We live in a world full of plastic, plastic is everywhere around us, our drink cups, shopping plastic bags, food packaging film, snack bags... Even the clothes we wear contain plastic. With the development of technology, it is becoming increasingly difficult to avoid the use of plastics in our daily lives.
Fabrics made of plastic, such as polyester, spandex, because of their durability, wear resistance, good formability, more and more used in industrial production, no longer limited to the traditional clothing industry, such as our experimental object - filter cloth. Industrial filter cloth is a filter medium woven by natural fiber or synthetic fiber, mainly made of polyester, widely used in automotive, machine tools, metallurgy, non-ferrous metal industry filtration and dust removal. In terms of industry background, more than a dozen large filter cloth production enterprises in the country are promoting the development of China's filter cloth industry with its vitality, but whether from the scale, or from the technical level, the development of these enterprises still can not meet the demand for filter cloth at home and abroad, China in the next decade will become the fastest growing country in the international filtration and separation industry. The market size ranks second in the world, and the filter cloth industry, an emerging environmental protection market, is booming, and the development of China's filter cloth market has immeasurable potential.
In addition to the above applications, around the key areas of safety, environmental protection, energy saving, etc., the filter cloth also uses new energy, new materials, and new processes to develop new products and occupy the commanding heights of technology. In the world's increasing attention to environmental protection, the broad application prospects of filter cloth can not be underestimated. With the gradual increase of the western development, landfill, sewage treatment and other projects have gradually increased, and the amount of filter cloth has gradually increased. To sum up, the filter cloth industry has a high advantage in terms of policy support, environmental protection demand, equipment supporting, raw material supply, or domestic and foreign market demand, and its market prospects are very considerable.
Our team learned that in Nanjing, many chemical, automotive, petroleum and other heavy industries and factories, such as Sinopec, China Construction, automobile new energy factories, etc., the filter cloth used in these factories will be discarded after a certain period of time, and transported to landfill like ordinary plastic waste, causing great pollution and harm to the environment. The polyester in the filter cloth is one of the main sources of pollution. As a widely used chemical fiber, polyester has accounted for more than 80% of the output of chemical fiber in China in recent years. According to our investigation, as shown in Figure 2, China's polyester production has been increasing in recent years, and exceeded 50 million tons of polyester production in2021.
Although the production of polyester is increasing, the pollution it brings is also increasing. Each kilogram of chemical fiber, polyester and other spinning requires an increase of 3.6 kilograms of carbon dioxide emissions, consumption of 6,000 liters of water, and the use of 0.3 kilograms of fertilizer and 0.2 kilograms of pesticides. Every year, a large number of waste polyester textiles are thrown into the trash, and the number is amazing. If the wasted resources are recycled, the annual supply of chemical and natural fibers is equivalent to saving 24 million tons of crude oil and reducing 80 million tons of carbon dioxide emissions.
Based on this background, our team intends to develop a new polyethylene terephthalate (PET) plastic degrading enzyme to solve this problem, based on the special experimental background of Escherichia coli with a wide expression system and IsPETase hydrolase showing excellent PET hydrolysis activity under mild conditions. We will use the expertise of synthetic biology to efficiently and green degrade PET in industrial filter cloth, make contributions to the city, promote the construction of ecological civilization, and promote sustainable green development.
The problems of modern biotechnology in the field of PET degradation
Although modern biotechnology has made some progress in the field of polyethylene terephthalate (PET) degradation, there are still some problems and challenges:
PETase soluble expression: In the pre-experiment, when we introduced pET-22b-LSPETase plasmid into E. coli for expression, we found that the expression of the target protein in Escherichia coli cells often formed inclusion bodies, and the reason for the formation of inclusion bodies was mostly that the expressed protein failed to fold correctly. In addition, the intracellular reductive environment of E. coli is not conducive to the formation of protein disulfide bonds, and proteins expressed at high levels can easily aggregate to form inclusion bodies. The extremely low expression of this enzyme leads to not further study its function later.
PET substrate binding: Due to the hydrophobicity of the PET substrate surface, it is difficult for PETase to bind to the substrate. The hydrophobic domain CBM usually has hydrophobic amino acid residues that interact with the PET surface, which can help PETase bind to the PET substrate more tightly and improve the reaction efficiency. If PETase is connected with hydrophobic CBM, PETase connected with hydrophobic CBM will be more easily bound to the PET surface under the force of water molecule movement.
Degradation efficiency: Currently discovered PET degrading enzymes are slow and have limited efficiency in degrading PET. The degradation process may take a long time to fully break down PET, which may not be efficient enough for practical applications.
Species adaptability: The identified PET-degrading enzymes are mainly derived from a few species of bacteria and fungi, and their adaptability is limited. Different types of PET (such as PET fibers and PET bottles) may have different structures and properties, so a wider range of enzyme resources are required to adapt to various PET materials.
Disposal of degradation products: Some compounds in PET degradation products can be toxic or potentially harmful to the environment. Therefore, it is necessary to ensure the safety of degradation products.
