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Novel strategies allowing environmentally friendly recycling of plastics are strongly needed. Enzymes have shown high potential, especially for the recovery of building blocks from multilayer materials which will be discussed in this paper. It has been shown that enzymes can specifically hydrolyze and solubilize certain components of blended packaging materials or mixed wastes. This allows a step—wise recovery of valuable building blocks which can be used for re-synthesis or for bioproduction (e.g. recovered glucose). However, despite the high potential of biocatalysts, even more efficient enzymes are required for economic industrial implementation. In this paper, which is based on a contribution to the SUM 2022 conference, we will consequently demonstrate how enzyme discovery can lead to more powerful tools for plastics recycling and provide some examples.

The study showed novel findings about changes in the fate and bioavailability of conazole fungicides (CFs) after biochar (BC) addition to soil. Two contrasting soils (low- and high-sorbing of CF; L soils, H soils) were amended by three BCs (low-, moderate-, and high-sorbing of CF; L-BC, M-BC, H-BC) at 0.2% and 2% doses. Epoxiconazole (EPC) and tebuconazole (TBC) were then added to the soil–BC mixtures, and their degradation, bioaccumulation in earthworms (Eisenia andrei), and bioconcentration in lettuce (Lactuca sativa) were studied for three months. Also, stir bar sorptive extraction (SBSE) was performed to determine CF (bio)accessibility. The EPC and TBC degradation in the soil–BC mixtures followed usually the first-order decay kinetics. The BC addition prevalently decreased the pesticides degradation in the L soil mixtures but often increased it in the H soil mixtures. In general, EPC degraded less than TBC. BC type and dose roles in the pesticides degradation were unclear. The BC addition significantly reduced pesticide uptake to the earthworms in the L soil mixtures (by 37–96%) and in the H soil mixtures (by 6–89%) with 2% BC. The BC addition reduced pesticide uptake to the lettuce roots and leaves significantly—up to two orders of magnitude, and this reduction was strong in H soil mixtures at 2% of BC. The BC addition reduced the CF (bio)accessibility measured by SBSE in all L soil mixtures and some H soil mixtures with 2% BC. Although not significant, it also seems that the pesticide bioaccumulation, bioconcentration, and (bio)accessibility were decreasing according to the BC type (L-BC > M-BC > H-BC). The pesticide concentrations in the earthworms and lettuce correlated significantly to the SBSE results, which indicates this technique as a possible predictor of biotic uptake. Our results showed that the interactions were hard to predict in the complex soil–BC–pesticide system.

Lignin is the second most abundant biopolymer on earth and accrues in large amounts in the pulp- and paper industry. While currently lignins are mainly used for low-value applications such as energy production, recently the potential of laccases for upgrading lignins has been demonstrated. In this study, two laccases of fungal and bacterial origin were characterized regarding their potential to polymerize lignosulfonate. The laccases MaL1 from Melanocarpus albomyces, and SrLA from Streptomyces rochei were heterologously expressed and showed typical characteristics of laccases, like acidic pH optima for ABTS at pH 4 and 5, respectively, and temperature optima at 50 °C and 80 °C. Polymerization of lignosulfonate with MaL1 led to an almost two-fold increase of the molecular weight, according to size exclusion (SEC) multiangle laser light scattering (MALLS) analysis. In contrast, SrLA showed considerably less activity on lignosulfonates, as measured based on oxygen consumption and SEC-MALLS.

By testing 92 different biochars, this study had the objective to determine the relations between simple physico-chemical characteristics of biochar (elemental composition, ash fraction, specific surface area, process parameters) and infrared sorption characteristics revealing the presence of specific functional groups. The results of Diffusive Reflection Fourier Transformation Infrared Spectroscopy were statistically analyzed with multiple linear regression techniques and Principal Component Analysis (PCA) with the biochar characterization parameters as model inputs. The dominant parameters affecting the functional group signals were the pyrolysis temperature, the H/C-ratio and specific surface area of the biochar and the ash fraction. Regression models were able to explain 60–90% of data variance for specific peaks in the DRIFTS-spectra. The application of PCA could further reduce the input parameters to three main factors explaining 70.8% of total data variance. Biplots of the first two main factors showed the similarity of infrared spectra of biochars produced at 300 and 450 °C, whereas a pyrolysis temperature of 600 °C lead to a partial and a 750 °C to a nearly complete loss of biochar surface functional groups.

