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1.
Sheng Wu Gong Cheng Xue Bao ; 37(9): 3268-3275, 2021 Sep 25.
Artigo em Chinês | MEDLINE | ID: mdl-34622634

RESUMO

Polyethylene terephthalate (PET) is a synthetic polymer consisting of ester bond-linked terephthalate and ethylene glycol. Tremendous amounts of PET have been produced and majority of them enters terrestrial and marine environment as wastes, posing serious threats to the global ecosystems. In 2016, a PET hydrolase from a PET-assimilating bacterium Ideonalla sakaiensis was reported and termed as IsPETase. This enzyme outperforms other PET-hydrolyzing enzymes in terms of its PET hydrolytic activity at ambient temperature, thus holds a great promise for PET biodegradation. In order to improve IsPETase activity, we conducted structure-based engineering to modify the putative substrate-binding tunnel. Among the several variants to the N233 residue of IsPETase, we discovered that the substitution of N233 with alanine increases its PET hydrolytic activity, which can be further enhanced when combined with a R280A mutation. We also determined the X-ray crystal structure of the IsPETase N233A variant, which shares nearly identical fold to the WT protein, except for an open end of subsite Ⅱ. We hypothesized that the smaller side chain of N233A variant might lead to an extended subsite Ⅱ for PET binding, which subsequently increases the enzymatic activity. Thus, this study provides new clues for further structure-based engineering of PETase.


Assuntos
Burkholderiales , Hidrolases , Polietilenotereftalatos/metabolismo , Burkholderiales/enzimologia , Hidrolases/genética , Engenharia de Proteínas
2.
FEBS J ; 288(16): 4728-4729, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34398531

RESUMO

With the current issue of The FEBS Journal, we are introducing a new category of invited review article contributions on Emerging Methods and Technologies. These articles provide an overview and discussion of recent, emerging methods that significantly advance and improve research efforts in the different fields of molecular and cellular research of our The FEBS Journal authors and readers. Deputy Editorial Manager Manuel Breuer and our Emerging Methods and Technologies Commissioning Editor Eric Chevet introduce the series.


Assuntos
Doenças Autoimunes/imunologia , Proteínas de Membrana/metabolismo , Neoplasias/imunologia , Polietilenotereftalatos/metabolismo , Polissacarídeos/imunologia , Humanos , Proteínas de Membrana/análise , Neoplasias/terapia , Polissacarídeos/química
3.
Appl Environ Microbiol ; 87(18): e0002021, 2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34260304

RESUMO

Poly(ethylene terephthalate) (PET) is a commonly used synthetic plastic; however, its nonbiodegradability results in a large amount of waste accumulation that has a negative impact on the environment. Recently, a PET-degrading bacterium, Ideonella sakaiensis 201-F6 strain, was isolated, and the enzymes involved in PET digestion, PET hydrolase (PETase), and mono(2-hydroxyethyl) terephthalic acid (MHET) hydrolase (MHETase) were identified. Despite the great potentials of I. sakaiensis in bioremediation and biorecycling, approaches to studying this bacterium remain limited. In this study, to enable the functional analysis of PETase and MHETase genes in vivo, we have developed a gene disruption system in I. sakaiensis. The pT18mobsacB-based disruption vector harboring directly connected 5'- and 3'-flanking regions of the target gene for homologous recombination was introduced into I. sakaiensis cells via conjugation. First, we deleted the orotidine 5'-phosphate decarboxylase gene (pyrF) from the genome of the wild-type strain, producing the ΔpyrF strain with 5-fluoroorotic acid (5-FOA) resistance. Next, using the ΔpyrF strain as a parent strain and pyrF as a counterselection marker, we disrupted the genes for PETase and MHETase. The growth of both Δpetase and Δmhetase strains on terephthalic acid (TPA; one of the PET hydrolytic products) was comparable to that of the parent strain. However, these mutant strains dramatically decreased the growth level on PET to that on a no-carbon source. Moreover, the Δpetase strain completely abolished PET degradation capacity. These results demonstrate that PETase and MHETase are essential for I. sakaiensis metabolism of PET. IMPORTANCE The poly(ethylene terephthalate) (PET)-degrading bacterium Ideonella sakaiensis possesses two unique enzymes able to serve in PET hydrolysis. PET hydrolase (PETase) hydrolyzes PET into mono(2-hydroxyethyl) terephthalic acid (MHET), and MHET hydrolase (MHETase) hydrolyzes MHET into terephthalic acid (TPA) and ethylene glycol (EG). These enzymes have attracted global attention, as they have potential to be used for bioconversion of PET. Compared to many in vitro studies, including biochemical and crystal structure analyses, few in vivo studies have been reported. Here, we developed a targeted gene disruption system in I. sakaiensis, which was then applied for constructing Δpetase and Δmhetase strains. Growth of these disruptants revealed that PETase is the sole enzyme responsible for PET degradation in I. sakaiensis, while PETase and MHETase play essential roles in its PET assimilation.


