Team:KEYSTONE/Description


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Overview

Rubber is widely used by humans for its high versatility. However, rubber waste is a pressing global issue. As COVID-19 struck the entire world, the demand for medical gloves has increased significantly and generated a large amount of rubber waste. The global rubber waste totals to 70 million tons in 2021. It is difficult to safely dispose of rubber waste because the current methods of managing rubber waste are risky, for which the landfilling and incineration of rubber fail to degrade rubber and threaten the environment and human health. Although the recycling of rubber waste is safe, its coverage and implementation are limited. Therefore, we have developed a new and preferable solution to tackle the growing rubber waste issue – biodegradation of rubber. By using Lcp (latex clearing protein) produced by E. coli, we aim to achieve safe biodegradation of natural rubber.

Human utilization of rubber

Natural rubber is an elastic substance that most commonly comprises the elastomer polyisoprene. Natural rubber is mostly derived from latex, a milky fluid produced by some plants, especially from the tropical rubber tree Hevea Brasiliensis. Natural rubber has high tear resistance, heat resistance, chemical resistance, and electrical resistance, and through the process of vulcanization – mixing the rubber with sulfur – it will become harder, more elastic, and more resistant to extreme temperatures. These properties make natural rubber a very useful material for a myriad of applications.

Humans has been utilizing rubber for over a thousand years. Around 3,500 years ago, people in Mexico and Central America were already harvesting and using natural rubber in liquid forms for medicinal applications and painting. Nowadays, however, more than half of all rubber produced goes into automobile tires, and the rest goes into mechanical parts, as well as consumer products. Rubber is in high demand by manufacturers due to its low cost and satisfactory performance. According to the Xinhua News Agency, the amount of waste tires in China is as high as 20 million tons. In addition, as COVID-19 struck the entire world, the demand of medical gloves especially steeply rose and generated a significant amount of sundry rubber waste globally. According to the Malaysian Rubber Glove Manufacturers Association (MARGMA), the increase in the demand of medical gloves during the COVID-19 pandemic is 57% higher than that of before the pandemic. This created a considerable amount of rubber gloves waste. Together with all other kinds of rubber waste the global rubber waste totals to 70 million tons.



Common Applications of Rubber:

  • Transportation
  • Military
  • Architecture
  • Electrical appliances
  • Healthcare
  • Sports
  • Science research
  • Other consumer products

Rubber waste disposal

The amount of rubber waste continues to grow at an alarming rate. If they are directly discarded into the environment and left untreated, rubber waste is dangerous as it causes serious pollution and damage to people’s physical health. Therefore, it is necessary to implement proper disposal methods in handling the waste rubber products. Nowadays, rubber waste is treated mainly with the following three methods:

Incineration

Combustion of rubber is a common method of rubber processing. In operation, the rubber is grounded into small particles and then placed in a uniform kiln system for incineration.

Take tire, a common rubber product as an example. Due to their higher heat content, and lower moisture and nitrogen content comparing to coal, tires are well suited for energy recovery through combustion. Therefore, the use of waste tires as a supplementary or alternative fuel in various industrial combustion facilities has become one of the most important alternatives for disposal.

However, burning rubber poses serious acute and chronic health hazards to people nearby. Depending on the duration and level of exposure, these health effects may include skin, eye and mucous membrane irritation, respiratory effects, central nervous system depression, and cancer.

Furthermore, tire incineration produces heavy metal and air pollutants, such as benzene, furan, and dioxin, which is highly toxic and can cause serious health problems, including infertility, learning disabilities, endometriosis, sexual reproductive disorders, birth defects, damage to the immune system, and cancer. Besides, dioxin is fat-soluble and once it is released into the environment, it can easily climb up the food chain, reaching people’s diet and increasing the risk of dioxin exposure. In fact, the now-closed tire incinerator in Modesto, California, released 0.0236 grams of dioxin in a single year. This, according to the EPA's risk-based emission standards, is more than the maximum dose a person should consume.

In addition, the incineration of rubber also causes hazardous environment risks. Each burning tire releases over two gallons of oil into the environment, damaging the soil and potentially contaminating the surface and underground water sources. From an environmental perspective, burning rubber also result in significant emissions of carbon dioxide, nitrogen oxide, and sulfur oxide – all of which are major contributors to global warming.



Fire and smoke rips through tire graveyard in Kuwait, 2021


Landfill

As one of the most common methods of rubber waste processing, burying is negative for being incompetent and environmentally devastating.

To begin with, burying cannot degrade rubber. Once being placed underground, rubber waste products, such as tires, imposes considerable damage on the environment through harmful chemicals. For instance, methane gas can be emitted into the air just under solar irradiation, escalating global warming as a greenhouse gas. Furthermore, the toxic gases leaking from waste rubber also has negative health effects, such as blood damage, liver damage, urinary system damage, nervous system damage, skin damage and allergic effects, carcinogenic, teratogenic, etc.

