A Novel Polyester Hydrolase From the Marine Bacterium Pseudomonas aestusnigri – Structural and Functional Insights

Frontiers in Microbiology, Journal Year: 2020, Volume and Issue: 11

Published: Feb. 12, 2020

Biodegradation of synthetic polymers, in particular polyethylene terephthalate (PET), is great importance, since environmental pollution with PET and other plastics has become a severe global problem. Here, we report on the polyester degrading ability novel carboxylic ester hydrolase identified genome marine hydrocarbonoclastic bacterium Pseudomonas aestusnigri VGXO14 T . The enzyme, designated PE-H, belongs to type IIa family hydrolytic enzymes as indicated by amino acid sequence homology. It was produced Escherichia coli, purified its crystal structure solved at 1.09 Å resolution representing first enzyme. shows typical α/β-hydrolase fold high structural homology known hydrolases. hydrolysis detected 30°C amorphous film (PETa), but not from commercial bottle (PETb). A rational mutagenesis study improve potential PE-H yielded variant (Y250S) which showed improved activity, ultimately also allowing PETb. this 1.35 allowed rationalize improvement enzymatic activity. oligomer binding model proposed molecular docking computations. Our results indicate significant P. for degradation.

Language: Английский

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Enzyme discovery and engineering for sustainable plastic recycling