Assuntos
Proteínas de Bactérias/genética , Burkholderiales/genética , Burkholderiales/metabolismo , Hidrolases/genética , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/metabolismo , Etilenoglicol/metabolismo , Genes Bacterianos , Hidrolases/metabolismo , Hidrólise , Engenharia Metabólica , Ácidos Ftálicos/metabolismo , Reciclagem
4.
J Biotechnol ; 334: 47-50, 2021 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-34044062

RESUMO

The large amounts of polyethylene terephthalate (PET) that enter and accumulate in the environment have posed a serious threat to global ecosystems and human health. A PET hydrolase from PET-assimilating bacterium Ideonella sakaiensis (IsPETase) that exhibits superior PET hydrolytic activity at mild conditions is attracting enormous attention in development of plastic biodegrading strategies. In order to enhance the PET hydrolysis capacity of IsPETase, we selected several polymer-binding domains that can adhere to a hydrophobic polymer surface and fused these to a previously engineered IsPETaseS121E/D186H/R280A (IsPETaseEHA) variant. We found that fusing a cellulose-binding domain (CBM) of cellobiohydrolase I from Trichoderma reesei onto the C-terminus of IsPETaseEHA showed a stimulatory effect on enzymatic hydrolysis of PET. Compared to the parental enzyme, IsPETaseEHA_CBM exhibited 71.5 % and 44.5 % higher hydrolytic activity at 30 ℃ and 40 ℃, respectively. The catalytic activity of IsPETaseEHA_CBM was increased by 86 % when the protein concentration was increased from 2.5 µg/mL to 20 µg/mL. These findings suggest that the fusion of polymer-binding module to IsPETase is a promising strategy to stimulate the enzymatic hydrolysis of PET.


Assuntos
Celulose 1,4-beta-Celobiosidase , Polietilenotereftalatos/metabolismo , Trichoderma , Burkholderiales , Celulose , Celulose 1,4-beta-Celobiosidase/genética , Ecossistema , Hidrólise , Hypocreales , Trichoderma/enzimologia
5.
Microb Cell Fact ; 20(1): 93, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33933097

RESUMO

Poly(ethylene terephthalate) (PET) is the world's most abundant polyester plastic, and its ongoing accumulation in nature is causing a global environmental problem. Currently, the main recycling processes utilize thermomechanical or chemical means, resulting in the deterioration of the mechanical properties of PET. Consequently, polluting de novo synthesis remains preferred, creating the need for more efficient and bio-sustainable ways to hydrolyze the polymer. Recently, a PETase enzyme from the bacterium Ideonella sakaiensis was shown to facilitate PET biodegradation, albeit at slow rate. Engineering of more efficient PETases is required for industrial relevance, but progress is currently hampered by the dependency on intracellular expression in Escherichia coli. To create a more efficient screening platform in E. coli, we explore different surface display anchors for fast and easy assaying of PETase activity. We show that PETases can be functionally displayed on the bacterial cell surface, enabling screening of enzyme activity on PET microparticles - both while anchored to the cell and following solubilization of the enzymes.