Most rubber wastes are highly flammable for high concentration of fossil fuel components. Fire can hardly be eliminated once being caught on, even a small quantity burns for months until running out of fuel. During this process, clouds of black poisonous smoke containing various chemicals incorporated in production, including nitrogen oxides, sulfur oxides, heavy metal particles, and furane, will be simultaneously released. The flame cannot be put out with water since it can seep into underground aquifers and pollute water sources like lakes and ponds, though this can also happen without letting water come in eliminating fire. Similarly, advantageous soil bacteria that nourish flora and fauna will be eliminated by these toxic chemicals, which damage the habitat of various plant and animal species and putting their survival at risk.

Recycling

Recycling is by far the safest method to dispose of waste rubber. Through devulcanization or different processes of retread, shredding, or pyrolysis, recycled rubber can be applied to a variety of other products, from shoes to mulch used for sports turfs. Rubber recycled into new products cost less and reduces energy consumption compared to using new rubber. Recycling also alleviates the pressure on natural rubber and the plantation of rubber trees and prevents spatial usage from landfills. Furthermore, it reduces environmental pollution, in which every four tires recycled reduces carbon dioxide emission by 343 pounds.

However, most of the wasted rubber cannot enter the recycling system. According to EPA, only 18.2 percent of the total rubber waste was recycled, while the others are being burnt or buried. Among the 242 million tires discarded in the United States annually, only less than 7 percent are recycled, 11 percent utilized as fuel, 5 percent exported, and the remaining 78 percent entering the landfilling system or illegally being dumped, creating serious environmental effects. In general, although recycling is a valuable waste rubber processing method, its coverage is limited.

What We Do

Seeing all the risks mentioned above, the current rubber recycling methods are not comprehensive enough to reach the benefits of all stakeholders. The rubber waste that cannot be recycled goes back to incineration or landfilling. However, incineration and landfilling aggravate a variety of pollutions, and creates potential dangers for the habitants and lives around their sites. On the other hand, the coverage of recycling is way too low to create actual effects. Therefore, a more satisfactory solution is urgently needed – biodegradation.

Our team has developed a safe method to biodegrade natural rubber, which utilizes the protein Lcp (latex-clearing protein) and is refined by adding several fusion proteins and signal peptides. For more information, please check out our "Proof of concept" and "Results" page.


What is Lcp?

In order to achieve a sustainable cleavage of the rubbers and tires, we introduce latex clearing protein. The latex clearing protein(Lcp) is belonged to the group of enzymes for oxidative cleavage of poly(cis-1,4-isoprene) ---which is the chemical basis for most of the synthesizing rubbers on the market. Lcp, which stands for latex clearing protein, is an extracellular dioxygenase which has the ability to decompose rubber by endo-cleaving the poly(cis-1, 4-isoprene) into pieces. Unlike other kinds of protein that can also degrade rubber in some ways, Lcp produces a mixture of cleaving products other than isoprene pieces alone. Therefore, it can be concluded the polymer can be better made use of with Lcp. (5) More specifically, the final product after the cleavage process, oligo(cis-1,4-isoprenoids) is a potentially valuable substance that can be reused in many fields.

Figure 1 cleavage reaction of poly(cis-1, 4-isoprene) by rubber cleaving enzymes.


There are mainly two types of commonly used Lcp: LcpK30 and Lcp1 VH2. In our project, we are looking for the best Lcp considering their rubber degrading ability. Although both Lcps come from the same category, there are still some slight differences between them two.

1. LcpK30.:

Lcp was first discovered in Streptomyces sp. strain K30, so the latex clearing protein from this strain was named LcpK30. We found out LcpK30 is the most commonly used one within all different teams according to the past iGem parts uploaded. The picture below is the three-dimensional structure of LcpK30. The amino acid sequence similarity between LcpK30 and LcpVH2 is said to be 50%.

Figure 2. A three-dimensional picture of LcpK30 colored from blue at the N-terminus to red at the C-terminus.


2. Lcp1 VH2:

Lcp1VH2 came from Gordonia polyisoprenivorans VH2. Study has shown that Lcp1VH2 can be grown heterogeneously in E. coli with a relatively high yield. An active degradation activity is observed in vitro experiment at the same time. In addition, Lcp1VH2 is also remarkable for its fast metabolization. Therefore, Lcp1VH2 was included into the target protein products we plan to focus on in our experiment.

