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Importance: In this study we described the discovery and analysis of new enzymes from microbial communities associated to plants (moss). The recovered enzymes show the capability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate-co-butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be applied as catalysts for selective recovery of valuable building blocks from this material.
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... There, the associated microbiota seems to employ some of the enzymatic mechanisms for deconstructing plant biomass to degrade synthetic plastics, based on some chemical and structural similarities between these polymers. Hence, plantdegrading and host-associated microbial communities have been investigated as source of enzymes and/or microbial consortia to potentially compose strategies for biodegrading plastic waste (Yang et al., 2015b(Yang et al., ,c, 2018Müller et al., 2017;Skariyachan et al., 2017Skariyachan et al., , 2021Peng et al., 2020;Quartinello et al., 2021). Esterases from cow (Bos taurus) rumen were able to partially hydrolyze the polyesters PET, polybutylene adipateco-terephthalate (PBAT, biodegradable) and polyethylene furanoate (PEF, biobased). ...
... btDSCE-01, Enterobacter cloacae btDSCE-02, and Pseudomonas aeruginosa btDSCE-CD03 partially degraded LDPE and PP (Skariyachan et al., 2021). Esterases from the microbiome associated with Sphagnum magellanicum moss hydrolyzed polybutylene adipate-co-butylene terephthalate (PBAT) and substrate bis(4-[benzoyloxy]butyl) terephthalate, highlighting the potential of plant-associated microbiomes as source of polymer degrading enzymes (Müller et al., 2017). ...
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Anthropogenic activities have extensively transformed the biosphere by extracting and disposing of resources, crossing boundaries of planetary threat while causing a global crisis of waste overload. Despite fundamental differences regarding structure and recalcitrance, lignocellulose and plastic polymers share physical-chemical properties to some extent, that include carbon skeletons with similar chemical bonds, hydrophobic properties, amorphous and crystalline regions. Microbial strategies for metabolizing recalcitrant polymers have been selected and optimized through evolution, thus understanding natural processes for lignocellulose modification could aid the challenge of dealing with the recalcitrant human-made polymers spread worldwide. We propose to look for inspiration in the charismatic fungal-growing insects to understand multipartite degradation of plant polymers. Independently evolved in diverse insect lineages, fungiculture embraces passive or active fungal cultivation for food, protection, and structural purposes. We consider there is much to learn from these symbioses, in special from the community-level degradation of recalcitrant biomass and defensive metabolites. Microbial plant-degrading systems at the core of insect fungicultures could be promising candidates for degrading synthetic plastics. Here, we first compare the degradation of lignocellulose and plastic polymers, with emphasis in the overlapping microbial players and enzymatic activities between these processes. Second, we review the literature on diverse insect fungiculture systems, focusing on features that, while supporting insects' ecology and evolution, could also be applied in biotechnological processes. Third, taking lessons from these microbial communities, we suggest multidisciplinary strategies to identify microbial degraders, degrading enzymes and pathways, as well as microbial interactions and interdependencies. Spanning from multiomics to spectroscopy, microscopy, stable isotopes probing, enrichment microcosmos, and synthetic communities, these strategies would allow for a systemic understanding of the fungiculture ecology, driving to application possibilities. Detailing how the metabolic landscape is entangled to achieve ecological success could inspire sustainable efforts for mitigating the current environmental crisis.
... This method could also miss new families of plastic-degrading enzymes with low sequence similarity to previously characterized ones. In addition, sequence similarities do not guarantee plastic-degrading activity, so further characterization and validation of enzyme functionality is needed [17]. ...
... For example, a novel thermostable cutinase homologue, leaf and branch compost cutinase (LCC), capable of PCL and PET degradation, was identified from the metagenome of a leaf-branch compost with copious natural plant-derived polymers via function-based screening [25]. Likewise, esterases capable of hydrolyzing poly(diethylene glycol adipate) (poly DEGA) and synthetic copolyester poly(butylene adipate-co-terephthalate) (PBAT) were identified in metagenomic libraries constructed from soil compost and Sphagnum moss, respectively [17,26]. In addition, the plastisphere is a promising source for plastic-degrading enzyme discovery because the environment can select ...