Assuntos
Biodegradação Ambiental , Escherichia coli/genética , Escherichia coli/metabolismo , Hidrolases/genética , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo , Hidrólise , Propriedades de Superfície
6.
FEBS J ; 288(16): 4730-4745, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33792200

RESUMO

The polyester PET (poly(ethylene terephthalate)) plastic is chemically inert and remarkably persistent, posing relevant and global pollution concerns due to its accumulation in ecosystems across the globe. In past years, research focused on identifying bacteria active on PET and on the specific enzymes responsible for its degradation. Here, the enzymatic degradation of PET can be considered as an 'erosion process' that takes place on the surface of an insoluble material and results in an unusual, substrate-limited kinetic condition. In this review, we report on the most suitable models to evaluate the kinetics of PET-hydrolyzing enzymes, which takes into consideration the amount of enzyme adsorbed on the substrate, the enzyme-accessible ester bonds, and the product inhibition effects. Careful kinetic analysis is especially relevant to compare enzymes from different sources and evolved variants generated by protein engineering studies as well. Furthermore, the analytical methods most suitable to screen natural bacteria and recombinant variant libraries generated by protein engineering have been also reported. These methods rely on different detection systems and are performed both on model compounds and on different PET samples (e.g., nanoparticles, microparticles, and waste products). All this meaningful information represents an optimal starting point and boosts the process of identifying systems able to biologically recycle PET waste products.


Assuntos
Enzimas/metabolismo , Polietilenotereftalatos/metabolismo , Biocatálise , Enzimas/análise , Cinética
7.
Artigo em Inglês | MEDLINE | ID: mdl-33847553

RESUMO

A novel Gram-stain-negative, aerobic, gliding, rod-shaped and carotenoid-pigmented bacterium, designated A20-9T, was isolated from a microbial consortium of polyethylene terephthalate enriched from a deep-sea sediment sample from the Western Pacific. Growth was observed at salinities of 1-8 %, at pH 6.5-8 and at temperatures of 10-40 °C. The results of phylogenetic analyses based on the genome indicated that A20-9T formed a monophyletic branch affiliated to the family Schleiferiaceae, and the 16S rRNA gene sequences exhibited the maximum sequence similarity of 93.8 % with Owenweeksia hongkongensis DSM 17368T, followed by similarities of 90.4, 90.1 and 88.8 % with Phaeocystidibacter luteus MCCC 1F01079T, Vicingus serpentipes DSM 103558T and Salibacter halophilus MCCC 1K02288T, respectively. Its complete genome size was 4 035 598 bp, the genomic DNA G+C content was 43.2 mol%. Whole genome comparisons indicated that A20-9T and O. hongkongensis DSM 17368T shared 67.8 % average nucleotide identity, 62.7 % average amino acid identity value, 46.6% of conserved proteins and 17.8 % digital DNA-DNA hybridization identity. A20-9T contained MK-7 as the major respiratory quinone. Its major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phospatidylcholine; and the major fatty acids were iso-C15 : 0 (37.5 %), iso-C16 : 0 3-OH (12.4 %), and summed feature 3 (C16 : 1ω7c /C16 : 1ω6c, 11.6 %). Combining the genotypic and phenotypic data, A20-9T could be distinguished from the members of other genera within the family Schleiferiaceae and represents a novel genus, for which the name Croceimicrobium hydrocarbonivorans gen. nov., sp. nov. is proposed. The type strain is A20-9T (=MCCC 1A17358T =KCTC 72878T).