Tyrosinases catalyse both the cresolase and catecholase reactions for the formation of reactive compounds which are very important for industrial applications. In this study, we describe a proteolytic activity of tyrosinases. Two different tyrosinases originating from mushroom and apple are able to cleave the carboxylesterase EstA. The cleavage reaction correlates with the integrity of the active site of tyrosinase and is independent of other possible influencing factors, which could be present in the reaction. Therefore, the cleavage of EstA represents a novel functionality of tyrosinases. EstA was previously reported to degrade synthetic polyesters, albeit slowly. However, the EstA truncated by tyrosinase shows higher degradation activity on the non-biodegradable polyester polyethylene terephthalate (PET), which is a well-established environmental threat. Tyrosinases are an industrially significant class of polyphenol oxidase. Here, two tyrosinases are shown to cleave a specific peptide bond in a carboxylesterase, yielding a truncated product with higher catalytic activity than the full-length enzyme.

Cutinases can play a significant role in a biotechnology-based circular economy. However, relatively little is known about the structure-function relationship of these enzymes, knowledge that is vital to advance optimized, engineered enzyme candidates. Here, two almost identical cutinases from Thermobifida cellulosilytica DSM44535 (Thc_Cut1 and Thc_Cut2) with only 18 amino acids difference were used for a rigorous biochemical characterization of their ability to hydrolyze poly(ethylene terephthalate) (PET), PET-model substrates, and cutin-model substrates. Kinetic parameters were compared with detailed in silico docking studies of enzyme-ligand interactions. The two enzymes interacted with, and hydrolyzed PET differently, with Thc_Cut1 generating smaller PET-degradation products. Thc_Cut1 also showed higher catalytic efficiency on long-chain aliphatic substrates, an effect likely caused by small changes in the binding architecture. Thc_Cut2, in contrast, showed improved binding and catalytic efficiency when approaching the glass transition temperature of PET, an effect likely caused by longer amino acid residues in one area at the enzyme's surface. Finally, the position of the single residue Q93 close to the active site, rotated out in Thc_Cut2, influenced the ligand position of a trimeric PET-model substrate. In conclusion, we illustrate that even minor sequence differences in cutinases can affect their substrate binding, substrate specificity, and catalytic efficiency drastically.

Environmentally friendly functionalization and recycling processes for synthetic polymers have recently gained momentum, and enzymes play a central role in these procedures. However, natural enzymes must be engineered to accept synthetic polymers as substrates. To enhance the activity on synthetic polyesters, the canonical amino acid methionine in Thermoanaerobacter thermohydrosulfuricus lipase (TTL) was exchanged by the residue-specific incorporation method for the more hydrophobic non-canonical norleucine (Nle). Strutural modelling of TTL revealed that residues Met-114 and Met-142 are in close vicinity of the active site and their replacement by the norleucine could modulate the catalytic activity of the enzyme. Indeed, hydrolysis of the polyethylene terephthalate model substrate by the Nle variant resulted in significantly higher amounts of release products than the Met variant. A similar trend was observed for an ionic phthalic polyester containing a short alkyl diol (C5). Interestingly, a 50% increased activity was found for TTL [Nle] towards ionic phthalic polyesters containing different ether diols compared to the parent enzyme TTL [Met]. These findings clearly demonstrate the high potential of non-canonical amino acids for enzyme engineering.

The study showed novel findings about changes in the fate and bioavailability of conazole fungicides (CFs) after biochar (BC) addition to soil. Two contrasting soils (low- and high-sorbing of CF; L soils, H soils) were amended by three BCs (low-, moderate-, and high-sorbing of CF; L-BC, M-BC, H-BC) at 0.2% and 2% doses. Epoxiconazole (EPC) and tebuconazole (TBC) were then added to the soil-BC mixtures, and their degradation, bioaccumulation in earthworms (Eisenia andrei), and bioconcentration in lettuce (Lactuca sativa) were studied for three months. Also, stir bar sorptive extraction (SBSE) was performed to determine CF (bio)accessibility. The EPC and TBC degradation in the soil-BC mixtures followed usually the first-order decay kinetics. The BC addition prevalently decreased the pesticides degradation in the L soil mixtures but often increased it in the H soil mixtures. In general, EPC degraded less than TBC. BC type and dose roles in the pesticides degradation were unclear. The BC addition significantly reduced pesticide uptake to the earthworms in the L soil mixtures (by 37-96%) and in the H soil mixtures (by 6-89%) with 2% BC. The BC addition reduced pesticide uptake to the lettuce roots and leaves significantly-up to two orders of magnitude, and this reduction was strong in H soil mixtures at 2% of BC. The BC addition reduced the CF (bio)accessibility measured by SBSE in all L soil mixtures and some H soil mixtures with 2% BC. Although not significant, it also seems that the pesticide bioaccumulation, bioconcentration, and (bio)accessibility were decreasing according to the BC type (L-BC M-BC H-BC). The pesticide concentrations in the earthworms and lettuce correlated significantly to the SBSE results, which indicates this technique as a possible predictor of biotic uptake. Our results showed that the interactions were hard to predict in the complex soil-BC-pesticide system.