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The drastically increasing amount of plastic waste is causing an environmental crisis that requires innovative technologies for recycling post-consumer plastics to achieve waste valorization while meeting environmental quality goals. Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment and recycling. A variety of plastic-degrading enzymes have been discovered from microbial sources. Meanwhile, protein engineering has been exploited to modify and optimize plastic-degrading enzymes. This review highlights the recent trends and up-to-date advances in mining novel plastic-degrading enzymes through state-of-the-art omics-based techniques and improving the enzyme catalytic efficiency and stability via various protein engineering strategies. Future research prospects and challenges are also discussed.
... The most important feature of biodegradable plastics is that they can be broken down into CO 2 and H 2 O by microbial actions in industrial or municipal composting facilities. Biodegradable plastics can be divided into bio-and petroleum-based plastics, depending on the synthesis methods and materials [134]. Biodegradable plastics have been made for decades to alleviate plastic waste overload and are considered a potential solution for plastic waste. ...
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The pollution of plastic waste has become an increasingly serious environmental crisis. Recently, plastic has been detected in various kinds of environments, even in human tissues, which is an increasing threat to the ecosystems and humans. In the ocean, the plastic waste is eventually fragmentized into microplastics (MPs) under the disruption of physical and chemical processes. MPs are colonized by microbial communities such as fungi, diatoms, and bacteria, which form biofilms on the surface of the plastic called “plastisphere”. In this review, we summarize the studies related to microorganisms in the plastisphere in recent years and describe the microbial species in the plastisphere, mainly including bacteria, fungi, and autotrophs. Secondly, we explore the interactions between MPs and the plastisphere. The depth of MPs in the ocean and the nutrients in the surrounding seawater can have a great impact on the community structure of microorganisms in the plastisphere. Finally, we discuss the types of MP-degrading bacteria in the ocean, and use the “seed bank” theory to speculate on the potential sources of MP-degrading microorganisms. Challenges and future research prospects are also discussed.
... Using the above approach, microbial polyester hydrolyzing enzymes have been identified from different ecosystems including leaf-branch compost (Sulaiman et al., 2012), marine (Pinnell and Turner, 2019), and moss-associated environments (Müller et al., 2017). The identified enzymes are mainly microbial cutinases able to hydrolyze the natural plant polyester cutin, a major component of the plant cell wall. ...
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Microorganisms, like bacteria and fungi, are becoming an emerging resource for the development of eco-sustainable plastic degradation and recycling processes. In this study, the rumen content from cattle (Bos taurus) was investigated regarding synthetic polyester hydrolyzing enzymes based on the fact that the diet of ruminants may contain natural plant polyesters. A screening with model substrates demonstrated hydrolytic activities of rumen fluid on p-NP-esters with four to eight carbon atoms. Rumen fluid hydrolyzed synthetic aromatic polyesters with higher amounts of terephthalic acid released from poly(butylene adipate-co-terephthalate) (PBAT) (0.75 and 0.5 mM for polymer powder and film, respectively) and thus exceeded when compared to the hydrolysis of the second terephthalic acid-based polymer—poly(ethylene terephthalate) (PET) (0.6 and 0.15 mM, for powder and film, reciprocally). Additionally, rumen fluid hydrolyzed the bio-based polyester poly(ethylene furanoate) (PEF) according to HPLC and SEM analysis. Shotgun metagenome analysis of the rumen microbiome revealed the real proportion of all domains of life, showing the dominance of bacteria (98%), followed by Eukaryota (1%) and finally Archaea. Among the most abundant genera encountered in this study, polyester hydrolysis activity has already been proven (e.g., Pseudomonas).
... The monomer Ta represented the most abundant compound detected at all experimental conditions and was proven not to be metabolised by the organism. This suggests the involvement of the fungus extracellular enzymes in the cleavage of aromatic ester bonds present in PBAT chains 17,45 , which are reportedly less prone to hydrolysis than the ester bond with aliphatic monomer (adipic acid) as also described for P. pseudoalcaligenes polyesterase PpEst 15 . Previous studies on single enzyme hydrolysis process of milled PBAT reported that BTaB and BTa were more abundant than the monomer, as well as that an increase of Ta concentration is given by higher pH than that selected in the current study 7 . ...
