Team:Estonia TUIT/Implementation


Oral Hygiene and Dental Caries

According to the World Health Organization, caries is the most common oral disease. It is estimated that 2.3 billion people, including 530 million children, suffer from this condition (Peres et al., 2019). Dental caries develops when bacteria, mainly Streptococcus mutans, accumulate on teeth, forming biofilm and metabolizing sugars. This results in the production of different acids that can cause tooth demineralization (Rathee & Sapra, 2021). If untreated, this condition can lead to severe toothache, tooth abscess, decay, or even the loss of teeth (Dental Caries: Symptoms Causes and Treatment of Tooth Caries| Oral-B India). Good oral hygiene and regular teeth brushing can significantly decrease the likelihood of dental caries. However, as around ⅓ of people suffer from caries worldwide, additional measures are necessary to improve oral hygiene.

Good oral hygiene and regular teeth brushing can significantly decrease the likelihood of dental caries. However, as around ⅓ of people suffer from caries worldwide, additional measures are necessary to improve oral hygiene.

SALSASMILE Proteolytic Solution

Our answer to this problem is SALSASMILE - a proteolytic enzyme solution that will detach bacteria from the teeth by precisely cutting their anchoring points. The protease mixture can be in the form of a spray, liquid, or even chewing gum. From our survey, we also found that people would prefer to have a spray, mouthwash, paste, or chewing gum to improve their oral hygiene. When stored in the right conditions, the protease can be kept in different forms. When looking for a suitable enzyme, we conducted research on different candidates’ toxicity and medical applications. We gathered the results into a table that can be found on our Modeling page.

Atlantic cod. Trypsin and its Safety

One of the best candidates was the Atlantic cod trypsin. It is not toxic and has been previously used in biomedical studies (Gudmundsdóttir et al., 2013). Moreover, it showed a promising efficiency in kinetic modeling of SALSA degradation (refer to the Modeling page). The optimal working pH of trypsin (pH 7-9) (Lam et al., 2012) differs significantly from the acidic gastrointestinal pH, which can reach 1.0-2.5 (Evans et al., 1988). As trypsin is fully inactivated at pH 4 (Review of Proteins & Enzymes), we can be sure that the protease will be inactive in the gastrointestinal tract. In the stability assay, it was found that less than 5% of trypsin stays active after incubation in the intestinal juice for half an hour (Wohlman et al., 1962). However, there is a difference between acidic pH in the stomach and the neutral environment (pH 7) of the esophagus (Tutuian & Castell, 2006). Since some portion of the enzyme could potentially be absorbed in the esophagus, additional safety measures could be considered (requires experimental confirmation). For example, the addition of non-toxic irreversible trypsin inhibitor α1-antitrypsin (Mordwinkin & Louie, 2007) to fully inactivate the enzyme may solve the problem. This way, the enzyme activity is limited to a short period in the mouth, reducing the risk of cleaving unwanted proteins. A potential drawback of trypsin is its broad target specificity - its cleavage sequence is only defined by the presence of lysine and arginine residues. This means that trypsin would target additional proteins in the mouth as well. However, we aim to overcome this drawback by two approaches: first, we dock the protease specifically to SALSA by addition of a peptide from S. mutans that binds tightly to SALSA. This will increase the local concentration, enabling us to use lower protease concentrations in our product. Secondly, we have set up an assay in yeast that allows engineering of proteases to target specific regions. We will apply the method with trypsin, using error-prone PCR to generate a wide library of trypsin mutants with different activity and slight changes in specificity and use the high-throughput screen to select for the optimal mutant to target the SALSA SID linkers.

Protease Storage Conditions

As we suggest an innovative way for dental care, we need to consider other challenges, such as finding the most suitable form for our protease and our final compound's efficiency and specificity. Another challenge is the form and storage conditions for the enzyme. Trypsin should be stored at a temperature of around -20 ℃ to avoid autolysis (Trypsin from Porcine Pancreas Proteomics Grade, BioReagent, Dimethylated). It raises a difficulty for correct product storage during transportation or at the customer's home. However, there are alternative ways for the storage of trypsin. For example, upon lyophilization, trypsin can be stored at 2-8 ℃ for two years (Trypsin from Porcine Pancreas Proteomics Grade, BioReagent, Dimethylated). Another possibility is keeping trypsin in an ammonium bicarbonate solution that can be stored at 2-8 ℃ for two weeks. Furthermore, low pH supports trypsin storage, as this suppresses the autolytic activity of trypsin.