Assuntos
Flavobacteriaceae/classificação , Sedimentos Geológicos/microbiologia , Consórcios Microbianos , Filogenia , Polietilenotereftalatos/metabolismo , Água do Mar/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Flavobacteriaceae/isolamento & purificação , Oceano Pacífico , Fosfolipídeos/química , Pigmentação , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Vitamina K 2/análogos & derivados , Vitamina K 2/química
8.
Int J Biol Macromol ; 180: 667-676, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33753197

RESUMO

Poly(ethylene terephthalate) (PET) is used widely by human beings, but is very difficult to degrade. Up to now, the PET degradation effect of PETase from Ideonella sakaiensis 201-F6 (IsPETase) variants with low stability and activity was not ideal. In this study, a mutation design tool, Premuse, was developed to integrate the sequence alignment and quantitative selection of the preferred mutations based on natural sequence evolution. Ten single point mutants were selected from 1486 homologous sequences using Premuse, and then two mutations (W159H and F229Y) with improved stability were screened from them. The derived double point mutant, W159H/F229Y, exhibited a strikingly enhanced enzymatic performance. Its Tm and catalytic efficiency values (kcat/Km) respectively increased by 10.4 °C and 2.0-fold using p-NPP as the substrate compared with wild type. The degradation activity for amorphous PET was increased by almost 40-fold in comparison with wild type at 40 °C in 24 h. Additionally, the variant could catalyze biodegradation of PET bottle preform at a mean rate of 23.4 mgPET/h/mgenzyme. This study allowed us to design the mutation more efficiently, and provides a tool for achieving biodegradation of PET pollution under mild natural environments.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiales/enzimologia , Biologia Computacional/métodos , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas/métodos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Burkholderiales/genética , Ensaios Enzimáticos/métodos , Hidrolases/classificação , Hidrolases/genética , Hidrólise , Internet , Cinética , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutação , Filogenia , Polietilenotereftalatos/química , Estabilidade Proteica , Temperatura de Transição
9.
Int J Biol Macromol ; 176: 157-164, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33561457

RESUMO

Poly(ethylene terephthalate) hydrolase (PETase) from Ideonella sakaiensis 201-F6 was expressed and purified from Escherichia coli to hydrolyze poly(ethylene terephthalate) (PET) fibers waste for its monomers recycling. Hydrolysis carried out at pH 8 and 30 °C was found to be the optimal condition based on measured monomer mono(2-hydroxyethyl) terephthalate (MHET) and terephthalic acid (TPA) concentrations after 24 h reaction. The intermediate product bis(2-hydroxyethyl) terephthalate (BHET) was a good substrate for PETase because BHET released from PET hydrolysis was efficiently converted into MHET. Only a trace amount of MHET could be further hydrolyzed to TPA. Class I hydrophobins RolA from Aspergillus oryzae and HGFI from Grifola frondosa were expressed and purified from E. coli to pretreat PET surface for accelerating PETase hydrolysis against PET. The weight loss of hydrolyzed PET increased from approximately 18% to 34% after hydrophobins pretreatment. The releases of TPA and MHET from HGFI-pretreated PET were enhanced 48% and 62%, respectively. The selectivity (TPA/MHET ratio) of the hydrolysis reaction was approximately 0.5.


Assuntos
Proteínas de Bactérias/metabolismo , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo , Reciclagem/métodos , Aspergillus oryzae/metabolismo , Biocatálise , Biodegradação Ambiental , Burkholderiales/enzimologia , Proteínas Fúngicas/metabolismo , Grifola/metabolismo , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Resíduos Industriais , Polietilenotereftalatos/química , Proteínas Recombinantes/metabolismo
10.
Methods Enzymol ; 648: 337-356, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33579411

RESUMO

The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.


Assuntos
Burkholderiales , Hidrolases , Biocatálise , Burkholderiales/metabolismo , Hidrolases/metabolismo , Polietilenotereftalatos/metabolismo
11.
Int J Biol Macromol ; 166: 251-258, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33122073

RESUMO

Hydrothermal degradation was used to pretreat terylene with an aim of noticeably improving the yield of fermentable monomers: terephthalic acid (TPA), mono (2- hydroxyethyl) terephthalic acid (MHET), bis-hydroxyethyl terephthalate (BHET), and ethylene glycol (EG). After 0.5 h of reaction time at 180 °C, hydrothermal degradation with ammonia led to almost complete conversion of the terylene to TPA, MHET, BHET and EG, which were then transformed by Taonella mepensis WT-6 to bacterial cellulose (BC). Furthermore, the optimum fermentation conditions with the maximum BC yield were 5.0 g/L yeast extract, 30.0 °C, pH 9.0, 8.0% inoculum, and hydrolysate TOC (5.02 g/L). Additionally, mechanical and thermal analysis revealed that the properties of BC produced from TAH medium were similar to those of BC produced with HS medium. Considering the substantial amount of global terylene waste being produced, this study provides an alternative solution for the biosynthesis of BC.


