN^- ions to detoxify the water used.

However, for many reasons that are clearly stated in the Engineering page, with the main one being the toxicity and the safety of our lab, the HCN Synthase Project was discontinued. It should be mentioned though that the design ,at that time, was in an advanced state

After elaboration and long discussions with one of our Instructors, Dr. Nikolaos Labrou, whose remarkable expertise in enzyme technology was immensely helpful during the course of our project, our team decided to opt for a bio-friendly approach and an innovative technique on the solubilization and binding of metals, harnessing the affinity between the latter and the tripeptide glutathione (GSH).

The Choice of Glutathione (GSH)

Glutathione (GSH - L-γ-glutamyl-L-cysteinylglycine) is a tripeptide, a non-protein compound and more specifically a low molecular weight thiol. It is a biological compound widespread in many organisms, including mammals and microorganisms, both eukaryotic and bacteria. The tripeptide is known mainly for its key role in the detoxification of xenobiotics, the cellular antioxidant defense as well as the regulation of intracellular metabolism. ^[11]

The abundance of glutathione in biological systems and more importantly, the fact that GSH belongs to a wider group of thiols that present high affinity in binding with metals, were the key advantages considered for choosing this tripeptide as a bioleaching agent for metals released by e-waste.

We were heavily inspired by the role of GSH in phytoextraction, an environmentally friendly approach towards removing toxic pollutants that contaminate and are accumulated in soil, using endogenous plant molecular mechanisms and plant-microbe interactions as a vehicle for environmental bioremediation. For this purpose, GSH serves as a chelator or even a metal hyper-accumulator, contributing to the uptake, translocation and detoxification of metals. Thus, the involvement of GSH on metal chelation mechanisms as part of phytoremediation approaches was a main source of inspiration for its use in bioleaching in e-waste.

Furthermore, the capability of Glutathione to act as a leaching agent was confirmed by model studies conducted by our group. More specifically, Glutathione was compared to Thiosulfate, the second most common leachate for gold and showed promising results, as shown by the table below. More details behind the computation chemistry modeling of GSH are available in the Model page.

Metal	ΔG for Thiosulfate (kcal)	ΔG for GSH (kcal)
Copper (Cu)	-51,78	-81,76
Silver (Ag)	-35,61	-41,44
Gold (Au)	-48,81	-83,63

The ΔG values refer to the following reactions:

M⁺ + S₂O₃^2- → [M-S₂O₃]^- for Thiosulfate

M⁺ + GS^- → [M-GS]^- for Glutathione

The interaction between Glutathione and inert gold is seen in the photo below :

Choosing the Biosynthesis of GSH through the Enzymatic System of GshF and PPK

We opted for the in vitro production of GSH through enzymatic catalysis, as it offers a multitude of benefits that suited well to the direction of our project in terms of synthetic biology. More specifically, biocatalysis could lead to the production of higher concentrations of GSH at a short time frame, using less precursor compounds, which in our case were the amino acids L-Glutamic Acid, L-Cysteine and Glycine. Furthermore, in vitro the application of enzyme purification methods could facilitate the isolation of our target proteins according to the needs of our project.

The biosynthesis of GSH in vivo takes place in the cytosol, involving a two-step ATP-dependent reaction of precursor amino-acids, L-Glutamic Acid, L-Cysteine and Glycine. In most organisms, these steps are catalyzed by two ATP-dependent enzymes, namely γ-GS Synthetase (γ-GCS) and GSH Synthetase (GS) as follows:^[13]

γ-glutamylcysteine synthetase γ-GCS (EC 6.3.2.2), otherwise known as glutamate-cysteine ligase is the enzyme responsible for catalyzing the production of the γ-GC dipeptide, while the second step is catalyzed by the GS or glutamine-synthetase (EC 6.3.1.2), which is the reaction between the γ-GC dipeptide and Glycine, to finally form glutathione in its reduced form, known as GSH. The first step of the reaction is limited to the overall reaction rate, due to a feedback inhibition mechanism on γ-GCS caused by the product of the biocatalysis, GSH. The inhibition mechanism occurs at both transcriptional and posttranslational levels, preventing the overaccumulation of GSH in living cells.^{[13] [14]}