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H2O2 to promote the epoxidation reaction. The proof‐of‐concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.

In the past years, several serine hydrolases such as cutinases, esterases and lipases have shown the ability to degrade not only natural polymers but also synthetic polyesters, even aromatic representatives like polyethylene terephthalate (PET). Hence, cutinases and related ester hydrolases have become very important to be applied in the biocatalytic plastic recycling as green alternative to chemical recycling as well as to the functionalization of polyester surfaces in order to change superficial properties like hydrophobicity or hydrophilicity. Sorption characteristics of the enzymes to the polymers have turned out to be a crucial process for efficient polymer hydrolysis. Hence, special attention was paid on tuning the sorption of the enzymes to the hydrophobic polymers. Engineering of the enzyme surface, fusion of hydrophobic substrate-binding domains or truncation of domains hindering the access of the polymer to the enzyme has led to significant improvement of sorption processes and consequently increased activity on the bulky substrate. Finally, the combination of engineering approaches has proved that they can bring additional advantages in improving the enzyme activity when used in a synergistic manner.

In the past years, several serine hydrolases such as cutinases, esterases and lipases have shown the ability to degrade not only natural polymers but also synthetic polyesters, even aromatic representatives like polyethylene terephthalate (PET). Hence, cutinases and related ester hydrolases have become very important to be applied in the biocatalytic plastic recycling as green alternative to chemical recycling as well as to the functionalization of polyester surfaces in order to change superficial properties like hydrophobicity or hydrophilicity. Sorption characteristics of the enzymes to the polymers have turned out to be a crucial process for efficient polymer hydrolysis. Hence, special attention was paid on tuning the sorption of the enzymes to the hydrophobic polymers. Engineering of the enzyme surface, fusion of hydrophobic substrate-binding domains or truncation of domains hindering the access of the polymer to the enzyme has led to significant improvement of sorption processes and consequently increased activity on the bulky substrate. Finally, the combination of engineering approaches has proved that they can bring additional advantages in improving the enzyme activity when used in a synergistic manner.

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 albicans. 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.

[EN] Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase-catalysed oxyfunctionali-zation reactions. Choline chloride-based NADES are of particularinterest as they can serve as solvent, enzyme-stabiliser and sacrificial electron donor for the in situ H2O2generation. This report provides the first proof-of-concept and basic character-isation of this new reaction system. Highly promising turnovernumbers for the biocatalysts of up to 200,000 have been achieved., This work was supported by the National Outstanding Youth Science Foundation of China (31725022), National Key R&D Program of China (2018YFC0311104), International Collaboration Base for Molecular Enzymology and Enzyme Engineering (2017 A050503001), and also supported by the 111 Project (B17018). F.H. gratefully acknowledges funding by the European Research Commission (ERC consolidator grant, No.648026), the European Union (H2020-BBI-PPP-2015-2-1-720297), and the Netherlands Organization for Scientific Research (VICI grant, No 724.014.003)

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

Certain members of the carboxylesterase superfamily can act at the interface between water and water-insoluble substrates. However, nonnatural bulky polyesters usually are not efficiently hydrolyzed. In the recent years, the potential of enzyme engineering to improve hydrolysis of synthetic polyesters has been demonstrated. Regions on the enzyme surface have been modified by using site-directed mutagenesis in order to tune sorption processes through increased hydrophobicity of the enzyme surface. Such modifications can involve specific amino acid substitutions, addition of binding modules, or truncation of entire domains improving sorption properties and/or dynamics of the enzyme. In this review, we provide a comprehensive overview on different strategies developed in the recent years for enzyme surface engineering to improve the activity of polyester-hydrolyzing enzymes.