Assuntos
Celulose/biossíntese , Polietilenotereftalatos/metabolismo , Rhodospirillaceae/metabolismo , Compostos de Amônio/química , Biodegradação Ambiental , Boehmeria/química , Fermentação , Hidrólise , Microbiologia Industrial/métodos , Resíduos Industriais , Polietilenotereftalatos/química
12.
Proteins ; 89(5): 502-511, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33340163

RESUMO

The cutinase-like enzyme from the thermophile Saccharomonospora viridis AHK190, Cut190, is a good candidate to depolymerize polyethylene terephthalate (PET) efficiently. We previously developed a mutant of Cut190 (S226P/R228S), which we designated as Cut190* that has both increased activity and stability and solved its crystal structure. Recently, we showed that mutation of D250C/E296C on one of the Ca2+ -binding sites resulted in a higher thermal stability while retaining its polyesterase activity. In this study, we solved the crystal structures of Cut190* mutants, Q138A/D250C-E296C/Q123H/N202H, designated as Cut190*SS, and its inactive S176A mutant, Cut190*SS_S176A, at high resolution. The overall structures were similar to those of Cut190* and Cut190*S176A reported previously. As expected, Cys250 and Cys296 were closely located to form a disulfide bond, which would assuredly contribute to increase the stability. Isothermal titration calorimetry experiments and 3D Reference Interaction Site Model calculations showed that the metal-binding properties of the Cut190*SS series were different from those of the Cut190* series. However, our results show that binding of Ca2+ to the weak binding site, site 1, would be retained, enabling Cut190*SS to keep its ability to use Ca2+ to accelerate the conformational change from the closed (inactive) to the open (active) form. While increasing the thermal stability, Cut190*SS could still express its enzymatic function. Even after incubation at 70°C, which corresponds to the glass transition temperature of PET, the enzyme retained its activity well, implying a high applicability for industrial PET depolymerization using Cut190*SS.


Assuntos
Actinobacteria/química , Proteínas de Bactérias/química , Cálcio/química , Hidrolases de Éster Carboxílico/química , Poluentes Ambientais/química , Polietilenotereftalatos/química , Actinobacteria/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Hidrolases de Éster Carboxílico/genética , Hidrolases de Éster Carboxílico/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Poluentes Ambientais/metabolismo , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Temperatura Alta , Hidrólise , Modelos Moleculares , Mutação , Polietilenotereftalatos/metabolismo , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
13.
mSphere ; 5(6)2020 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-33361127

