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Plastic pollution is becoming a major health issue due to the recent discovery of microplastics and nanoplastics in living organisms and the environment, calling for advanced technologies to remove plastic waste. Here we review enzymes that degrade plastics with focus on plastic properties, protein engineering and polymers such as poly(ethylene terephthalate), poly(butylene adipate-co-terephthalate), poly(lactic acid), polyamide and polyurethane. The mechanism of action of natural and engineered enzymes has been probed by experimental and computation approaches. The performance of polyester-degrading enzymes has been improved via directed evolution, structure-guided rational design and machine learning-aided strategies. The improved enzymes display higher stability at elevated temperatures, and tailored substrate-binding sites. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion. Polyethylene terephthalate (PET) can be depolymerized by the Ideonella sakaiensis PETase enzyme, however, questions remain about the precise catalytic mechanism. Here, the authors use unbiased QM/MM MD simulations to determine optimal mechanistic descriptions of the acylation and deacylation reactions, revealing the rate-limiting step and key interactions within the catalytic triad and Trp185 conformation. [ABSTRACT FROM AUTHOR]

The application of enzymes is expanding across diverse industries due to their nontoxic and biodegradable characteristics. Another advantage is their cost-effectiveness, reflected in reduced processing time, water, and energy consumption. Although Gram-positive bacteria, Bacillus, and Streptomyces spp. are successfully used for production of industrially relevant enzymes, they still lag far behind Escherichia coli as hosts for recombinant protein production. Generally, proteins secreted by Bacillus and Streptomyces hosts are released into the culture medium; their native conformation is preserved and easier recovery process enabled. Given the resilience of both hosts in harsh environmental conditions and their spore-forming capability, a deeper understanding and broader use of Bacillus and Streptomyces as expression hosts could significantly enhance the robustness of industrial bioprocesses. This mini-review aims to compare two expression hosts, emphasizing their specific advantages in industrial surroundings such are chemical, detergent, textile, food, animal feed, leather, and paper industries. The homologous sources, heterologous hosts, and molecular tools used for the production of recombinant proteins in these hosts are discussed. The potential to use both hosts as biocatalysts is also evaluated. Undoubtedly, Bacillus and Streptomyces spp. as production hosts possess the potential to take on a more substantial role, providing superior (bio-based) process robustness and flexibility. Key points: • Bacillus and Streptomyces spp. as robust hosts for enzyme production. • Industrially relevant enzyme groups for production in alternative hosts highlighted. • Molecular biology techniques are enabling easier utilization of both hosts. [ABSTRACT FROM AUTHOR]

The flexibility and the low production costs offered by plastics have made them crucial to society. Unfortunately, due to their resistance to biological degradation, plastics remain in the environment for an extended period of time, posing a growing risk to life on earth. Synthetic treatments of plastic waste damage the environment and may cause damage to human health. Bacterial and fungal isolates have been reported to degrade plastic polymers in a logistic safe approach with the help of their microbial cell enzymes. Recently, the bacterial strain Ideonella sakaiensis (201-F6) was discovered to break down and assimilate polyethylene terephthalate (PET) plastic via metabolic processes at 30 °C to 37 °C. PETase and MHETase enzymes help the bacterium to accomplish such tremendous action at lower temperatures than previously discovered enzymes. In addition to functioning at low temperatures, the noble bacterium's enzymes have amazing qualities over pH and PET plastic degradation, including a shorter period of degradation. It has been proven that using the enzyme PETase, this bacterium hydrolyzes the ester linkages of PET plastic, resulting in production of terephthalic acid (TPA), nontoxic compound and mono-2-hydroxyethyl (MHET), along with further depolymerization of MHET to release ethylene glycogen (EG) and terephthalic acid (TPA) by the second enzyme MHETase. Enzymatic plastic degradation has been proposed as an environmentally friendly and long-term solution to plastic waste in the environment. As a result, this review focuses on the enzymes involved in hydrolyzing PET plastic polymers, as well as some of the other microorganisms involved in plastic degradation. [ABSTRACT FROM AUTHOR]