Enzyme cascade systems can be widely applied for the industrial production of compounds of biological importance, such as GSH. The method involves the use of purified enzymes, crude cell extracts, as well as lyophilized cells that can be used to catalyze multi-step reactions within a common environment. It possesses the advantages of overcoming limitations of conventional catalysis methods, leading to enhanced product yield, lower bioprocessing cost in a shorter time period through faster reaction rates. What's more, multi-enzymatic systems in vitro constitute an environmentally friendly solution for the synthesis and later the easier isolation of important biological products, which can be harnessed for the purposes of bioremediation and biomanufacturing applications in the near future.^[11]

For this purpose, we chose to study an in vitro synthetic biosystem based on purified enzymes instead of a whole-cell catalysis. Since the biosynthesis of GSH takes place in two steps and relies on ATP as an energy supply, we drew valuable inspiration from the research paper of Zhang et. al (2017) for a combined system to synthesize GSH and regenerating ATP. More specifically, in the case of GSH biosynthesis, given the limitations of feedback inhibition mechanisms, a bi-functional synthetase known as GshF (EC 6.3.2.2) present in microorganisms (Gram positive bacteria, e.g. Streptococcus agalactiae and Listeria monocytogenes as well as Gram negative bacteria, such as Pasteurella multocida), drew our attention as a candidate solution for the construction of the enzyme-based GSH production system. This enzyme seems to remain unaffected by inhibition mechanisms, achieving the production of GSH in high levels, which can be potentially applied in the industrial production of the tripeptide through biocatalysis. On the other hand, the high cost of ATP as a cofactor essential for the in vitro biosynthesis of GSH created the need for an economically viable ATP regeneration solution for later use through bioremediation or industrial processes. This need could be covered by Polyphosphate kinase, otherwise known as PPK (EC 2.7.4.1), which efficiently catalyzes the regeneration of ATP by ADP and phosphate groups using the PolyP inorganic polymer as substrate, thus making the system independent from external ATP addition.^[11]

Choosing the Genes

Given the fact that the enzymatic system of GshF and PPK has been already well studied by many research groups, we dedicated ourselves to study a variety of papers during our initial design and brainstorming process, in order to conclude which genes would be more suited for the purposes of molecular cloning, heterologous overexpression in bacterial systems and later the purification of the produced proteins.^{[11] [14] [15] [16] [19] [20] [21] [24]}

Given this fact, our choice of genes relied on the basis of efficient product yield specifically for the case of GshF - taking into account the amount of GSH produced. For the regeneration of ATP, we considered the compatibility of the chosen PPK enzyme to the GSH production process. Furthermore, we took into account in both cases the feasibility of our gene's choice, given the time and resource constraints of an iGEM team project.

According to the work of research groups of Zhang et. al (2017)^[11] and Cui et. al (2021)^[14], the bi-functional glutathione synthetase of a Streptococcus strain, GshFSs (Streptococcus sanguinis SK36) was valued as an optimal solution for the efficient production of GSH. More specifically, both groups proudly reported the highest yield of GSH synthesis ever reported by the enzyme GshFSs of this particular strain. We were impressed by the fact that through the enzymatic system of GshFSs with a PPK enzyme for ATP regeneration, production of GSH was reported in a molar yield of 0.96 mol/mol with a productivity of 49.95 mM per hour Cui et. al (2021)^[14]. The choice of PPK was based on the feasibility of the project, taking into consideration the compatibility of the enzyme originated from Thermosynechococcus strain, namely Thermosynechococcus vestitus (nee. elongatus) BP-1, which was previously successfully tested and implemented in a one-pot enzymatic system with GshFSs by Zhang et. al (2017)^[11].

Overall, why was the enzyme system of GshF Synthetase - PPK Polyphosphate Kinase selected, and how does it address the previous challenges?

• Glutathione is an abundant tripeptide, a non-toxic product, as are the by-products of the biosynthetic reaction.
• The PPK enzyme serves in the regeneration of ATP, a costly enzyme co-factor necessary for the GSH biosynthesis reaction as an important energy source.
• While bioleaching may not directly compete with hydrometallurgy and pyrometallurgy, this idea incorporates upcycling and the synthesis of very valuable nanoparticles, providing a competitive advantage.
• The project directly showcases the value of synthetic biology in creating in vitro enzymatic systems with potential for environmental and industrial applications.
• Glutathione forms less stable complexes that can break down more easily.
• Protein purification is significantly easier for both enzymes, facilitating their in vitro application for metal binding and solubilisation.