Trends in biotechnology, Journal Year: 2021, Volume and Issue: 40(1), P. 22 - 37

Published: March 3, 2021

Biocatalytic depolymerization mediated by enzymes has emerged as an efficient and sustainable alternative for plastic treatment recycling, which aims to reduce adverse environmental effects recover valuable components from waste.Metagenomic proteomic approaches can be harnessed powerful tools in mining capable of a wide variety environments ecosystems.Plastic-degrading optimized protein engineering improved performance, including enhancement enzyme thermostability, reinforcement the binding substrate active site, interaction between surface, refinement catalytic capacity. The drastically increasing amount waste is causing crisis that requires innovative technologies recycling post-consumer plastics achieve valorization while meeting quality goals. recycling. A plastic-degrading have been discovered microbial sources. Meanwhile, exploited modify optimize enzymes. This review highlights recent trends up-to-date advances novel through state-of-the-art omics-based techniques improving efficiency stability via various strategies. Future research prospects challenges are also discussed. Plastic materials play revolutionary role modern world, although enormous manufacture extensive use commodities inevitably generate extraordinary waste. Around 12 000 million metric tons predicted accumulate landfills natural environment 2050 [1.Geyer R. et al.Production, use, fate all ever made.Sci. Adv. 2017; 3e1700782Crossref PubMed Scopus (3209) Google Scholar]. Improper handling caused grand challenge. debris waste, especially microplastics (see Glossary), impose hazardous on organisms eventually threaten human well-being [2.Redondo-Hasselerharm P.E. al.Nano- affect composition freshwater benthic communities long term.Sci. 2020; 6eaay4054Crossref (14) Scholar, 3.Koelmans A.A. al.Microplastics freshwaters drinking water: critical assessment data quality.Water Res. 2019; 155: 410-422Crossref (256) 4.Seeley M.E. sedimentary nitrogen cycling.Nat. Commun. 11: 2372Crossref (27) 5.Boots B. al.Effects soil ecosystems: above below ground.Environ. Sci. Technol. 53: 11496-11506Crossref (63) In addition, degradation resistance further escalates their impacts [6.Chamas A. al.Degradation rates environment.ACS Sustain. Chem. Eng. 8: 3494-3511Crossref (230) Therefore, it urgent develop plastics, both protection. Enzymatic biocatalysis gained attention eco-friendly conventional methods (Box 1) [7.Wei al.Possibilities limitations biotechnological recycling.Nat. Catal. 3: 867-871Crossref To date, discovered, representing promising biocatalyst candidates depolymerization. Considering ubiquity different ecosystems tremendous metabolic genetic diversity microorganisms, habitats likely evolved capabilities decomposition utilization. identified so far might only account small portion relevant environment. ever-growing interest explore diverse discover new with desirable properties functionalities. However, naturally occurring not well suited synthetic industrial applications due poor thermostability low activity. Particularly, usually possess distinct physical chemical (e.g., high crystallinity) render them more resistant enzymatic attack than biogenic polymers. increasingly utilized construct better stability. Recent efforts made significant discovering enzymes, showing great promise progress discovery using optimization article timely provides holistic view current stage emerging obtaining effective biocatalysts degradation, will inspire future address Metagenomics demonstrated potential facilitate ecological habitats. culture-dependent method applied most known [8.Satti S.M. Shah Polyester-based biodegradable plastics: approach towards development.Lett. Appl. Microbiol. 70: 413-430Crossref (3) Scholar,9.Wierckx N. al.Plastic biodegradation: opportunities.in: Steffan Consequences Microbial Interactions Hydrocarbons, Oils, Lipids: Biodegradation Bioremediation. Springer International Publishing, 2018: 1-29Crossref method, microorganisms expressing desired first enriched isolated under proper cultivation conditions, followed strain taxonomical classification, identification putative molecular biological or computational (Figure 1A ) [10.Kawai F. al.A Ca2+-activated, thermostabilized polyesterase hydrolyzing polyethylene terephthalate Saccharomonospora viridis AHK190.Appl. Biotechnol. 2014; 98: 10053-10064Crossref (112) 11.Taniguchi I. al.Biodegradation PET: status application aspects.ACS 9: 4089-4105Crossref (106) 12.Yoshida S. bacterium degrades assimilates poly(ethylene terephthalate).Science. 2016; 351: 1196-1199Crossref (705) seriously limits scope finding because estimated less 1% total planet cultured. By contrast, culture-independent metagenomic tool vast majority As summarized Table 1, many genes encoding depolymerizing retrieved wealth metagenome samples. this section we discuss deciphering huge reservoir techniques. overall workflow metagenomics illustrated Figure 1B. Among these steps, selecting appropriate screening pivotal mining. Generally, there two commonly used screen library, sequence-based function-based [13.Ufarte L. al.Metagenomics pollutant degrading enzymes.Biotechnol. 2015; 33: 1845-1854Crossref (0) Scholar,14.Sankara Subramanian S.H. al.RemeDB: rapid prediction involved bioremediation high-throughput sets.J. Comput. Biol. 27: 1020-1029Crossref (2) Sequence-based takes advantage sequence similarity comparison functional gene annotation searching bioinformatic databases [14.Sankara For example, terephthalate) (PET) hydrolytic (PET2) was uncovered silico search algorithm powered hidden Markov model [15.Danso D. al.New insights into function global distribution (PET)-degrading bacteria marine terrestrial metagenomes.Appl. Environ. 2018; 84: e02773-e02817Crossref (50) More recently, number sequences similar ones activity degrade polyurethane (PU) were landfill-derived metagenomes [16.Gaytan recalcitrant xenobiotic additives selected landfill community its biodegradative revealed proximity lgation-based analysis.Front. 10: 2986Crossref (8) relatively cost-effective success limited size could miss families previously characterized ones. similarities do guarantee activity, characterization validation functionality needed [17.Muller C.A. al.Discovery polyesterases moss-associated microorganisms.Appl. 83: e02641-e02716Crossref Alternatively, uses assays phenotypes libraries 1B). particularly advantageous over screening, completely groups divergent existing homologous multiple phylogenetically belonging entirely esterase screened agar plate assays, exhibited polyesters, poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL), poly(butylene succinate-co-adipate) (PBSA) [18.Hajighasemi M. al.Screening against polyesters.Environ. 