Synthetic polyesters are today the second-largest class of ingredients in household products and are entering wastewater treatment plants (WWTPs) after product utilization. One approach to improve polymer biodegradation in wastewater would be to complement current processes with polyester-hydrolyzing enzymes and their microbial producers. In this study, the hydrolysis of poly(oxyethylene terephthalate) polymer by hydrolases from wastewater microorganisms was investigated in vitro and under realistic WWTP conditions. An esterase and a cutinase from Pseudomonas pseudoalcaligenes and a lipase from Pseudomonas pelagia were heterologously expressed in Escherichia coli BL21-Gold(DE3) and were purified by a C-terminal His6 tag. The hydrolases were proven to hydrolyze the polymer effectively, which is a prerequisite for further biodegradation. The hydrolases maintained high activity up to 50 % upon lowering the temperature from 28 to 15 °C to mimic WWTP conditions. The hydrolases were also not inhibited by the wastewater matrix. Polyester-hydrolyzing enzymes active under WWTP conditions and their microbial producers thus have the potential to improve biological treatment of wastewater rich in synthetic polymers.

Carbonic anhydrase (CA) is an enzyme of high interest due to its high implications relative to the medical and environmental sectors. In the current paper, an enzyme assay for the determination of CA activity is proposed and it is characterized by its simplicity and high practicability. It permits the straightforward comparison of CAs performance in physiological conditions. The methodology and the theoretical background of the evaluation method are explained in detail. Moreover, the presumed advantages over alternative assays are discussed. The assay has proven to be particularly useful for the screening of CA activity with respect to their application in CO2, capturing processes for further utilization or storage.

We have investigated the structures of two native cutinases from Thermobifida cellulosilytica, namely Thc_Cut1 and Thc_Cut2 as well as of two variants, Thc_Cut2_DM (Thc_Cut2_ Arg29Asn_Ala30Val) and Thc_Cut2_TM (Thc_Cut2_Arg19Ser_Arg29Asn_Ala30Val). The four enzymes showed different activities towards the aliphatic polyester poly(lactic acid) (PLLA). The crystal structures of the four enzymes were successfully solved and in combination with Small Angle X-Ray Scattering (SAXS) the structural features responsible for the selectivity difference were elucidated. Analysis of the crystal structures did not indicate significant conformational differences among the different cutinases. However, the distinctive SAXS scattering data collected from the enzymes in solution indicated a remarkable surface charge difference. The difference in the electrostatic and hydrophobic surface properties could explain potential alternative binding modes of the four cutinases on PLLA explaining their distinct activities. Biotechnol. Bioeng. 2017;114: 2481-2488. © 2017 Wiley Periodicals, Inc.

The carboxylesterase from Clostridium botulinum (Cbotu_EstA) has been shown to hydrolyze the surface of the polyester poly(butylene adipate-co-terephthalate) (PBAT) releasing the monomeric building blocks. Cbotu_EstA contains a zinc ion, tetrahedrally coordinated by two histidine and two aspartic acid residues, which is buried inside an extra domain typical for members of the I.5 lipase family. To elucidate the role of this extra domain with regard to polyester hydrolysis, variants of the zinc-binding domain were constructed and expressed in E. coli BL21-Gold(DE3). These enzyme variants were characterized with respect to their specific activity, kinetic parameters and thermostability on soluble substrates as well as on PBAT. All the variants exhibited a similar affinity towards the small substrate para-nitrophenyl butyrate (pNPB), with KM values between 0.4 and 1.2 mM, while the catalytic efficiency decreased approximately 1000-fold for the zinc-binding variants and 5-fold for the zinc cavity variants. Moreover, all four variants of the zinc-coordination site (D130L, H150F, H156F, and D302L) showed a loss of thermostability. However, H156F and D302L revealed a drastic loss of thermostability compared to D130L and H150F. Nevertheless, compared to Cbotu_EstA, variants carrying substitutions of amino acids in the zinc-binding domain were able to release up to 10 times more soluble products from the polymeric substrate PBAT. The thermostability at 50 °C was increased in the case of F154Y and W274H, carrying more hydrophilic residues. These data clearly demonstrate the importance of different regions of the zinc-binding domain for the hydrolysis of polyesters like PBAT.

A novel esterase, PpEst, that hydrolyses the co-aromatic-aliphatic polyester poly(1,4-butylene adipate-co-terephthalate) (PBAT) was identified by proteomic screening of the Pseudomonas pseudoalcaligenes secretome. PpEst was induced by the presence of PBAT in the growth media and had predicted arylesterase (EC 3.1.1.2) activity. PpEst showed polyesterase activity on both whole and milled PBAT film releasing terephthalic acid and 4-(4-hydroxybutoxycarbonyl)benzoic acid while end product inhibition by 4-(4-hydroxybutoxycarbonyl)benzoic acid was observed. Modelling of an aromatic polyester mimicking oligomer into the PpEst active site indicated that the binding pocket could be big enough to accommodate large polymers. This is the first report of a PBAT degrading enzyme being identified by proteomic screening and shows that this approach can contribute to the discovery of new polymer hydrolysing enzymes. Moreover, these results indicate that arylesterases could be an interesting enzyme class for identifications of polyesterases. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7992-8) contains supplementary material, which is available to authorized users.