When it comes to the design of the molecular cloning experiments, there were several questions that needed to be answered. Here we will go through them:

1) Which expression vector (plasmid)?

Much of the inspiration for our experiments came from the paper by Zhang, X. et al (2017). In this paper they used pET28a(+) for the expression of both genes and their results showed great promise that their system would be suitable for us as well. In addition, pET28a(+) was available to us by the Department of Biology of the National and Kapodistrian University of Athens. After considering the time as well as the cost required to obtain any other of the potential expression vectors studied in the bibliography, we decided to use pET28a(+).

pET28a(+) has several features that are of interest to our project:

• A strong bacteriophage T7 RNA promoter: This allows for high levels of expression of our protein, enough to satisfy the goals of our project.
• A 6xHis-tag: This tag allows for the purification of our recombinant proteins through Ni-NTA affinity chromatography.
• A lac promoter and a lac operator: This allows for the controlled expression of our proteins, induced by the addition of IPTG.
• Kanamycin Resistance Gene: This allows us to separate E.coli transformed with our plasmid from the ones that have not been transformed.

All of these features can be seen in the plasmid map as shown through the Snapgene Visualization:

2) Which antibiotic we would use to separate transformed and untransformed E.coli?

This is rather straightforward. Kanamycin, as that is what our plasmid provides resistance to.

3) Which E.coli strain would be used for the multiplication of the recombinant plasmid after the ligation reaction?

For this part, Zhang X, et al. use and recommend E. coli Top10. After careful consideration of the strain characteristics and in order to comply with iGEM Safety Rules, we decided not to proceed with Top10. Instead we used E. coli DH5a. This strain is E.coli K12 derived and therefore in accordance with iGEM Safety Rules. It was also made available to us by the Department of Biology of the National and Kapodistrian University of Athens. It fulfills all the fundamental criteria for our use. For more extensive research, we attach the E.coli DH5a genotype: fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17.

4) How would the genes be cloned into pET28a(+)?

For a more extensive description of how the gene DNA sequence was adapted for our molecular cloning experiments, please refer to the Engineering page. In this particular section, we will refer to the choices of restriction sites and how they were integrated with the DNA Sequence.

Once again, we took guidelines from the paper by Zhang, X et al. They recommended the use of BamHI (5' end) and HindIII (3' end) for the cloning of the GshFss gene and EcoRI (5' end) and SalI (3' end) for the PPKTe gene. All these enzymes were made available to us or were easily obtainable, therefore the choice was made to use them. In addition, all these enzymes were from New England Biolabs (NEB), which heavily simplified the protocols that needed to be executed in order to achieve the double restriction of our genes and our plasmid.

Let us now examine the restriction sites in more detail: In our plasmid, these sites are unique and are placed in an appropriate position to facilitate gene expression and purification:

In our gene DNA Sequences, the enzyme restriction sites needed to be added according to specific parameters. To achieve that, we used Snapgene Software, and specifically the Primer and PCR features of Snapgene.

a) For GshFss:

For BamHI the required sequence (5'-GGATCC-3') was added right before the first coding ATG. However, for efficient cleavage, NEB requires at least one base to be added at the 5' DNA end. After consulting our professors, we added 4 bases as can be seen in the picture. For HindII the required sequence (5'-AAGCTT-3') was added right after the last codon. However, for efficient cleavage, NEB requires at least three base to be added at the 3' DNA end. After consulting our professors, we added 4 bases as can be seen in the picture. The right positioning of these restriction sites was then tested with Snapgene Software using the Cloning Feature:

Top: The BamHI restriction site, Middle: The HindIII restriction site, Bottom: In silico Cloning, Snapgene software

b) For PPKTe:

For EcoRI the required sequence (5'-GAATTC-3') was added right before the first coding ATG (it was changed before gene ordering). However, for efficient cleavage, NEB requires at least a 5 bases to be added at the 5' DNA end. After consulting our professors, we added 5 bases as can be seen in the picture. For SalI the required sequence (5'-GTCGAC-3') was added right after the last codon. However, for efficient cleavage, NEB requires at least three base to be added at the 3' DNA end. After consulting our professors, we added 5 bases as can be seen in the picture. The right positioning of these restriction sites was then tested with Snapgene Software using the Cloning Feature:

Top: The EcoRI restriction site, Middle: The SalI restriction site, Bottom: In silico Cloning, Snapgene software

Chemical Engineering Design

In the chemical engineering design part, we wanted to advance from the production of Glutathione to the leaching process. As it was mentioned, the conditions and the industrial process was modified according to the result from our models.In order to increase the leaching rate of our process we decided to implement the following plan.

