# Team:SMS Shenzhen/Proof Of Concept

Proof of Concept
Enzyme Activities Laccase LCP Hardware Spray Mop Refined Water Cannon Cost Estimation Conclusion

Our technology integrates two kinds of enzymes, Lcp and Laccase CotA, and Laccase enzyme is modified by using amonial-acid mutation for rational design. Then we designed two hardware products, spray mop and refined water cannon, to apply our technology to the field. The separation of the two hardware products is primarily attributed to very different customers and usage scenarios.

Enzyme Activities

In order to develop an enzyme-based cleaner, we determined enzymes utilized to degrade the main ingredients of gum base at first. Latex clearing protein (LCP) is used to degrade natural rubber. And laccase CotA is used to degrade natural ingredients and synthetic ingredients of gum base. Moreover, we decided to enhance the activity of Laccase CotA by optimizing the cultural medium and thermal stimulus, respectively, in the second phase. After that, two approaches, rational design and directed evolution, were applied to improve the substrate affinity and thermal stability of Laccase CotA, respectively.

Laccase

Heterologous Expression

Laccase CotA is from Bacillus sp. HR03 and we expressed Laccase CotA in E.coli BL21 successfully. Next, we purified the expressed Laccase CotA and quantitively determined the enzyme activities of the original Laccase CotA, which is 0.079U/mg after being preincubated at 60 ℃ for 30 min. The enzyme activity data will be used in the following experiment to judge if the modifications to laccase CotA are effective.

Figure 1 | CotA enzymatic activity in different preincubation conditions.

In order to determine the optimal temperature for modified laccase CotA, we conducted a temperature gradient experiment. We found that 60℃ is the optimal temperature of the original Laccase CotA, which is close to the normal temperature of the ground in semitropic and tropic zones, suggesting that Laccase CotA can degrade PE in the gum that stuck to the ground effectively.

Rational Design

We used Homology Modeling, Molecular Docking, Multiple Sequence Alignments to find the two best simulated amino acid mutation. By comparing the abosorbances of ABTS that were oxidized Laccase CotA enzymes at 420nm, we could understand its enzyme activity because oxidized ABTS's absorbance at 420nm is directedly proportional to its enzyme activity. We managed to express 386-mutated Laccase CotA, 497-mutated Laccase CotA, and 386-497-mutated Laccase CotA in BL21. We used purified enzymes to conduct the enzyme activity assay. Although 497-mutated Laccase CotA and 497-386-mutated Laccase CotA managed to present satisfying enzyme activity, 386-mutated Laccase CotA failed to present any enzyme activity. Despite these failures, we still hold hopes for 386-mutated Laccase CotA that its enzyme activities would be improved.

Directed-Evolution

We used EP-PCR to provide multiple opportunities for Laccase CotA to mutate spontaneously. However, we still managed to get the enzyme activity of Laccase CotA-2-12 and Laccase CotA-2-8 using roughly purified enzymes, which suggesting promising enzyme activity.

Figure 2 | enzymatic Activities of original and mutation CotA.

Latex Clearing Protei (LCP)

We successfully expressed Lcp in E.coli BL21, but we failed to purified Lcp in a larger scale. As a result, crude-purified Lcp would be used for the following experiments.

Longterm Functionality

We put Lcp into different temperature conditions to see the effect of the length of time of thermal activation of the enzyme activity of Lcp. We found that after being thermal activated for 180min, which indicates its potential to function for a long time after being sprayed on the gum that sticks to the ground.

Figure 3 | Enzymatic Activity of LCP.

Hardware

Based on the experiment results of Laccase enzyme and Lcp enzyme and the need for gum cleaning in different environments, we designed the spray mop, which is supposed to be used to clean gum in indoor environments, and the refined water cannon, which is supposed to be used to clean gum in outdoor areas.

Spray mop

The spray mop is designed to be used inside the buildings. It could be roughly divided into two parts, the srayer and the ordinary mop. The sprayer contains a nozzle, a liquid storage tank, a long thin tube and a spraying trigger. It could be considered that this sprayer is a lengthened one. It is necessary to fill the liquid tank with some liquid before pulling the trigger that is located underneath the handle. Once the trigger is pulled, the liquid would be sprayed out from the nozzle that locates a few inches up from the bottom of the mop pole. It is proven that the actual spray mop can successfully spray enzyme solution on the gum stuck to the ground, and the gum can be cleaned if the cleaner wipes the floor with some strength.

For specific details of hardware construction and user manual script, please visit Hardware.

Refined water cannon

The refined water cannon is designed to be used in outdoor enviroment. It can also be roughly divided into two parts, the ordinary water cannon and the double-pipe connection joint. This joint would allow street cleaners to connect the water cannon with two seperate pipes at the same time, and the cleaners can easily control which type of liquid in which pipe they want to jet out of the water cannon and scour the sticky rubbish on the street. In the case of using our hardware products to clean gum on the streets, the two different kinds of liquid are referred to as water and our enzyme solution, and the sticky rubbish is referred to as gum. It has been proven that the double-pipe connection joint can function expectedly, and the refined water cannon was able to clean the gum stuck in the street.The refined water cannon is designed to be used in outdoor enviroment. It can also be roughly divided into two parts, the ordinary water cannon and the double-pipe connection joint. This joint would allow street cleaners to connect the water cannon with two seperate pipes at the same time. The cleaners can easily control which type of liquid in which pipe they want to jet out of the water cannon and scour the sticky rubbish on the street. Using our hardware products to clean gum on the streets, the two different kinds of liquid are referred to as water and our enzyme solution, and the sticky rubbish is referred to as gum. It has been proven that the double-pipe connection joint can function expectedly, and the refined water cannon was able to clean the gum stuck in the street.

For specific details of hardware construction and user manual script, please visit Hardware.

Refined water cannon

The spray mob can be directed purchased from the internet, and the average price of it ranges from 10 $to 20$. A normal water cannon would cost from 5 $to 38$. The additional part of double-pipe connection joint would cost no more than 5 $. The average price of 1 g commercial Laccase is 50$. The average price of 1 g commercial ABTS is 36 $. The average price of 1 g of Lcp is 45$. The average price of 1L citrate buffer is approximately 25.6 $. Last but not least, 1L of our pre-mixed enzyme solution would approximately cost 125.28$. All in all, the total price of our product with 1L pre-mixed enzyme solution should range from 14.28 $to 150.28$.

Conclusion

Based on the experiment results, both Laccase and Lcp have expected enzyme activities, thought they might not be quantitatively determined, and both the enzyme managed to function in the both of the hardware products. Additionally, the cost of our hardware is very affordable. Therefore, it is proven that our project is both theoractically successful and practically successful.

References

[1]Osma, J. F., Toca-Herrera, J. L., & Rodríguez-Couto, S. (2011). Cost analysis in laccase production. Journal of environmental management, 92(11), 2907–2912. https://doi.org/10.1016/j.jenvman.2011.06.052