For decades, plastic waste management has been one of the major ecological challenges of our society. Despite the introduction of biodegradable alternatives such as polylactic acid (PLA), their beneficial environmental impact is limited by the requirement of specific compost facility as biodegradation of PLA in natural environment occurs at a very slow rate. In this work, a plastic-degrading enzyme was utilized to facilitate degradation process. Genomic and proteomic tools were employed to identify a new biodegradable plastic-degrading enzyme from Cryptococcus nemorosus TBRC2959. The new enzyme, Cr14CLE, functions optimally under mild conditions with temperature range of 30 to 40 °C and suffers no significant loss of enzymatic activity at pH ranging from 6 to 8. In addition to PLA, Cr14CLE is capable to degrade other types of biodegradable plastic such as polybutylene succinate (PBS) and polybutylene adipate terephthalate (PBAT) as well as composite bioplastic. Applications of Cr14CLE have been demonstrated through the preparation of enzyme-coated PLA film and laminated PLA film with enzyme layer. PLA films prepared by both approaches exhibited capability to self-degrade in water. Key points: • Novel plastic-degrading enzyme (Cr14CLE) was identified and characterized. • Cr14CLE can degrade multiple types of biodegradable plastics under mild conditions. • Applications of Cr14CLE on self-degradable plastic were demonstrated. [ABSTRACT FROM AUTHOR]

Recently developed enzymes for the depolymerization of polyethylene terephthalate (PET) such as FAST-PETase and LCC-ICCG are inhibited by the intermediate PET product mono(2-hydroxyethyl) terephthalate (MHET). Consequently, the conversion of PET enzymatically into its constituent monomers terephthalic acid (TPA) and ethylene glycol (EG) is inefficient. In this study, a protein scaffold (1TQH) corresponding to a thermophilic carboxylesterase (Est30) was selected from the structural database and redesigned in silico. Among designs, a double variant KL-MHETase (I171K/G130L) with a similar protein melting temperature (67.58 °C) to that of the PET hydrolase FAST-PETase (67.80 °C) exhibited a 67-fold higher activity for MHET hydrolysis than FAST-PETase. A fused dual enzyme system comprising KL-MHETase and FAST-PETase exhibited a 2.6-fold faster PET depolymerization rate than FAST-PETase alone. Synergy increased the yield of TPA by 1.64 fold, and its purity in the released aromatic products reached 99.5%. In large reaction systems with 100 g/L substrate concentrations, the dual enzyme system KL36F achieved over 90% PET depolymerization into monomers, demonstrating its potential applicability in the industrial recycling of PET plastics. Therefore, a dual enzyme system can greatly reduce the reaction and separation cost for sustainable enzymatic PET recycling. A study on the computational design of enzymes for plastic (MHET and PET) degradation yielded an efficient dual enzyme degradation system for PET recycling with a substantially increased activity and a near pure concentration of product TPA. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate hydrolases (PETases) are a newly discovered and industrially important class of enzymes that catalyze the enzymatic degradation of polyethylene terephatalate (PET), one of the most abundant plastics in the world. The greater enzymatic efficiencies of PETases compared to close relatives from the cutinase and lipase families have resulted in increasing research interest. Despite this, further characterization of PETases is essential, particularly regarding their possible activity against other kinds of plastic. In this study, we exploited for the first time the use of the microalgal chloroplast for more sustainable synthesis of a PETase enzyme. A photosynthetic-restoration strategy was used to generate a marker-free transformant line of the green microalga Chlamydomonas reinhardtii in which the PETase from Ideonella sakaiensis was constitutively expressed in the chloroplast. Subsequently, the activity of the PETase against both PET and post-consumer plastics was investigated via atomic force microscopy, revealing evidence of degradation of the plastics. [ABSTRACT FROM AUTHOR]