RESUMO

Plastics, such as polyethylene terephthalate (PET) from water bottles, are polluting our oceans, cities, and soils. While a number of Pseudomonas species have been described that degrade aliphatic polyesters, such as polyethylene (PE) and polyurethane (PUR), few from this genus that degrade the semiaromatic polymer PET have been reported. In this study, plastic-degrading bacteria were isolated from petroleum-polluted soils and screened for lipase activity that has been associated with PET degradation. Strains and consortia of bacteria were grown in a liquid carbon-free basal medium (LCFBM) with PET as the sole carbon source. We monitored several key physical and chemical properties, including bacterial growth and modification of the plastic surface, using scanning electron microscopy (SEM) and attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) spectroscopy. We detected by-products of hydrolysis of PET using 1H-nuclear magnetic resonance (1H NMR) analysis, consistent with the ATR-FTIR data. The full consortium of five strains containing Pseudomonas and Bacillus species grew synergistically in the presence of PET and the cleavage product bis(2-hydroxyethyl) terephthalic acid (BHET) as sole sources of carbon. Secreted enzymes extracted from the full consortium were capable of fully converting BHET to the metabolically usable monomers terephthalic acid (TPA) and ethylene glycol. Draft genomes provided evidence for mixed enzymatic capabilities between the strains for metabolic degradation of TPA and ethylene glycol, the building blocks of PET polymers, indicating cooperation and ability to cross-feed in a limited nutrient environment with PET as the sole carbon source. The use of bacterial consortia for the biodegradation of PET may provide a partial solution to widespread planetary plastic accumulation.IMPORTANCE While several research groups are utilizing purified enzymes to break down postconsumer PET to the monomers TPA and ethylene glycol to produce new PET products, here, we present a group of five soil bacteria in culture that are able to partially degrade this polymer. To date, mixed Pseudomonas spp. and Bacillus spp. biodegradation of PET has not been described, and this work highlights the possibility of using bacterial consortia to biodegrade or potentially to biorecycle PET plastic waste.


Assuntos
Bacillus/metabolismo , Plásticos/metabolismo , Polietilenotereftalatos/metabolismo , Pseudomonas/metabolismo , Biodegradação Ambiental , Ácidos Ftálicos
14.
Enzyme Microb Technol ; 141: 109656, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33051015

RESUMO

Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETaseS242T and IsPETaseN246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETaseWT. We also developed the IsPETaseS121E/D186H/S242T/N246D variant by integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETaseWT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETaseWT.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiales/enzimologia , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Biodegradação Ambiental , Burkholderiales/genética , Biologia Computacional , Estabilidade Enzimática , Mutação , Polietilenotereftalatos/química , Engenharia de Proteínas , Temperatura
15.
Proc Natl Acad Sci U S A ; 117(41): 25476-25485, 2020 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-32989159

RESUMO

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.


Assuntos
Proteínas de Bactérias/metabolismo , Burkholderiales/enzimologia , Plásticos/metabolismo , Engenharia de Proteínas/métodos , Modelos Moleculares , Mutação , Plásticos/química , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Conformação Proteica , Domínios Proteicos , Especificidade por Substrato
17.
Lett Appl Microbiol ; 71(3): 235-241, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32394501

RESUMO

The polyethylene terephthalate hydrolase (PETase) has been proved to have a high activity to degrade polyethylene terephthalate (PET), but few studies have been carried on its secretion in Bacillus subtilis. In this study, the coding gene of PETase, which was isolated from the Ideonella sakaiensis, was synthesized and expressed in B. subtilis. Then, we evaluated the ability of five Bacillus signal peptides to enhance PETase secretion by B. subtilis. The results indicated that the SPamy -induced secretion of PETase was the highest, and its activity against p-Nitrophenyl palmitate was about fourfold that of the natural signal peptide SPPETase . The weak promoter P43 provided sufficient time for translation and folding of PETase, resulting in increased extracellular expression. Use of P43 and SPamy in combination yielded the greatest bis-(2-hydroxyethyl) terephthalate degradation and PET-film etching activity due to maximized secretion of PETase by B. subtilis. Our findings will facilitate biodegradation of PET plastic. SIGNIFICANCE AND IMPACT OF THE STUDY: High-level expression of polyethylene terephthalate hydrolase (PETase) facilitates biodegradation of PET. In this study, the expression elements, signal peptide and promoter, in the secretory expression system, were optimizing for maximizing secreted expression of PETase in Bacillus subtilis. The constructed strains yielded the greatest bis-(2-hydroxyethyl) terephthalate degradation and PET-film etching activities.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Biodegradação Ambiental , Hidrolases/genética , Plásticos/metabolismo , Polietilenotereftalatos/metabolismo , Proteínas de Bactérias/genética , Burkholderiales/genética , Hidrolases/metabolismo , Palmitatos/metabolismo , Sinais Direcionadores de Proteínas/fisiologia
18.
Nature ; 580(7802): 216-219, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32269349