Despite these problems and challenges, scientists and engineers are still working to solve the technical challenges of PET degradation. With further research and technological innovation, it is hoped that these problems can be overcome and more efficient, viable and sustainable PET degradation technologies can be achieved.
C. Experimental object
PET, also known as polyethylene terephthalate, is a plastic commonly used in electronic appliances, which can be spun into polyester fibers, made into films, beverage bottles, and other electrical components. Although PET is widely used, it also brings a lot of harm to the environment, especially in the case of imperfect waste incineration technology, and the toxic substances produced by thermal decomposition will pollute air and water sources. Technically, PET can be rapidly degraded by enzymes into molecular monomers and then repolymerized or transformed/value-added into other products to achieve the circular carbon economy of PET.
In 2005, enzymes that can degrade PET were first reported and 19 different PET hydrolases (PHEs) were preliminarily demonstrated. However, most of these enzymes can only exhibit significant hydrolytic activity at high reaction temperatures and highly processed substrates. Most other PET hydrolases are less active at moderate temperatures and neutral pH. This greatly limits in-situ/microbial degradation solutions for PET waste.
To realize the efficient enzymatic degradation of PET, enzyme preparation is the first key point. E. coli is commonly used for the PETase expression. However, when foreign proteins (ie. LSPETase identified from Oleispira antarctica in our lab) are expressed in E. coli, the expressed proteins often form inclusion bodies. To solve this common problem, we tried to harness molecular molecular chaperones, fusion tags, and signal peptides with LSPETase as an example to realize the soluble expression.
Further, to obtain a more efficient PETase, the typical IsPETase was chosen as the original template. Different carbohydrate-binding modules were fused to IsPETase to facilitate the binding of substrate PET and enhance the degradation. Additionally, a mutation strategy was also carried out in IsPETase to obtain more efficient mutants for PET degradation. During the preparation of superior IsPETase, the soluble expression strategies were also harnessed to solve the problem of inclusion bodies.
The project plans to design an efficient IsPETase to bsignal preak down PET in the environment, typically filter cloth, for the benefit of humans.
D. Experimental content
4.1 Soluble expression
E. coli is often used as a host bacterium for protein expression due to its simple system and wide range of options for expression systems. We use E. coli to efficiently produce IsPETase, and when foreign proteins are expressed in E. coli, the expressed proteins often form inclusion bodies or are degraded by proteases, mostly because the expressed proteins are not folded correctly, which is not conducive to studying protein function. Therefore, we combine LSPET with some molecular molecular chaperones, fusion tags, and signal peptides for soluble expression.
4.1.1 Molecular chaperone
The Chaperone Plasmid Set contains three types of plasmids, each of which efficiently expresses a different type of chaperone proteome that works synergistically to participate in protein folding to increase the recovery of soluble proteins.
4.1.2 Fusion tags
1、 TrxA:It is thermally stable, highly soluble and has solid folding properties. Often used as a fusion protein to improve the expression of small peptides. It increases solubility and expression rate, aiding in the refolding of target proteins.
2、NusA:It is a highly soluble protein that binds and isolates the aggregation-prone folding intermediates of its target protein, preventing its self-association and aggregation. Placing highly soluble proteins into proteins with low solubility can promote protein synthesis. It states that fusing targeted enzymes into highly soluble proteins can increase the yield of recombinant proteins, prevent proteolysis, and improve their solubility
4.1.3 Signal peptide
1、DsbA:Used to transfer the resulting protein into the periplasm, the accumulation of expressed proteins in the periplasm can be used for protein purification and prevent interaction with cytoplasmic components.
2、OmpA: Outer membrane protein A (OmpA) is an N-terminal signaling peptide,which is a small protein with a structural length of 20-30 amino acids. It can transfer the produced proteins to the periplasmics pace and utilize the oxidizing environment in the periplasmic space to promote the formation of dis ulfide bonds, thereby facilitating the proper folding of soluble proteins and enhancing soluble expre ssion.
4.2.1 Mutation
According to the literature research, we found that most of the scholars used the mutation strategy to modify IsPETase. Therefore, we also decided to apply the mutation strategy to improve the activity of IsPETase. By analyzing the structure and mechanism of IsPETase, we selected 10 mutation sites, namely IsPETaseS93_I94insE, IsPETaseT116R, IsPETaseQ119F, IsPETaseS121E, IsPETaseQ126L, IsPETaseM157A, IsPETaseM157A, IsPETaseM157A, IsPETaseM157F, and IsPETaseM157A. IsPETaseM157A, IsPETaseM157S, IsPETaseW159H, IsPETaseW185F, and IsPETaseA240_S242del.
4.2.2 Hydrophobic structural domains
In addition to using a mutation strategy to enhance the activity of IsPETase, which is essentially a change in the structure of the protein, we have also considered altering the extrinsic properties of the enzyme in order to enhance its activity. We decided to attach a hydrophobic domain to the highly efficient IsPETase mutant to enhance the hydrophobicity of the IsPETase mutant and to facilitate the binding of the IsPETase mutant to the hydrophobic surface of PET. We chose five structural domains: CBM3, CBM4, CBM11, LSChiCBM4, and LSChiCBM5.