Firstly, it is advised to use a combination of Glutathione and Thiosulfate. Thiosulfate has a strong leaching ability with metals with an oxidation state of 0, in contrast to glutathione, as it may be seen in the table below.

Comparison of complexes between Au(0), glutathione and thiosulfate

Leaching Agent	ΔG (kcal/mol)	K
Glutathione	+20,63	3*10-15
Thiosulfate	-52,78	2*1037

In this design a small quantity of Thiosulfate will be added to increase the solubility of Glutathione. Then, Glutathione will substitute Thiosulfate based on this reaction :

In this way, Thiosulfate will be recycled and the metals will be even more stabilized. This reaction will be irreversible as it is supported by the data gained from the thermodynamic analysis.

Reaction	Value of K
Thiosulfate-Cu + GS- → GS-Cu + Thiosulfate	4*1063
Thiosulfate-Ag + GS- → GS-Ag + Thiosulfate	1*1025
Thiosulfate-Au + GS- → GS-Au + Thiosulfate	3*1034

In our design, we will also include the reductive agent sulfuric sodium since it will increase the percentage of the reactive and necessary GSH. Otherwise, Glutathione will be oxidized and form disulfide bonds (GS-SG).

From the model, it was clear that the pH should be regulated at 7. Therefore, our design should include the regulation of the pH of the solution during the leaching process.

Glutathione Coated Metal Nanoparticles

Glutathione in a reductive environment like the one described in our design has the capability to form Glutathione Coated Metal Nanoparticles (NPs). The synthesis of these nanoparticles does not demand high temperature and high pressure and will take place simultaneously with the leaching process. Actually, the formation of these nanoparticles increases the solubility of metals like Cu and Ag. After the end of the leaching experiments the isolation of these nanoparticles should follow. The most common technique used experimentally is the ultracentrifugation of the solution. ^{[26] [27]}