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Knufia chersonesos is an ascomycotal representative of black fungi, a morphological group of polyextremotolerant melanotic fungi, whose ability to resort to recalcitrant carbon sources makes it an interesting candidate for degradation purposes. A secretome screening towards polyesterases was carried out for the fungus and its non-melanized mutant, grown in presence of the synthetic copolyester polybutylene adipate terephthalate (pBAt) as additional or sole carbon source, and resulted in the identification of 37 esterolytic and lipolytic enzymes across the established cultivation conditions. Quantitative proteomics allowed to unveil 9 proteins being constitutively expressed at all conditions and 7 which were instead detected as up-regulated by PBAT exposure. Protein functional analysis and structure prediction indicated similarity of these enzymes to microbial polyesterases of known biotechnological use such as MHetase from Ideonella sakaiensis and calA from Candida antarctica. for both strains, pBAt hydrolysis was recorded at all cultivation conditions and primarily the corresponding monomers were released, which suggests degradation to the polymer's smallest building block. the work presented here aims to demonstrate how investigations of the secretome can provide new insights into the eco-physiology of polymer degrading fungi and ultimately aid the identification of novel enzymes with potential application in polymer processing, recycling and degradation.
... Functional metagenomics has been applied successfully for the discovery of a myriad of enzymes, especially of hydrolases (Kimura, 2018). For example, novel polyesterases were identified in a plant-associated metagenome using this strategy (Müller et al., 2017). Certainly, activity-based metagenomics is limited by the availability of highthroughput screening methods for particular enzymatic activities or enzyme classes, as is the case for oxidoreductases. ...
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Environmental pollution caused by polyesters has become a major ecological safety concern that needs to be managed urgently. One way to resolve this problem is giving the spotlight to current emerging research of microbial biocatalysts. During the last two decades many researchers have reported the ability to break down and modify natural and synthetic polyesters using different microbial carboxyl ester hydrolases (lipases, esterases, cutinases, PETases, etc.) also called polyesterases, and contribution of these enzymes towards the reduction of plastic levels in the future. Continuous search of such lipolytic biocatalysts and their improvement via protein engineering strategies results in beneficial findings making the use of polyesterases in the biodegradation of plastics increasingly more realistic. The present review provides a comprehensive insight into the structural properties enabling microbial lipolytic‐type carboxyl ester hydrolases to effectively catalyze the cleavage of ester linkages in different polyester plastics. Moreover, the management of extensively used polyester plastics using different lipolytic enzymes as innovative eco‐friendly solution is presented in this report. Furthermore, improvement of polyesterases via protein engineering for the development of more effective and suitable polyester‐degrading lipolytic biocatalysts is summarized in this review as well. This article is protected by copyright. All rights reserved.
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Biodegradable mulch films have been developed as a suitable alternative to conventional nondegradable polyethylene films. However, the key factors controlling the degradation speed of biodegradable mulch films in soils remain unclear. Here, we linked changes in the soil microbiome with the degradation rate of a promising biodegradable material poly(butylene adipate-co-terephthalate) (PBAT) in four soil types, a lou soil (LS), a fluvo-aquic soil (CS), a black soil (BS), and a red soil (RS), equivalent to Inceptisols (the first two soils), Mollisols, and Ultisols, using soil microcosms. The PBAT degradation rate differed with the soil type, with PBAT mineralization levels of 16, 9, 0.3, and 0.9% in LS, CS, BS, and RS, respectively, after 120 days. Metagenomic analysis showed that the microbial community in LS was more responsive to PBAT than the other three soils. PBAT hydrolase genes were significantly enriched in LS but were not significantly stimulated by PBAT in CS, BS, or RS. Several members of Proteobacteria were identified as novel potential degraders, and their enrichment extent was significantly positively correlated with PBAT degradation capacity. Overall, our results suggest that soil environments harbored a range of PBAT-degrading bacteria and the enrichment of potential degraders drives the fate of PBAT in the soils.
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Poly(ethylene terephthalate) (PET) is used extensively worldwide in plastic products, and its accumulation in the environment has become a global concern. Because the ability to enzymatically degrade PET has been thought to be limited to a few fungal species, biodegradation is not yet a viable remediation or recycling strategy. By screening natural microbial communities exposed to PET in the environment, we isolated a novel bacterium, Ideonella sakaiensis 201-F6, that is able to use PET as its major energy and carbon source. When grown on PET, this strain produces two enzymes capable of hydrolyzing PET and the reaction intermediate, mono(2-hydroxyethyl) terephthalic acid. Both enzymes are required to enzymatically convert PET efficiently into its two environmentally benign monomers, terephthalic acid and ethylene glycol.
