Overview
Inshore pollution has been concerned for scientists and governments in recent years. Although the inshore ocean only is small part of entire ocean, the inshore ecology plays an important role of the entire marine ecology. That inshore ocean neighbors to land provides abundant nutrients and sunlight for marine organisms, which offers an excellent habitat for benthic organisms and the juvenile stages of a large number of marine organisms, such as corals and sea anemones. Therefore, inshore ocean attracts many organisms to migrate to live during their breeding seasons and complete reproduction activities. The seagrass ecosystem that supports a large number of marine consumers also requires the support of the inshore ecosystem. The inshore area is where a large number of halobios activities initial and live. These factors determine its significance.
However, since the inshore ocean is adjacent to land and very close to human activities, the inshore ecology suffers more pollution problems than other sea areas. Pollution such as chemical pollution, agricultural pollution, medical pollution, and domestic pollution has caused very serious damage to the inshore ecology. Fortunately, with the efforts of the government and many institutions in education and propaganda for many years, the public's awareness of marine protection has been arousing year by year. Factory owners, agricultural workers, and the public will pay more attention to reducing pollution emissions in their daily lives to avoid water pollution and thus try their best to prevent inshore areas from pollution. Due to long-term education and propaganda, the public has already known the harms of many pollutants, such as heavy metal pollution, plastic pollution, petroleum pollution, some types of organic pollution and inorganic pollution, etc, and can spontaneously avoid these pollutants from entering nature. However, there are still some pollutants that are easily neglected in daily life, medical activities, agricultural production, or industrial production, which can also deteriorate the marine ecosystem. The consequences of these troubles become manufest gradually and slowly: it is difficult for people to react to those pollutants before bad consequences become manifest.
In this time, let us introduce a category of pollutants, phenolic compounds, which is being neglected in our daily lives.
Phenol is a organic compound containing a phenolic group. Such as phenol, cresol, chlorophenol, and estrogen. Some of these compounds can directly damage the cell structure of organisms, some will affect the growth and reproductive activities of organisms, and the other will cause damage to the nervous system and respiratory system of marine organisms. What's more severe is that most of these compounds are difficult in degradation and are easily accumulated in various organisms, which leads to more serious problems for the inshore marine ecology. Besides. Although most of these pollutants come from agricultural production, human daily activities, and medical activities, most people do not know the existence and harm of phenolic pollutants except for researchers and ecological workers , since the names of most phenolic pollutants are hard to memorize and learn.
Therefore, as a iGEM participant, QS-FMI, will try solve the phenols pollution problem in our first participation in iGEM. We plan to design a synthetic biological pathway for this problem and see if our first attempt at synthetic biology can bring some new solutions to this overlooked problem!
Sources of phenolic compounds
Industrial emissions
Factories related to Petrochemicals, pharmaceuticals, and printing and dyeing usually produce a large amount of phenolic wastewater.
Urban domestic sewage
Urban domestic sewage also contain phenolic compounds, mainly from chemicals used in household and commercial activities and landfill leachate.
Phenolic compound pollution in agriculture
Pesticides and fertilizers may contain phenolic compounds. They will enter nearby water source if raining. Eventually, the phenolic compounds will enter ocean through water cycle.
Stocking breeding waste
In Stocking breeding industry, animal feces, urine, and the blood shed by animals in slaughterhouses contain sex hormones, which become a source of pollution source of phenolic compounds.
Impact of phenolic compounds on marine creature
I. Physiological impacts
1.Hinder growth and development
1.Phenolic compounds can interfere with the metabolic processes of marine organisms. For example, they can deteriorate the photosynthetic efficiency of marine algae, leading to a slower growth rate and smaller individuals. For marine shellfish and other organisms, phenolic compounds may interfere with calcium absorption and shell formation, affecting their growth and development.
2.Impair reproduction
1.Phenolic compounds have adverse effects on the reproductive systems of marine organisms. They may cause abnormal gonad development in fish, affecting the quality and quantity of germ cells and deteriorate the success of reproduction. For marine invertebrates such as shrimp and crabs, phenolic compounds may interfere with their reproductive endocrine systems; it affect their mating behavior and spawning amount.
II. Behavioral impacts
1.Decline athletic ability
1.Marine organisms exposed to an environment polluted by phenolic compounds may experience a weakened activity ability. For example, the swimming speed of fish may slow down, and their ability to escape predators and find food may decrease. Shellfish may reduce filter-feeding activities and lower their food intake.
2. Abnormal behavior
1.Phenolic compounds can also induce abnormal behaviors of marine organisms: some marine organisms may behave abnormally with excessive excitement, or excessive inhibition, which affecting their survival and ecological functions.