1. Macedo, K. B., Sorato, D. L., Macedo, J. L., Moura, C. F., & Angyalossy-Almeida, M. (2024). Assessment of smartphones’ components and materials for a recycling perspective: tendencies of composition and target elements. Journal of Material Cycles and Waste Management
2. Xu, T., Zheng, X., Ji, B., Xu, Z., Bao, S., Zhang, X., Li, G., Mei, J., & Li, Z. (2024). Green recovery of rare earth elements under sustainability and low carbon: A review of current challenges and opportunities. Separation and Purification Technology, 330, 125501.
3. Fornari, P., Abbruzzese, C., & Massacci, P. (2022). Leaching of copper contained in waste printed circuit boards, using the thiosulfate–oxygen system: A kinetic approach. Materials, 15(7), 2354.
4. Chen, S., Yang, Y., Liu, C., Dong, F., & Liu, B. (2015). Column bioleaching copper and its kinetics of waste printed circuit boards (WPCBs) by Acidithiobacillus ferrooxidans. Chemosphere, 141, 162-168.
5. Arshadi, M., & Mousavi, S. M. (2015). Multi-objective optimization of heavy metals bioleaching from discarded mobile phone PCBs: Simultaneous Cu and Ni recovery using Acidithiobacillus ferrooxidans. Separation and Purification Technology, 147, 210-219.
6. Jones, S., & Santini, J. M. (2023). Mechanisms of bioleaching: Iron and sulfur oxidation by acidophilic microorganisms. Essays in Biochemistry, 67(4), 685-699.
7. Lee, J., & Pandey, B. D. (2012). Bio-processing of solid wastes and secondary resources for metal extraction – A review. Waste Management, 32(1), 3-18.
8. Laville, J., Blumer, C., Von Schroetter, C., Gaia, V., Défago, G., Keel, C., & Haas, D. (1998). Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0. Journal of Bacteriology, 180(12), 3187-3196.
9. Işıldar, A., van de Vossenberg, J., Rene, E. R., van Hullebusch, E. D., & Lens, P. N. L. (2016). Two-step bioleaching of copper and gold from discarded printed circuit boards (PCB). Waste Management, 57, 149-157.
10. Yuan, Z., Yuan, Y., Liu, W., Ruan, J., Li, Y., Fan, Y., & Qiu, R. (2019). Heat evolution and energy analysis of cyanide bioproduction by a cyanogenic microorganism with the potential for bioleaching of precious metals. Journal of Hazardous Materials, 377, 284-289.
11. Zhang, X., Wu, H., Huang, B., Li, Z., & Ye, Q. (2017). One-pot synthesis of glutathione by a two-enzyme cascade using a thermophilic ATP regeneration system. Journal of Biotechnology, 241, 163–169.
12. Seth, C. S., Remans, T., Keunen, E., Jozefczak, M., Gielen, H., Opdenakker, K., Weyens, N., Vangronsveld, J., & Cuypers, A. (2012). Phytoextraction of toxic metals: a central role for glutathione. Plant, cell & environment, 35(2), 334–346.
13. Xiong, Z., Kong, L., Wang, G., Xia, Y., Zhang, H., Yin, B., & Ai, L. (2018). Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. Journal of Dairy Science, 101(8), 6937–6945.
14. Cui, Xiangwei & Li, Zhimin (2021). High production of glutathione by in vitro enzymatic cascade after thermostability enhancement. AIChE Journal, Volume 67 – Issue 1.
15. Wang, C., Zhang, J., Wu, H., Li, Z., & Ye, Q. (2015). Heterologous gshF gene expression in various vector systems in Escherichia coli for enhanced glutathione production. Journal of biotechnology, 214, 63–68.
16. Wang, D., Wang, C., Wu, H., Li, Z., & Ye, Q. (2016). Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase. Journal of industrial microbiology & biotechnology, 43(1), 45–53.
17. Janowiak, B. E., & Griffith, O. W. (2005). Glutathione synthesis in Streptococcus agalactiae. One protein accounts for gamma-glutamylcysteine synthetase and glutathione synthetase activities. The Journal of biological chemistry, 280(12), 11829–11839.
18. Zhang, X., Cui, X., & Li, Z. (2020). Characterization of Two Polyphosphate Kinase 2 Enzymes Used for ATP Synthesis. Applied biochemistry and biotechnology, 191(2), 881–892.
19. Mori, H., Matsui, M., Bamba, T., Hori, Y., Kitamura, S., Toya, Y., Hidese, R., Yasueda, H., Hasunuma, T., Shimizu, H., Taoka, N., & Kobayashi, S. (2024). Engineering Escherichia coli for efficient glutathione production. Metabolic engineering, 84, 180–190.
20. Cao, H., Li, C., Zhao, J., Wang, F., Tan, T., & Liu, L. (2018). Enzymatic Production of Glutathione Coupling with an ATP Regeneration System Based on Polyphosphate Kinase. Applied biochemistry and biotechnology, 185(2), 385–395.
21. [Cui, C., Kong, M., Wang, Y., Zhou, C., & Ming, H. (2021). Characterization of polyphosphate kinases for the synthesis of GSH with ATP regeneration from AMP. Enzyme and microbial technology, 149, 109853.
22. ExplorEnz: the Enzyme Database - International Union of Biochemistry and Molecular Biology (EC 6.3.2.1, EC 6.3.1.2, EC 6.3.2.2, EC 2.7.4.1)
23. Xiong, Z., Kong, L., Wang, G., Xia, Y., Zhang, H., Yin, B., & Ai, L. (2018). Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. Journal of Dairy Science, 101(8), 6937–6945. doi:0.3168/jds.2017-14142
24. Li, W., Li, Z., Yang, J., & Ye, Q. (2011). Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. Journal of biotechnology, 154(4), 261–268. doi:10.1016/j.jbiotec.2011.06.001
25. Jiang, Y., Tao, R., Shen, Z., Sun, L., Zhu, F., & Yang, S. (2016). Enzymatic Production of Glutathione by Bifunctional γ-Glutamylcysteine Synthetase/Glutathione Synthetase Coupled with In Vitro Acetate Kinase-Based ATP Generation. Applied biochemistry and biotechnology, 180(7), 1446–1455. doi:10.1007/s12010-016-2178-5
26. Mao, X., Li, ZP. & Tang, ZY. One pot synthesis of monodispersed L-glutathione stabilized gold nanoparticles for the detection of Pb2+ ions. Front. Mater. Sci. 5, 322–328 (2011)
27. Sahu, M., Ganguly, M. & Doi, A. Role of Glutathione Capping on Copper Nanoclusters and Nanoparticles: A Review. J Clust Sci 35, 1667–1685 (2024)