As there is a universal need for laundry detergents, our project addresses an issue that is relevant for everyone. In the following, we want to investigate how well our product approaches the initial problem of inhibiting proteases in laundry detergents in comparison to other solutions, depict the considerations why replacing boric acid is necessary and address further challenges that our project could help to overcome.

Discussion

Currently, there are many attempts to reversibly inactivate proteases like subtilisin E.
Small molecules or peptides are being used as inhibitors. However, there are some obvious disadvantages to those solutions.

Firstly, subtilisin E digests peptides unspecifically. As a consequence, peptide inhibitors are doomed to be destroyed by the protease that they are supposed to inactivate.
Also, small molecules are used to establish an inactivation. A prominent example for a small molecule inhibitor for proteases is boric acid.

Boric acid is widely used in laundry detergents to temporarily inactivate the containing proteases. It works as a competitive inhibitor in the laundry detergent and as soon as the liquid is diluted in the washing machine, boric acid dissociate and the proteases become active again.

In contrast to other solutions, boric acid can be produced chemically in bulk. Unfortunately, high amounts are needed to achieve an inactivation, as the boric acid has to compete with the substrate for the protease and is not covalently attached to it. About 450,000 molecules of it are needed to inactivate one molecule of protease. With our photocaged protease, only one molecule of a photo-labile, non-canonical amino acid per enzyme would be needed. While these amino acids are currently very expensive, higher demand due to extended usage and optimized production methods would dramatically reduce the costs both for the costumer as well as the producer. In the long term, the perfect solution would be a biotechnological production method through pathway engineering to generate the non-canonical amino acid directly inside the host.
Although boric acid can be produced for a comparatively lower price, it will enter the sewages during the washing process. Currently, boric acid will not be filtered from the wastewater treatment plant [1] and therefore it accumulates in the water cycle.
This does not seem to be a very big issue yet, as only small amounts of it are released during the washing process. But higher doses of it have been proven to be harmful to the environment and to humans and even toxic for fertility [2], [3]. Hence, it is listed as a substance of very high concern by the European Chemicals Agency [4].
The cleavage products that emerge after irradiation of either ONB-tyrosine or DMNB-serine are nitrosobenzenes. While acute and chronic toxicity data is unavailable, similar chemicals react in the human blood circulation with hemoglobin and minimizes its ability of O₂ uptake [5]. However, many other photo-labile derivatives of the 20 canonical amino acids are currently in existence [6] and many more could be developed, if actual problems would occur with the existing one. But the probability of this is very low, as the used quantities are infinitesimal. Unfortunately, we were unable to use any of those in the course of our project, as neither orthogonal tRNA/synthetase pairs had been evolved to allow the incorporation of these amino acids in S. cerevisiae or E. coli nor had the chemicals been available for sale to us. Hence, there had been no alternative to using ONB-tyrosine and DMNB-serine for us at the moment. While the method of photocaging develops, more and more harmless photo-labile protection groups for canonical amino acids will be designed.
Photocaging is a promising method as it utilizes the factor light which is not only greatly available but can also be controlled, more conveniently than anything else, in terms of intensity, location and timing. As a result, it allows precise spatial and temporal control of protein function to such a high degree that no other method can compete with it.
Simultaneously, activation by irradiation with light is non-invasive. Therefore, photocaging has already been put to usage in intra-cellular studies [7]-[9] and as no toxicity occurred when the protection groups were cleaved of in vivo, can not only be applied in scientific research, but – with appropriate improvement - could also be used in medicine and even therapeutics. Furthermore, photocaging could reduce the amount of inhibitors needed and thus could be of high value in industrial processes.

Concluding, photocaging could be easily transferred to other challenges if established once and thus provides a great opportunity not only for science but also for industry and medicine.

Real World Application

To further investigate the challenges and opportunities of a photo-caged protease, we examined which effects it would cause if transferred into reality.

Costumer
As the dangers of boric acid are not yet widely known to the public, the need for an alternative way of inhibiting proteases and therefore the necessity of a newly developed laundry detergent might not be of obvious importance. Especially, because the connection between photocaging, proteases and laundry detergents is not obvious for people outside of this field of research. The only change visible to the costumer could possibly be an increase in the prize of laundry detergents and also the need for the additional step of light-activation before the washing process.
But as the common interest in ecological issues and eco-friendly alternatives rises, boric acid might not be tolerated by consumers anymore. Furthermore, the results of our survey showed that the majority would invest up to 20 minutes more in the washing process if that would allow using a more ecological product.
Therefore, our improved laundry detergent could be of interest for costumers.