III. Impacts on the immune system
1.Compromised immunity
1.Phenolic compounds can suppress the immune system function of marine organisms. This makes marine organisms more susceptible to pathogens such as bacteria, viruses, and parasites, increasing the risk of disease.
Our Solution
Laccase
Laccase is a copper-containing polyphenol oxidase, widely existing in plants, fungi and insects. It plays an important role in the biodegradation of phenolic compounds.
The catalytic active site of laccase contains multiple copper ions and is capable of catalyzing the oxidation reactions of phenolic compounds, aromatic amines and some non-phenolic aromatic compounds. These reactions are usually single-electron transfer reactions, oxidizing the substrate to the corresponding quinones or other oxidation products.
Laccase has wide applications in fields such as environmental science, biomedicine and food industry. In wastewater treatment, laccase can be used to degrade toxic pollutants such as phenols and anilines.
Mechanism of degradation of phenols by laccase
Substrate Binding and Electron Transfer:
The Role of Copper Ions: The catalytic active site of laccase contains a cluster composed of four copper atoms. Among them, the type I copper ion is in an oxidized state and is capable of absorbing a single electron from the reduced phenolic substrate. After the phenolic compound as the substrate approaches the active site of laccase, it transfers one electron to the type I copper ion in the laccase molecule, and is oxidized itself to form a free radical. This process is the key initial step for laccase to decompose phenolic compounds, and the substrate undergoes an oxidation reaction through the transfer of electrons.
Electron Transfer Pathway: After the type I copper ion absorbs the electron, it transfers the single electron to the trinuclear copper cluster center through the Cys-His pathway.
Oxygen Reduction
Formation of Transients: At the trinuclear copper cluster center, the oxygen molecule that acquires the electron is reduced. This reduction process occurs step-wise, and a super-oxide transient is formed initially. This is an intermediate state where the oxygen molecule undergoes a preliminary reduction reaction after acquiring the electron but has not yet been completely converted into water.
Production of Water: The superoxide transient further reacts and is ultimately reduced to water. Throughout the reaction process, the oxygen molecule acts as an electron acceptor, continuously accepting electrons transferred from the substrate, and is reduced to water. In this way, laccase uses molecular oxygen to oxidize and decompose phenolic compounds, and it is not consumed in the reaction process but only plays a catalytic role.
Secondary Reactions Initiated by Free Radicals
Subsequent Reaction Changes: After the phenolic substrate is oxidized to form a free radical, the free radical will trigger various non-enzymatic secondary reactions, such as hydroxylation, disproportionation, and polymerization. These secondary reactions will cause further changes in the structure and properties of the phenolic compound, and ultimately decompose the phenolic compound into relatively stable products. For example, polymerization reactions may occur between multiple phenolic free radicals to form larger molecules; or the free radical may react with other molecules to introduce functional groups such as hydroxyl groups and change the properties of the molecule.
Overall, through its unique copper ion catalytic core, laccase achieves the oxidation and decomposition of phenolic compounds, converting them into other substances that are relatively easier to handle or less harmful to the environment. This property makes laccase have important application value in fields such as environmental remediation and wastewater treatment.
Synthetic Biology Utilization
The laccase gene derived from rhizobia was integrated onto the pET-6xHis vector by using synthetic biology methods. Subsequently, the vector carrying the laccase gene was introduced into BL21(DE3) super competent bacteria to make them our bacterial components and become the source of our laccase production factory.
The device carrying laccase
Seawater is flowing. If we put specific enzymes into the flowing water to treat phenolic compound pollution, the cost will be extremely high due to the fluidity of seawater. The enzymes themselves do not participate in chemical reactions, but the flowing seawater will cause our laccase to fail to function on where it is needed for treatment. Therefore, we should put the laccase in a container to prevent it from leaving its original position.
UHWPE Membrane-enzyme-membrane
We embeds the enzymes in UHWPE membranes to form a membrane-enzyme-membrane structure. This membrane has high strength and can resist deterioration from complex environments at the bottom of the water to avoid plastic pollution from self disintegration of membrane. The device is replaceable and has an alarm device to facilitate timely replacement of the membrane. This technology can purify water bodies in flowing water sources and has the following advantages:
1.The size of device is flexible.
2.It does not damage effective compounds. Easily settled in the water , it purifies pollution as water flows through.
3.No infrastructure construction is needed. Just replace the built-in membrane regularly.
4.Sensors can be added to detect water quality in real time.
5.No biological toxicity: It does not affect the existing ecological safety of the water body.
6.Different kinds and proportions of embedded proteins can be added to respond to the pollution situation of the specific water.
The detailed information about our hardware can view our hardware page.