Surface functionalization such as hydrophilization is an important step in the polymer manufacturing process and a key requirement for application of polyesters. Conventional methods like chemical or plasma treatment are often toxic, expensive and adversely affect the mechanical properties of the polymer. Enzymes have proven to be an attractive alternative for surface hydrolysis and functionalization of synthetic polymers since they work under mild and environmental friendly process conditions while preserving the mechanical properties. This chapter deals with the enzymatic surface treatment of polyesters and in particular with current methods for the analysis of polymer hydrolysis and of changes of surface properties.

The recalcitrance of plastics like nylon and other polyamides contributes to environmental problems (

Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Surface functionalization such as hydrophilization is an important step in the polymer manufacturing process and a key requirement for application of polyesters. Conventional methods like chemical or plasma treatment are often toxic, expensive and adversely affect the mechanical properties of the polymer. Enzymes have proven to be an attractive alternative for surface hydrolysis and functionalization of synthetic polymers since they work under mild and environmental friendly process conditions while preserving the mechanical properties. This chapter deals with the enzymatic surface treatment of polyesters and in particular with current methods for the analysis of polymer hydrolysis and of changes of surface properties.

The aliphatic-aromatic copolyester poly(butylene adipate-co-butylene terephthalate) (PBAT), 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]. Keywords: Oligomer synthesis, Oligomer analysis, Polyester model substrates

Sialic acid groups of protein N-glycans are important determinants of biological activity. Exposed at the end of the glycan chain, they are potential targets for glycan remodeling. Sialyltransferases (STs; EC 2.4.99) are the enzymes that catalyze the sialic acid transfer from a CMP-activated donor on to a carbohydrate acceptor in vivo. Recombinant expression of the full-length human β-galactoside α2,6 sialyltransferase I (ST6Gal-I) was hampered and therefore variants with truncated N-termini were investigated. We report on the distinct properties of two N-terminally truncated versions of ST6Gal-I, namely Δ89ST6Gal-I and Δ108ST6Gal-I, which were successfully expressed in human embryonic kidney cells. The different properties of these enzymes result most probably from the loss of interactions from helix α1 in the Δ108ST6Gal-I variant, which plays a role in acceptor substrate binding. The Km for N-acetyl-d-lactosamine was 10-fold increased for Δ108ST6Gal-I (84 mM) as compared to Δ89ST6Gal-I (8.3 mM). The two enzyme variants constitute a suitable tool box for the terminal modification of N-glycans. While the enzyme Δ89ST6Gal-I exhibited both ST (di-sialylation) and sialidase activity on a monoclonal antibody, the enzyme Δ108ST6Gal-I showed only ST activity with specificity for mono-sialylation.

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.

Recently, a variety of biodegradable polymers have been developed as alternatives to recalcitrant materials. Although 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. On the basis of reverse-phase high-performance liquid chromatography (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 micro-organisms in anaerobic habitats can degrade manmade PBAT.

Two novel esterases from the anaerobe Clostridium botulinum ATCC 3502 (Cbotu_EstA and Cbotu_EstB) were expressed in Escherichia 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 (sixfold) 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.

Cutinases have shown potential for hydrolysis of the recalcitrant synthetic polymer polyethylene terephthalate (PET). We have shown previously that the rate of this hydrolysis can be enhanced by the addition of hydrophobins, small fungal proteins that can alter the physicochemical properties of surfaces. Here we have investigated whether the PET-hydrolyzing activity of a bacterial cutinase from Thermobifida cellulosilytica (Thc_Cut1) would be further enhanced by fusion to one of three Trichoderma hydrophobins, i.e., the class II hydrophobins HFB4 and HFB7 and the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibited decreased k cat values on soluble substrates ( p -nitrophenyl acetate and p -nitrophenyl butyrate) and strongly decreased the hydrophilicity of glass but caused only small changes in the hydrophobicity of PET. When the enzyme was fused to HFB4 or HFB7, the hydrolysis of PET was enhanced >16-fold over the level with the free enzyme, while a mixture of the enzyme and the hydrophobins led only to a 4-fold increase at most. Fusion with the non-class II hydrophobin HFB9b did not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performed best when PET was preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion enzymes differed from that of Thc_Cut1 as the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid, increased. Small-angle X-ray scattering (SAXS) analysis revealed an increased scattering contrast of the fusion proteins over that of the free proteins, suggesting a change in conformation or enhanced protein aggregation. Our data show that the level of hydrolysis of PET by cutinase can be significantly increased by fusion to hydrophobins. The data further suggest that this likely involves binding of the hydrophobins to the cutinase and changes in the conformation of its active center.