Industrial perspective
As boric acid has been listed as a substance of very high concern and has even been proven to be toxic for reproduction, it might be banned from the markets soon. Therefore, a million-dollar industry is looking for an alternative solution to an old problem that now demands an urgent answer. A lot of different methods have been tried to inactivate proteases reversibly, but none of them showed the desired effect to a reasonable price.
Though we are aware of the fact that our product is currently not the optimal answer to these demands, we are profoundly convinced that the method of photocaging enzymes could become the optimal solution. It offers the advantage of reducing the risk of being exposed to potentially dangerous chemicals by fractions, or even to none after the appropriate improvements.
Summarizing, our product addresses very urgent questions of ecological and personal health impact. Thus it paves the way for a great industrial application.

Environmental perspective
As outlined before, boric acid bears environmental dangers. First and foremost, it is toxic and has been proven to potentially harm the unborn child and affect reproduction organs. This becomes even more alarming, as humans are exposed to boric acid in many different ways: Primarily, humans get in touch with boric acid during the process of washing clothes. But as residues remain in fabrics the chemical is able to circulate in the household. Additionally, people working in the protease production process are exposed to the chemical, and have to wear additional safety clothing. Furthermore, as boric acid is flooded in the sewage system, it is able to enter the water cycle and thus spreads in the environment uncontrolled. Especially, as enormous quantities of washing detergents are being consumed regularly and boric acid gets into waters in additional ways like fertilizers in agriculture, alternatives have to be found as soon as possible.
Until now, the future harms of boric acid in our environment can only be estimated approximately, but it has now become very obvious that strong regulations are needed to prohibit the further spread of the chemical.
As our product would render the usage of boric acid in laundry detergents unnecessary, it would contribute to the process of limiting the danger to our environment.

Workflow

In the course of our project we also took in considerations how our product would be applied in reality. Therefore, we want to show as a final conclusion that our modified laundry detergent would barely affect the process of washing.

References

[1] Barufke et al., “Bor: Ableitung einer Geringfügigkeitsschwelle zur Beurteilung von Grundwasserverunreinigungen“, LUBW. Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Feb. 2012 [2] Human Health and Environment Task Force, “Human and Environmental Risk Assessment on ingredients of Household Cleaning Products”, 2015. [Online]. Available: http://www.heraproject.com. [Accessed: 19-Oct-2016]. [3] R. E. Chapin and W. W. Ku, “The reproductive toxicity of boric acid.,” Environ. Health Perspect., vol. 102, no. Suppl 7, pp. 87–91, Nov. 1994. [4] European Chemicals Agency, “Comments and Response to Comments on Annex XV SVHC: Proposal and Justification”, 2010. [Online]. Available: https://echa.europa.eu. [Accessed: 19-Oct-2016]. [5]H. U. Käfferlein et al., “Bildung von Methämoglobin durch Anilin”, IPA-Journal, pp.26, Jan. 2014. [6]P. Klán et al., “Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy,” Chem. Rev., vol. 113, no. 1, pp. 119–191, Jan. 2013. [7] D. P. Nguyen, M. Mahesh, S. J. Elsässer, S. M. Hancock, C. Uttamapinant, and J. W. Chin, “Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells,” J. Am. Chem. Soc., vol. 136, no. 6, pp. 2240–2243, Feb. 2014. [8] D. P. Nguyen, M. Mahesh, S. J. Elsässer, S. M. Hancock, C. Uttamapinant, and J. W. Chin, “Genetic Encoding of Photocaged Cysteine Allows Photoactivation of TEV Protease in Live Mammalian Cells,” J. Am. Chem. Soc., vol. 136, no. 6, pp. 2240–2243, Feb. 2014. [9]E. A. Lemke, D. Summerer, B. H. Geierstanger, S. M. Brittain, and P. G. Schultz, “Control of protein phosphorylation with a genetically encoded photocaged amino acid,” Nat. Chem. Biol., vol. 3, no. 12, pp. 769–772, Dec. 2007.