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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

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.

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.

The growing pollution of the environment with plastic debris is a global threat which urgently requires biotechnological solutions. Enzymatic recycling not only prevents pollution but also would allow recovery of valuable building blocks. Therefore, we explored the existence of microbial polyesterases in microbial communities associated with the Sphagnum magellanicum moss, a key species within unexploited bog ecosystems. This resulted in the identification of six novel esterases, which were isolated, cloned, and heterologously expressed in Escherichia coli . The esterases were found to hydrolyze the copolyester poly(butylene adipate- co -butylene terephthalate) (PBAT) and the oligomeric model substrate bis[4-(benzoyloxy)butyl] terephthalate (BaBTaBBa). Two promising polyesterase candidates, EstB3 and EstC7, which clustered in family VIII of bacterial lipolytic enzymes, were purified and characterized using the soluble esterase substrate p -nitrophenyl butyrate ( K m values of 46.5 and 3.4 μM, temperature optima of 48°C and 50°C, and pH optima of 7.0 and 8.5, respectively). In particular, EstC7 showed outstanding activity and a strong preference for hydrolysis of the aromatic ester bond in PBAT. Our study highlights the potential of plant-associated microbiomes from extreme natural ecosystems as a source for novel hydrolytic enzymes hydrolyzing polymeric compounds. IMPORTANCE In this study, we describe the discovery and analysis of new enzymes from microbial communities associated with plants (moss). The recovered enzymes show the ability to hydrolyze not only common esterase substrates but also the synthetic polyester poly(butylene adipate- co -butylene terephthalate), which is a common material employed in biodegradable plastics. The widespread use of such synthetic polyesters in industry and society requires the development of new sustainable technological solutions for their recycling. The discovered enzymes have the potential to be used as catalysts for selective recovery of valuable building blocks from this material.

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.

Certain α/β hydrolases have the ability to hydrolyze synthetic polyesters. While their partial hydrolysis has a potential for surface functionalization, complete hydrolysis allows recycling of valuable building blocks. Although knowledge about biodegradation of these materials is important regarding their fate in the environment, it is currently limited to aerobic organisms. A lipase from the anaerobic groundwater organism Pelosinus fermentans DSM 17108(PfL1) was cloned and expressed in Escherichia coli BL21-Gold (DE3) and purified from the cell extract. Biochemical characterization with small substrates showed thermoalkalophilic properties (Topt=50 °C, pHopt=7.5) and higher activity towards para-nitrophenyl octanoate (12.7 U mg(-1)) compared to longer and shorter chain lengths (C14 0.7 U mg(-1) and C2 4.3 U mg(-1), respectively). Crystallization and determination of the 3-D structure displayed the presence of a lid structure and a zinc ion surrounded by an extra domain. These properties classify the enzyme into the I.5 lipase family. PfL1 is able to hydrolyze poly(1,4-butylene adipate-co-terephthalate) (PBAT) polymeric substrates. The hydrolysis of PBAT showed the release of small building blocks as detected by liquid chromatography mass spectrometry (LC-MS). Protein dynamics seem to be involved with lid opening for the hydrolysis of PBAT by PfL1.

Alkyd resins are polyesters containing unsaturated fatty acids that are used as binding agents in paints and coatings. Chemical drying of these polyesters is based on heavy metal catalyzed cross-linking of the unsaturated fatty acid moieties. Among the heavy-metal catalysts, cobalt complexes are the most effective, yet they have been proven to be carcinogenic. Therefore, strategies to replace the cobalt-based catalyst by environmentally friendlier and less toxic alternatives are under development. Here, we demonstrate for the first time that a laccase–mediator system can effectively replace the heavy-metal catalyst and cross-link alkyd resins. Interestingly, the biocatalytic reaction does not only work in aqueous media, but also in a solid film, where enzyme diffusion is limited. Within the catalytic cycle, the mediator oxidizes the alkyd resin and is regenerated by the laccase, which is uniformly distributed within the drying film as evidenced by confocal laser scanning microscopy. During gradual build-up of molecular weight, there is a concomitant decrease of the oxygen content in the film. A new optical sensor to follow oxygen consumption during the cross-linking reaction was developed and validated with state of the art techniques. A remarkable feature is the low sample amount required, which allows faster screening of new catalysts.