52: 12388-12401Crossref (11) Scholar] (Table 1). Traditional capability large-sized libraries. studies developing accelerate microbes [19.Weinberger al.High throughput fungal polyester enzymes.Front. 554Crossref Scholar,20.Bunzel H.A. al.Speeding up ultrahigh-throughput methods.Curr. Opin. Struct. 48: 149-156Crossref (54) When approach, important select host cell constructing heterologous expression level library representativeness. Escherichia coli widely convenient manipulation [21.Lorenz P. Eck J. applications.Nat. Rev. 2005; 510-516Crossref (370) systems employed ensure expression. instance, eukaryotic cells, such yeast Pichia pastoris, disulfide bonds, they unsuitably expressed common E. [22.Fecker T. al.Active site flexibility hallmark PET sakaiensis PETase.Biophys. 114: 1302-1312Abstract Full Text PDF (84) 23.Urbanek A.K. al.Biochemical polyester-type plastics.Biochim. Biophys. Acta Proteins Proteom. 1868140315Crossref (13) 24.Chen al.Contribution bond Thermobifida fusca cutinase.Food Biosci. 6-10Crossref It type successful screening. chosen determined factors; coverage. Due short length insert plasmid harbor, plasmid-based large but coverage, unfavorable longer DNA fragments inserted phage fosmid Moreover, phage-based some toxic target concomitant lysis cells directly plaques. Besides methods, sampling sources determining discovery. Most investigated showed hit rate related 1), major challenge analysis worldwide broad extremely frequency indicating slow evolution indigenous utilize anthropogenic likelihood greater abundant biopolymeric substances. thermostable cutinase homologue, leaf branch compost (LCC), PCL leaf-branch copious plant-derived polymers [25.Sulaiman al.Isolation homolog terephthalate-degrading approach.Appl. 2012; 78: 1556-1562Crossref (155) Likewise, esterases poly(diethylene glycol adipate) (poly DEGA) copolyester adipate-co-terephthalate) (PBAT) constructed Sphagnum moss, respectively Scholar,26.Kang C.H. family VII library.Microb. Cell Factories. 2011; 41Crossref (38) plastisphere source compounds survival growth [27.Roager Sonnenschein E.C. Bacterial colonization debris.Environ. 11636-11643Crossref (25) 28.Jacquin al.Microbial ecotoxicology debris: biodegradation ‘plastisphere.Front. 865Crossref 29.Amaral-Zettler L.A. al.Ecology plastisphere.Nat. 18: 139-151Crossref currently underexplored growing Techniques targeted stable-isotope probing (SIP) helpful increase Targeted stimulate presence functions before extraction, situ habitat. pre-incubation native activated prevalence species raised [30.Mayumi al.Identification poly(DL-lactic depolymerases metagenome.Appl. 2008; 79: 743-775Crossref (34) Additionally, SIP technique integrated [31.Coyotzi al.Targeted populations probing.Curr. 41: 1-8Crossref (39) Scholar,32.Chen Y. Murrell J.C. meets probing: perspectives.Trends 2010; 157-163Abstract Recently, 13C-labeled developed [33.Sander al.Assessing transformation nanoplastic 13C-labelled polymers.Nat. Nanotechnol. 14: 301-303Crossref (7) Scholar,34.Zumstein M.T. soils: tracking carbon CO2 biomass.Sci. 4eaas9024Crossref (52) Using would help pinpoint participating processes. proteomics-based detects quantifies proven repertoire [35.Bers K. hydrolase genomic-proteomic phenylurea herbicide mineralization Variovorax sp. SRS16.Appl. 77: 8754-8764Crossref (48) Scholar,36.Sturmberger al.Synergism proteomics mRNA sequencing discovery.J. 235: 132-138Crossref (9) 1C shows First, pure consortia grown without substrate, differentially induce express produced cultures extracted digested peptides, subjected sequencing, analysis. Typically, exoproteome principal when insoluble unable enter engaged secreted extracellularly [23.Urbanek effectiveness already identifying plant biopolymer inspiring implementation [37.Schneider al.Proteome bacterial involvement litter decomposition.Proteomics. 1819-1830Crossref (64) Comparative frequently based presumption incubation comparatively analyzing Pseudomonas pseudoalcaligenes fungus Knufia chersonesos, several PBAT identified, demonstrating unavailable annotated genomic [38.Tesei al.Shotgun reveals secretome rock-inhabiting chersonesos.Sci. Rep. 9770Crossref (1) Scholar,39.Wallace P.W. al.PpEst pseudoalcaligenes.Appl. 101: 2291-2303Crossref (16) another study, polyhydroxybutyrate (PHB) depolymerase ALC24_4107 Alcanivorax 24 comparative exoproteomic [40.Zadjelovic V. al.Beyond oil degradation: 22: 1356-1369Crossref Proteomics-guided still infancy, reported conducted cultures. Direct metaproteomics complex samples challenging, difficulty high-quality extraction availability downstream [41.Biswas Sarkar ‘Omics’ microbiology: state art.in: Adhya T.K. Advances Soil Microbiology: Trends Prospects. Singapore, 35-64Crossref Leveraging improve performance recently topic. Protein categories general; rational design directed evolution. Rational modifies knowledge structure mechanistic characteristics, simulation, modeling. Almost reports available structural information lack main barrier attempt far, employing direct engineer PHB Ralstonia pickettii T1, failed acquire any variant [42.Tan L.T. al.Directed poly[(R)-3-hydroxybutyrate] surface display system: importance asparagine at position 285.Appl. 2013; 97: 4859-4871Crossref focus discussing strategies 2 examples 2. Thermostability highly depolymerization, glass transition temperature (Tg) ~65–70°C PET). reaction gets close Tg polymeric chains considerably increased mobility, facilitating accessibility [43.Wei Zimmermann W. petroleum-based how we?.Microb. 1308-1322Crossref (208) one bottleneck practical applications. Inspired unique features thermophilic proteins, designed detailed later. Introduction bonds salt bridges beneficial 2A) [44.Rigoldi al.Review: applications.APL Bioeng. 2011501Crossref 45.Son H.F. al.Structural bioinformatics-based thermo-stable PETase Ideonella sakaiensis.Enzym. Microb. 141109656Crossref 46.Oda al.Enzymatic hydrolysis roles three Ca2+ ions bound cutinase-like enzyme, Cut190*, activity.Appl. 102: 10067-10077Crossref (17) 47.Zhong-Johnson E.Z.L. al.An absorbance kinetics films.Sci. 2021; 928Crossref Disulfide crucial folding correct local conformation confer thermal resistance. residues metal responsible replaced introduce bond. D204C E253C mutations calcium TfCut2 formed bond, melting [48.Then bridge increases terephthalate.FEBS Open Bio. 6: 425-432Crossref (47) formation negatively-charged N246D residue positively-charged Arg280 contribute engineered PETaseN246D [45.Son construction work synergistically benefit ag