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The aliphatic-aromatic copolyester PBAT (poly(butylene adipate-co-butylene terephthalate), also known as ecoflex, contains adipic acid, 1,4-butanediol and terephthalic acid and is proven to be compostable [1–3]). We describe here data for the synthesis and analysis of poly(butylene adipate-co-butylene terephthalate variants with different adipic acid: terephatalic acid ratios and 6 oligomeric PBAT model substrates. Data for the synthesis of the following oligomeric model substrates are described: mono(4-hydroxybutyl) terephthalate (BTa), bis(4-(hexanoyloxy)butyl) terephthalate (HaBTaBHa), bis(4-(decanoyloxy)butyl) terephthalate (DaBTaBDa), bis(4-(tetradecanoyloxy)butyl) terephthalate (TdaBTaBTda), bis(4-hydroxyhexyl) terephthalate (HTaH) and bis(4-(benzoyloxy)butyl) terephthalate (BaBTaBBa). Polymeric PBAT variants were synthesized with adipic acid: terephatalic acid ratios of 100: 0, 90: 10, 80: 20, 70: 30, 60: 40 and 50: 50. These polymeric and oligomeric substances were used as ecoflex model substrates in enzymatic hydrolysis experiments in the article “Substrate specificities of cutinases on aliphatic-aromatic polyesters and on their model substrates” [4].
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Certain α/β hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108 (PfL1) was cloned and expressed in Escherichia coli BL21-Gold(DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (T opt = 50 °C, pHopt = 7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg−1) compared to longer and shorter chain lengths (C14 0.7 U mg−1 and C2 4.3 U mg−1, respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography-mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.
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There is growing demand for new bioactive compounds and biologicals for the pharmaceutical, agro- and food industries. Plant-associated microbes present an attractive and promising source to this end, but are nearly unexploited. Therefore, bioprospecting of plant microbiomes is gaining more and more attention. Due to their highly specialized and co-evolved genetic pool, plant microbiomes host a rich secondary metabolism. This article highlights the potential detection and use of secondary metabolites and enzymes derived from plant-associated microorganisms in biotechnology. As an example we summarize the findings from the moss microbiome with special focus on the genus Sphagnum and its biotechnological potential for the discovery of novel microorganisms and bioactive molecules. The selected examples illustrate unique and yet untapped properties of plant-associated microbiomes, which are an immense treasure box for future research.
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Natural biodiversity undoubtedly inspires biocatalysis research and innovation. Biotransformations of interest also inspire the search for appropriate biocatalysts in nature. Indeed, natural genetic resources have been found to support the hydrolysis and synthesis of not only common but also unusual synthetic scaffolds. The emerging tool of metagenomics has the advantage of allowing straightforward identification of activity directly applicable as biocatalysis. However, new enzymes must not only have outstanding properties in terms of performance but also other properties superior to those of well-established commercial preparations in order to successfully replace the latter. Esterases (EST) and lipases (LIP) from the α/β-hydrolase fold superfamily are among the enzymes primarily used in biocatalysis. Accordingly, they have been extensively examined with metagenomics. Here we provided an updated (October 2015) overview of sequence and functional data sets of 288 EST–LIP enzymes with validated functions that have been isolated in metagenomes and (mostly partially) characterized. Through sequence, biochemical, and reactivity analyses, we attempted to understand the phenomenon of variability and versatility within this group of enzymes and to implement this knowledge to identify sequences encoding EST–LIP which may be useful for biocatalysis. We found that the diversity of described EST–LIP polypeptides was not dominated by a particular type of protein or highly similar clusters of proteins but rather by diverse nonredundant sequences. Purified EST–LIP exhibited a wide temperature activity range of 10–85 °C, although a preferred bias for a mesophilic temperature range (35–40 °C) was observed. At least 60% of the total characterized metagenomics-derived EST–LIP showed outstanding properties in terms of stability (solvent tolerance) and reactivity (selectivity and substrate profile), which are the features of interest in biocatalysis. We hope that, in the future, the search for and utilization of sequences similar to those already encoded and characterized EST–LIP enzymes from metagenomes may be of interest for promoting unresolved biotransformations in the chemical industry. Some examples are discussed in this review.