Polyethylene terephthalate (PET) is the most abundantly produced plastic due to its excellent performance, but is also the primary source of poorly degradable plastic pollution. The development of environment-friendly PET biodegradation is attracting increasing interest. The leaf-branch compost cutinase mutant ICCG (F243I/D238C/S283C/Y127G) exhibits the best hydrolytic activity and thermostability of all known PET hydrolases. However, its superior PET degradation is highly dependent on its preparation as a purified enzyme, which critically reduces its industrial utility. Herein, we report the use of rational design and combinatorial mutagenesis to develop a novel ICCG mutant RITK (D53R/R143I/D193T/E208K) that demonstrated excellent whole-cell biocatalytic activity. Whole cells expressing RITK showed an 8.33-fold increase in biocatalytic activity compared to those expressing ICCG. Thermostability was also improved. After reacting at 85 °C for 3 h, purified RITK exhibited a 12.75-fold increase in depolymerization compared to ICCG. These results will greatly enhance the industrial utility of PET hydrolytic enzymes and further the progress of PET recycling. [ABSTRACT FROM AUTHOR]

Biodegradable plastics can solve the problem of unwanted plastics accumulating in the environment if they can be given the contradictory properties of durability in use and rapid degradation after use. Commercially available agricultural biodegradable mulch films are made from formulations containing polybutylene adipate-co-terephthalate (PBAT) to provide mechanical and UV resistance during the growing season. Although used films are ploughed into the soil using a tiller to promote decomposition, it is difficult if they remain durable. We showed that an enzyme produced by the leaf surface yeast Pseudozyma antarctica (PaE) degrades PBAT-containing films. In laboratory studies, PaE randomly cleaved the PBAT polymer chain and induced erosion of the film surface. In the field, commercial biodegradable films containing PBAT placed on ridges were weakened in both the warm and cold seasons by spraying the culture filtrate of P. antarctica. After the field was ploughed the next day, the size and total weight of residual film fragments decreased significantly (p < 0.05). Durable biodegradable plastics used in the field are degraded using PaE treatment and are broken down into small fragments by the plough. The resultant degradation products can then be more readily assimilated by many soil microorganisms. [ABSTRACT FROM AUTHOR]

Biodegradable plastics can solve the problem of unwanted plastics accumulating in the environment if they can be given the contradictory properties of durability in use and rapid degradation after use. Commercially available agricultural biodegradable mulch films are made from formulations containing polybutylene adipate-co-terephthalate (PBAT) to provide mechanical and UV resistance during the growing season. Although used films are ploughed into the soil using a tiller to promote decomposition, it is difficult if they remain durable. We showed that an enzyme produced by the leaf surface yeast Pseudozyma antarctica (PaE) degrades PBAT-containing films. In laboratory studies, PaE randomly cleaved the PBAT polymer chain and induced erosion of the film surface. In the field, commercial biodegradable films containing PBAT placed on ridges were weakened in both the warm and cold seasons by spraying the culture filtrate of P. antarctica. After the field was ploughed the next day, the size and total weight of residual film fragments decreased significantly (p < 0.05). Durable biodegradable plastics used in the field are degraded using PaE treatment and are broken down into small fragments by the plough. The resultant degradation products can then be more readily assimilated by many soil microorganisms. [ABSTRACT FROM AUTHOR]

Global warming and plastic pollution result from the massive use of fossil fuels, calling for the development of sustainable bioplastics derived from modern biomass. Bioplastic production is expected to increase to 2.43 million tons globally in 2024. In particular, the production of biopolymers from bioresources and organic waste should highly reduce waste accumulation. Biopolymers can be produced by employing microbes or by using monomers from agro-resources. Here we review the use of lignin and polylactic acid for polymer production using enzymes and microorganisms. Carnobacterium, Enterococcus and Aspergillus niger show high lactic acid productivity of about 0.4–1.4 g/L/h. Lignin and polylactic acid can also be transformed into value-added chemicals such as muconic acid, hydrouronic acid, adipic acid and terephthalic acid. [ABSTRACT FROM AUTHOR]