End-Users and Survey Results

Nevertheless, the surveys that we conducted as a part of our Integrated Human Practices both among the public and dentists showed considerable interest and a need for new approaches in dental care. The public survey revealed that many people would be interested in an easier alternative tooth care approach, especially when having braces or while traveling. We believe that our end-users will be all people that need an efficient way to improve their oral hygiene. We envision that together with toothbrushing, our product will help keep teeth free from cariogenic bacteria, relieving people from the most common oral disease.

Novel Research Tool for Protease Engineering

Our project also develops a valuable research tool to engineer proteases with distinct specificity. We set up a method based on a yeast two-hybrid system where the proteolytic cleavage efficiency of a linker sequence between the DNA binding domain and the activation domain determines yeast growth rate (Figure 1).

Figure 1. Schematic representation of our modified yeast two-hybrid system for the selection of efficient protease.

This method can be used for at least two purposes. First, it can be used to map the specificity determinants of distinct proteases. For this, a library of potential cleavage sequences can be introduced as the linker sequence between the DNA binding and activation domains, and when expressed together with the protease of interest, the cleavage efficiency is translated to the yeast growth rate. The protease specificity determinants can be obtained by deep sequencing of the linker region. The sequences increasing in abundance will be the preferred substrates of the protease. Secondly, as in our project, the method can be used to engineer proteases that cleave the desired sequence. In this case, either random mutagenesis or rational design is applied to the protease, followed by a competitive growth assay to select the proteases that most efficiently cut the desired sequence (Figure 2). Alternatively, a library of various proteases can be used to screen for the best enzyme to cleave the desired sequence. The method can be widely used, as the ability to design proteases with distinct specificity would greatly benefit the field of synthetic biology.

Figure 2. The applications of our high-throughput protease specificity selection method.

Dental Caries: Symptoms Causes and Treatment of Tooth Caries| Oral-B India. Retrieved October 21, 2021, from

Evans, D. F., Pye, G., Bramley, R., Clark, A. G., Dyson, T. J., & Hardcastle, J. D. (1988). Measurement of gastrointestinal pH profiles in normal ambulant human subjects. Gut, 29(8), 1035–1041.

Gudmundsdóttir, Á., Hilmarsson, H., & Stefansson, B. (2013). Potential Use of Atlantic Cod Trypsin in Biomedicine. BioMed Research International, 2013, 11.

Lam, M. P. Y., Lau, E., Liu, X., Li, J., & Chu, I. K. (2012). 3.15 - Sample Preparation for Glycoproteins. In J. Pawliszyn (Ed.), Comprehensive Sampling and Sample Preparation (pp. 307–322). Academic Press.

Mordwinkin, N. M., & Louie, S. G. (2007). Aralast: an α1-protease inhibitor for the treatment of α-antitrypsin deficiency. Expert Opinion on Pharmacotherapy, 8(15), 2609–2614.

Peres, M. A., Macpherson, L. M. D., Weyant, R. J., Daly, B., Venturelli, R., Mathur, M. R., Listl, S., Celeste, R. K., Guarnizo-Herreño, C. C., Kearns, C., Benzian, H., Allison, P., & Watt, R. G. (2019). Oral diseases: a global public health challenge. The Lancet, 394(10194), 249–260.

Rathee, M., & Sapra, A. (2021). Dental Caries. StatPearls.

Review of Proteins & Enzymes. Retrieved October 21, 2021, from

Trypsin from porcine pancreas Proteomics Grade, BioReagent, Dimethylated. Retrieved October 21, 2021, from

Tutuian, R., & Castell, D. O. (2006). Gastroesophageal reflux monitoring: pH and impedance. GI Motility Online, Published Online: 16 May 2006; | Doi:10.1038/Gimo31.

Wohlman, A., Kabacoff, B. L., & Avakian, S. (1962). Comparative Stability of Trypsin and Chymotrypsin in Human Intestinal Juice. Proceedings of the Society for Experimental Biology and Medicine, 109(1), 26–28.

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