Language: Английский

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Current State and Perspectives Related to the Polyethylene Terephthalate Hydrolases Available for Biorecycling

ACS Sustainable Chemistry & Engineering, Journal Year: 2020, Volume and Issue: 8(24), P. 8894 - 8908

Published: May 22, 2020

Polyethylene terephthalate (PET) hydrolase is a challenging target as PET commonly used plastic that extremely resistant to enzymatic attack. Since the discovery of from Thermobifida fusca in 2005, novel hydrolases and their availability toward waste have been investigated. At present, at least four thermophilic cutinases are known could be for management amorphous waste, such packaging materials. Heat-labile PETase Ideonella sakaiensis its homologues mesophilic bacteria exist environment. However, can efficiently hydrolyzed with hydrolases. This Review focuses on current state potential application. Contrary an PET, hydrolysis crystalline (particularly bottles) remains fully elucidated. It cannot assured whether biorecycling general would put into practice near future, but plan getting closer goal. versatile polyesterases they hydrolyze not only also other polyesters. Additionally, thermostability advantageous application terms reaction speed durability.

Language: Английский

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Plastics and the microbiome: impacts and solutions

Environmental Microbiome, Journal Year: 2021, Volume and Issue: 16(1)

Published: Jan. 20, 2021

Abstract Global plastic production has increased exponentially since manufacturing commenced in the 1950’s, including polymer types infused with diverse additives and fillers. While negative impacts of plastics are widely reported, particularly on marine vertebrates, microbial life remain poorly understood. Plastics impact microbiomes directly, exerting toxic effects, providing supplemental carbon sources acting as rafts for colonisation dispersal. Indirect consequences include environmental shading, altered compositions host communities disruption organism or community health, hormone balances immune responses. The isolation application plastic-degrading microbes substantial interest yet little evidence supports biodegradation most high molecular weight synthetic polymers. Over 400 species have been presumptively identified capable degradation, but degradation highly prevalent polymers polypropylene, nylon, polystyrene polyvinyl chloride must be treated caution; studies fail to differentiate losses caused by leaching monomers, Even where is demonstrated, such polyethylene terephthalate, ability microorganisms degrade more crystalline forms used commercial appears limited. Microbiomes frequently work conjunction abiotic factors heat light structural integrity accessibility enzymatic attack. Consequently, there remains much scope extremophile explored a source enzymes microorganisms. We propose best-practice workflow isolating reporting taxa from microbiomes, which should multiple lines supporting changes structure, mass loss, detection presumed products, along confirmation strains (and their associated genes) responsible degradation. Such approaches necessary degraders differentiated organisms only degrading labile within predominantly amorphous plastics,

Language: Английский

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Efficient Degradation of Poly(ethylene terephthalate) with Thermobifida fusca Cutinase Exhibiting Improved Catalytic Activity Generated using Mutagenesis and Additive-based Approaches

Scientific Reports, Journal Year: 2019, Volume and Issue: 9(1)

Published: Nov. 5, 2019

Abstract Cutinases are promising agents for poly(ethylene terephthalate) (PET) bio-recycling because of their ability to produce the PET monomer terephthalic acid with high efficiency under mild reaction conditions. In this study, we found that low-crystallinity (lcPET) hydrolysis activity thermostable cutinase from Thermobifida fusca (TfCut2), was increased by addition cationic surfactant attracts enzymes near lcPET film surface via electrostatic interactions. This approach applicable mutant TfCut2 G62A/F209A, which designed based on a sequence comparison PETase Ideonella sakaiensis . As result, degradation rate in presence 31 ± 0.1 nmol min −1 cm −2 , 12.7 times higher than wild-type absence surfactant. The long-duration showed (200 μm) 97 1.8% within 30 h, fastest biodegradation thus far. We therefore believe our would expand possibility enzyme utilization industrial biodegradation.