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Recently a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. While many studies on polyester biodegradability have focused on aerobic environments, there is much less known on biodegradation of polyesters in natural and artificial anaerobic habitats. Consequently, the potential of anaerobic biogas sludge to hydrolyze the synthetic compostable polyester PBAT (poly(butylene adipate-co-butylene terephthalate) was evaluated in this study. Based on RP-HPLC analysis, accumulation of terephthalic acid (Ta) was observed in all anaerobic batches within the first 14 days. Thereafter a decline of Ta was observed, which occurred presumably due to consumption by the microbial population. The esterase Chath_Est1 from the anaerobic risk 1 strain Clostridium hathewayi DSM-13479 was found to hydrolyze PBAT. Detailed characterization of this esterase including elucidation of the crystal structure was performed. The crystal structure indicates that Chath_Est1 belongs to the α/β-hydrolases family. This study gives a clear hint that also microorganisms in anaerobic habitats can degrade manmade PBAT.
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Knowledge of bacterial lipolytic enzymes is increasing at a rapid and exciting rate. To obtain an overview of this industrially very important class of enzymes and their characteristics, we have collected and classified the information available from protein and nucleotide databases. Here we propose an updated and extensive classification of bacterial esterases and lipases based mainly on a comparison of their amino acid sequences and some fundamental biological properties. These new insights result in the identification of eight different families with the largest being further divided into six subfamilies. Moreover, the classification enables us to predict (1) important structural features such as residues forming the catalytic site or the presence of disulphide bonds, (2) types of secretion mechanism and requirement for lipase-specific foldases, and (3) the potential relationship to other enzyme families. This work will therefore contribute to a faster identification and to an easier characterization of novel bacterial lipolytic enzymes.
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The enzymatic hydrolysis of the biodegradable polyester ecoflex and of a variety of oligomeric and polymeric ecoflex model substrates was investigated. For this purpose, substrate specificities of two enzymes of typical compost inhabitants, namely a fungal cutinase from Humicola insolens (HiC) and a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) were compared. Model substrates were systematically designed with variations of the chain length of the alcohol and the acid as well as with varying content of the aromatic constituent terephthalic acid (Ta). HPLC/MS identification and quantification of the hydrolysis products terephthalic acid (Ta), benzoic acid (Ba), adipic acid (Ada), mono(4-hydroxybutyl) terephthalate (BTa), mono-(2-hydroxyethyl) terephthalate (ETa), mono-(6-hydroxyhexyl) terephthalate (HTa) and bis(4-hydroxybutyl) terephthalate (BTaB) indicated that these enzymes indeed hydrolyze the tested esters. Shorter terminal chain length acids but longer chain length alcohols in oligomeric model substrates were generally hydrolyzed more efficiently. Thc_Cut1 hydrolyzed aromatic ester bonds more efficiently than HiC resulting in up to 3-fold higher concentrations of the monomeric hydrolysis product Ta. Nevertheless, HiC exhibited a higher overall hydrolytic activity on the tested polyesters, resulting in 2-fold higher concentration of released molecules. Thermogravimetry and Differential Scanning Calorimetry (TG-DSC) of the polymeric model substrates revealed a general trend that a lower difference between melting temperature (Tm) and the temperature at which the enzymatic degradation takes place resulted in higher susceptibility to enzymatic hydrolysis.
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Two novel esterases from the anaerobic Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in E. coli BL21-Gold(DE3) and were found to hydrolyze the polyester poly(butylene adipate-co-butylene terephthalate) (PBAT). The active site residues (triad Ser, Asp, His) are present in both enzymes at the same location only with some amino acid variations near the active site at the surrounding of aspartate. Yet, Cbotu_EstA showed higher kcat values on para-nitrophenyl butyrate and para-nitrophenyl acetate and was considerably more active (6 fold) on PBAT. The entrance to the active site of the modeled Cbotu_EstB appears more narrowed compared to the crystal structure of Cbotu_EstA and the N-terminus is shorter which could explain its lower activity on PBAT. The Cbotu_EstA crystal structure consists of two regions that may act as movable cap domains and a zinc metal binding site. This article is protected by copyright. All rights reserved.