RESUMO

Present estimates suggest that of the 359 million tons of plastics produced annually worldwide1, 150-200 million tons accumulate in landfill or in the natural environment2. Poly(ethylene terephthalate) (PET) is the most abundant polyester plastic, with almost 70 million tons manufactured annually worldwide for use in textiles and packaging3. The main recycling process for PET, via thermomechanical means, results in a loss of mechanical properties4. Consequently, de novo synthesis is preferred and PET waste continues to accumulate. With a high ratio of aromatic terephthalate units-which reduce chain mobility-PET is a polyester that is extremely difficult to hydrolyse5. Several PET hydrolase enzymes have been reported, but show limited productivity6,7. Here we describe an improved PET hydrolase that ultimately achieves, over 10 hours, a minimum of 90 per cent PET depolymerization into monomers, with a productivity of 16.7 grams of terephthalate per litre per hour (200 grams per kilogram of PET suspension, with an enzyme concentration of 3 milligrams per gram of PET). This highly efficient, optimized enzyme outperforms all PET hydrolases reported so far, including an enzyme8,9 from the bacterium Ideonella sakaiensis strain 201-F6 (even assisted by a secondary enzyme10) and related improved variants11-14 that have attracted recent interest. We also show that biologically recycled PET exhibiting the same properties as petrochemical PET can be produced from enzymatically depolymerized PET waste, before being processed into bottles, thereby contributing towards the concept of a circular PET economy.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Plásticos/química , Plásticos/metabolismo , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Engenharia de Proteínas , Reciclagem , Actinobacteria/enzimologia , Burkholderiales/enzimologia , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Fusarium/enzimologia , Modelos Moleculares , Ácidos Ftálicos/metabolismo , Polimerização , Thermobifida
19.
Microb Cell Fact ; 19(1): 97, 2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345276

RESUMO

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


Assuntos
Hidrolases/genética , Microalgas/enzimologia , Polietilenotereftalatos/metabolismo , Biocatálise , Hidrolases/metabolismo , Hidrólise , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Polietilenotereftalatos/química , Propriedades de Superfície
20.
Chemosphere ; 238: 124560, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31437632

RESUMO

Plastics are the most abundant marine debris globally dispersed in the oceans and its production is rising with documented negative impacts in marine ecosystems. However, the chemical-physical and biological interactions occurring between plastic and planktonic communities of different types of microorganisms are poorly understood. In these respects, it is of paramount importance to understand, on a molecular level on the surface, what happens to plastic fragments when dispersed in the ocean and directly interacting with phytoplankton assemblages. This study presents a computer-aided analysis of electron paramagnetic resonance (EPR) spectra of selected spin probes able to enter the phyoplanktonic cell interface and interact with the plastic surface. Two different marine phytoplankton species were analyzed, such as the diatom Skeletonema marinoi and dinoflagellate Lingulodinium polyedrum, in absence and presence of polyethylene terephthalate (PET) fragments in synthetic seawater (ASPM), in order to in-situ characterize the interactions occurring between the microalgal cells and plastic surfaces. The analysis was performed at increasing incubation times. The cellular growth and adhesion rates of microalgae in batch culture medium and on the plastic fragments were also evaluated. The data agreed with the EPR results, which showed a significant difference in terms of surface properties between the diatom and dinoflagellate species. Low-polar interactions of lipid aggregates with the plastic surface sites were mainly responsible for the cell-plastic adhesion by S. marinoi, which is exponentially growing on the plastic surface over the incubation time.


Assuntos
Diatomáceas/metabolismo , Dinoflagelados/metabolismo , Microalgas/crescimento & desenvolvimento , Fitoplâncton/metabolismo , Plásticos/metabolismo , Polietilenotereftalatos/metabolismo , Ecossistema , Espectroscopia de Ressonância de Spin Eletrônica , Microalgas/metabolismo , Oceanos e Mares , Água do Mar/química , Resíduos/análise
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