Language: Английский

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Mechanism-Based Design of Efficient PET Hydrolases

ACS Catalysis, Journal Year: 2022, Volume and Issue: 12(6), P. 3382 - 3396

Published: Feb. 28, 2022

Polyethylene terephthalate (PET) is the most widespread synthetic polyester, having been utilized in textile fibers and packaging materials for beverages food, contributing considerably to global solid waste stream environmental plastic pollution. While enzymatic PET recycling upcycling have recently emerged as viable disposal methods a circular economy, only handful of benchmark enzymes thoroughly described subjected protein engineering improved properties over last 16 years. By analyzing specific material reaction mechanisms context interfacial biocatalysis, this Perspective identifies several limitations current degradation approaches. Unbalanced enzyme-substrate interactions, limited thermostability, low catalytic efficiency at elevated temperatures, inhibition caused by oligomeric intermediates still hamper industrial applications that require high efficiency. To overcome these limitations, successful studies using innovative experimental computational approaches published extensively recent years thriving research field are summarized discussed detail here. The acquired knowledge experience will be applied near future address contributed other mass-produced polymer types (e.g., polyamides polyurethanes) should also properly disposed biotechnological

Language: Английский

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Microbial synthesis of vanillin from waste poly(ethylene terephthalate)

Green Chemistry, Journal Year: 2021, Volume and Issue: 23(13), P. 4665 - 4672

Published: Jan. 1, 2021

An engineered biosynthetic pathway in Escherichia coli enables the one-pot upcycling of post-consumer plastic waste into vanillin.

Language: Английский

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Functional expression of polyethylene terephthalate-degrading enzyme (PETase) in green microalgae

Microbial Cell Factories, Journal Year: 2020, Volume and Issue: 19(1)

Published: April 28, 2020

For decades, plastic has been a valuable global product due to its convenience and low price. example, polyethylene terephthalate (PET) was one of the most popular materials for disposable bottles beneficial properties, namely impact resistance, high clarity, light weight. Increasing demand resulted in indiscriminate disposal by consumers, causing severe accumulation wastes. Because this, scientists have made great efforts find way biologically treat As result, novel degradation enzyme, PETase, which can hydrolyze PET, discovered Ideonella sakaiensis 201-F6 2016. A green algae, Chlamydomonas reinhardtii, produces developed this study. Two representative strains (C. reinhardtii CC-124 CC-503) were examined, we found that could express PETase well. To verify catalytic activity produced C. cell lysate transformant PET samples co-incubated at 30 °C up 4 weeks. After incubation, terephthalic acid (TPA), i.e. fully-degraded form detected performance liquid chromatography analysis. Additionally, morphological changes, such as holes dents on surface film, observed using scanning electron microscopy. hydrolyzing successfully expressed demonstrated. best our knowledge, is first case expression algae.

Language: Английский

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Recent advances in biocatalysts engineering for polyethylene terephthalate plastic waste green recycling

Environment International, Journal Year: 2020, Volume and Issue: 145, P. 106144 - 106144

Published: Sept. 25, 2020

The massive waste of poly(ethylene terephthalate) (PET) that ends up in the landfills and oceans needs hundreds years for degradation has attracted global concern. poor stability productivity available PET biocatalysts hinder their industrial applications. Active can provide a promising avenue bioconversion recycling. Therefore, there is an urgent need to develop new strategies could enhance stability, catalytic activity, solubility, productivity, re-usability these under harsh conditions such as high temperatures, pH, salinity. This raised great attention using bioengineering improve biocatalysts' robustness behavior. Herein, historical forecasting data plastic production disposal were critically reviewed. Challenges facing process be used solve them highlighted summarized. In this review, we also discussed recent progress enzyme approaches discovering biocatalysts, elucidating mechanism, improving performance, assess strength weakness highlighting gaps data. Discovery more potential hydrolases studying molecular mechanism extensively via solving crystal structure will widen research area move forward application. A deeper knowledge mechanisms give insight into future identification related enzymes. reported during review reduce crystallinity increase operational temperature hydrolyzing

Language: Английский

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Biotechnological upcycling of plastic waste and other non-conventional feedstocks in a circular economy

Current Opinion in Biotechnology, Journal Year: 2019, Volume and Issue: 62, P. 212 - 219

Published: Dec. 24, 2019

Language: Английский

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