However, Lcp, as introduced in the articles, is flawed. The main problem of Lcp is its insolubility. Solubility is usually a great problem in most protein production, because insoluble protein will not allow any further processing of protein. As a result, the insolubility of protein will contribute to a low concentration of target protein after purification process. In order to overcome this problem a series of research and experiments were done.

Fusion partner- NusA

In order to increase the overall yield of Lcp in the experiment, we found a fusion partner, which called NusA. In general, NusA can work as a solubility enhancer when it is connected with Lcp1VH2, and consequently increase the concentration of Lcp1VH2 produced after being cultivated in E.coli C41::pET23a. In the article, a composite sequence is constructed with Lcp1VH2 and NusA. His6 tag was added due to further purification purpose. The configurations below show the structure of the plasmids. In the end, the highest solubility was achieved by NusA-His- Lcp1VH2 with an enhancement of 5.7-fold compared to the original Lcp1VH2. Considering the prominent increment in the yield due to the contribution of NusA, we decided to use it in our experiment as a result.

Figure 3 Vector with the genes encoding of NusA and Lcp1VH2.


Signal peptide designs

1. Signal peptides

In order to further solve the problem of productivity of Lcp, we introduce the use of several signal peptides. In the traditional fermentation process, protein needed to be centrifuged and broken before it can be used or purified. However, according to our research, the solubility of Lcp is poor due to the formation of inclusion body, and a large part of Lcp production is lost in the purification process. Therefore, it seems like if we can solve the problem of inclusion body and avoid the purification process to some extent, we will be able to get higher concentration of soluble protein in the production.

Three types of signal peptides included in the design of our experiment are---Hlya、GeneIII and DsbA. With the signal peptides, we should be able to induce E.coli to excrete the protein produced in its body to the surrounding environment from the cell cytoplasm. While the protein gets to be transported to the outside, it makes direct contact with rubber. Instead of breaking the cells and obtaining protein from them in the form of inclusion body, secretion of protein to the surrounding is definitely better, since protein can have more effective reaction with its substrate in this way. Additionally, using signal peptide will also help to relieve the burden of E. coli. Lcp is initially a toxic substance for E. coli. Without the help from signal peptides to excrete the Lcp that the E. coli produces, Lcp will accumulate inside its body, and eventually destroy it. Therefore, the use of peptide will increase the solubility as well as the sustainability of rubber degrading bacteria.

2. GFP and Laccase as reporters of Lcp secretion

Even though the yield and degrading ability of Lcp can be measured in some ways, we need a visual indicator to show the secretion of Lcp more clearly. GFP and laccase are the two reporters that have been used in our experiment. Vector p47 with GFP is used to examine the effectiveness of different peptides at first. However, we soon realized that it was not the most visual and straight-forward way to see the secretion of Lcp. As the result, laccase was used.

Laccase will only react with a specific substrate---ABTS. As we encode laccase into the cell, it will produce laccase along with Lcp. Since ABTS is filled into the LB, it will react with laccase that produced by the cell and soon be oxidized, turning into the color of bullish-green. Therefore, by using laccase, we will be allowed to observe the secretion of Lcp according to the color change.

Reference

Andler, R., Heger, F., Andressen, C., Steinbüchel, A., 2019a. Enhancing the synthesis of latex clearing protein by different cultivation strategies. J. Biotechnol 297,32-40. doi: 10.1016/j.jbiotec.2019.03.019

Altenhoff A-Lena, Thierbach S, Steinbu ̈chel A, High yield production of the latex clearing protein from Gordonia polyisoprenivorans VH2 in fed batch fermentations using a recombinant strain of Escherichia coli, Journal of Biotechnology (2019), doi: https://doi.org/10.1016/j.jbiotec.2019.12.013

Altenhoff, A. L., Thierbach, S., & Steinbüchel, A. (2021). In vitro studies on the degradation of common rubber waste material with the latex clearing protein (Lcp1 VH2) of Gordonia polyisoprenivorans VH2. Biodegradation, 32(2), 113-125.

Hiessl, S., Böse, D., Oetermann, S., Eggers, J., Pietruszka, J., & Steinbüchel, A. (2014). Latex clearing protein—an oxygenase cleaving poly (cis-1, 4-isoprene) rubber at the cis double bonds. Applied and environmental microbiology, 80(17), 5231-5240.

Ilcu, L., Röther, W., Birke, J., Brausemann, A., Einsle, O., & Jendrossek, D. (2017). Structural and functional analysis of latex clearing protein (Lcp) provides insight into the enzymatic cleavage of rubber. Scientific reports, 7(1), 1-11.

Nayanashree, G., & Thippeswamy, B. (2015). Natural rubber degradation by laccase and manganese peroxidase enzymes of Penicillium chrysogenum. International journal of environmental science and technology, 12(8), 2665-2672.