A series of copolyesters based on furanic acid and sulfonated isophthalic acid with various polyols were synthetized and their susceptibility to enzymatic hydrolysis by cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) investigated. All copolyesters consisted of 30 mol % 5-sulfoisophthalate units (NaSIP) and 70 mol % 2,5-furandicarboxylic acid (FDCA), while the polyol component was varied, including 1,2-ethanediol, 1,4-butanediol, 1,8-octanediol, diethylene glycol, triethylene glycol, or tetraethylene glycol. The composition of the copolyesters was confirmed by 1H-NMR and the number average molecular weight (Mn) was determined by GPC to range from 2630 to 8030 g/mol. A DSC analysis revealed glass-transition temperatures (Tg) from 84 to 6 °C, which were decreasing with increasing diol chain length. The crystallinity was below 1% for all polyesters. The hydrolytic stability increased with the chain length of the alkyl diol unit, while it was generally higher for the ether diol units. Thc_Cut1 was able to hydrolyze all of the copolyesters containing alkyl diols ranging from two to eight carbon chain lengths, while the highest activities were detected for the shorter chain lengths with an amount of 13.6 ± 0.7 mM FDCA released after 72 h of incubation at 50 °C. Faster hydrolysis was observed when replacing an alkyl diol by ether diols, as indicated, e.g., by a fivefold higher release of FDCA for triethylene glycol when compared to 1,8-octanediol. A positive influence of introducing ionic phthalic acid was observed while the enzyme preferentially cleaved ester bonds associated to the non-charged building blocks.

Biodegradable polyesters have a large potential to replace persistent polymers in numerous applications and to thereby reduce the accumulation of plastics in the environment. Ester hydrolysis by extracellular carboxylesterases is considered the rate-limiting step in polyester biodegradation. In this work, we systematically investigated the effects of polyester and carboxylesterase structure on the hydrolysis of nanometer-thin polyester films using a quartz-crystal microbalance with dissipation monitoring. Hydrolyzability increased with increasing polyester-chain flexibility as evidenced from differences in the hydrolysis rates and extents of aliphatic polyesters varying in the length of their dicarboxylic acid unit and of poly(butylene adipate-co-terephthalate) (PBAT) polyesters varying in their terephthalate-to-adipate ratio by Rhizopus oryzae lipase and Fusarium solani cutinase. Nanoscale nonuniformities in the PBAT films affected enzymatic hydrolysis and were likely caused by domains with elevated terephthalate contents that impaired enzymatic hydrolysis. Yet, the cutinase completely hydrolyzed all PBAT films, including films with a terephthalate-to-adipate molar ratio of one, under environmentally relevant conditions (pH 6, 20 °C). A comparative analysis of the hydrolysis of two model polyesters by eight different carboxylesterases revealed increasing hydrolysis with increasing accessibility of the enzyme active site. Therefore, this work highlights the importance of both polyester and carboxylesterase structure to enzymatic polyester hydrolysis.

Water-soluble polyesters are used in a range of applications today and enter wastewater treatment plants after product utilization. However, little is known about extracellular enzymes and aquatic microorganisms involved in polyester biodegradation and mineralization. In this study, structurally different ionic phthalic acid based polyesters (the number-average molecular weights (Mn) 1770 to 10 000 g/mol and semi crystalline with crystallinity below 1%) were synthesized in various combinations. Typical wastewater microorganisms like Pseudomonas sp. were chosen for in-silico screening toward polyester hydrolyzing enzymes. Based on the in-silico search, a cutinase from Pseudomonas pseudoalcaligenes (PpCutA) and a putative lipase from Pseudomonas pelagia (PpelaLip) were identified. The enzymes PpCutA and PpelaLip were demonstrated to hydrolyze all structurally different polyesters. Activities on all the polyesters were also confirmed with the strains P. pseudoalcaligenes and P. pelagia. Parameters identified...