Hydrolysis of Antimicrobial Peptides by Extracellular Peptidases in Wastewater.

Several antimicrobial peptides (AMPs) are emerging as promising novel antibiotics. When released into wastewater streams after use, AMPs might be hydrolyzed and inactivated by wastewater peptidases─resulting in a reduced release of active antimicrobials into wastewater-receiving environments. A key step towards a better understanding of the fate of AMPs in wastewater systems is to investigate the activity and specificity of wastewater peptidases. Here, we quantified peptidase activity in extracellular extracts from different stages throughout the wastewater treatment process. For all four tested municipal wastewater treatment plants, we detected highest activity in raw wastewater. Complementarily, we assessed the potential of enzymes in raw wastewater extracts to biotransform 10 selected AMPs. We found large variations in the susceptibility of AMPs to enzymatic transformation, indicating substantial substrate specificity of extracted enzymes. To obtain insights into peptidase specificities, we searched for hydrolysis products of rapidly biotransformed AMPs and quantified selected products using synthetic standards. We found that hydrolysis occurred at specific sites and that these sites were remarkably conserved across the four tested wastewaters. Together, these findings provide insights into the fate of AMPs in wastewater systems and can inform the selection and design of peptide-based antibiotics that are hydrolyzable by wastewater peptidases.

Environmental Biodegradation of Water-Soluble Polymers: Key Considerations and Ways Forward.

Water-soluble polymers (WSPs) have unique properties that are valuable in diverse applications ranging from home and personal care products to agricultural formulations. For applications that result in the release of WSPs into natural environments or engineered systems, such as agricultural soils and wastewater streams, biodegradable as opposed to nonbiodegradable WSPs have the advantage of breaking down and, thereby, eliminating the risk of persistence and accumulation. In this Commentary, we emphasize central steps in WSP biodegradation, discuss how these steps depend on both WSP properties and characteristics of the receiving environment, and highlight critical requirements for testing WSP biodegradability.

Uptake, Metabolism, and Accumulation of Tire Wear Particle-Derived Compounds in Lettuce.

Tire wear particle (TWP)-derived compounds may be of high concern to consumers when released in the root zone of edible plants. We exposed lettuce plants to the TWP-derived compounds diphenylguanidine (DPG), hexamethoxymethylmelamine (HMMM), benzothiazole (BTZ), N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine (6PPD), and its quinone transformation product (6PPD-q) at concentrations of 1 mg L-1 in hydroponic solutions over 14 days to analyze if they are taken up and metabolized by the plants. Assuming that TWP may be a long-term source of TWP-derived compounds to plants, we further investigated the effect of leaching from TWP on the concentration of leachate compounds in lettuce leaves by adding constantly leaching TWP to the hydroponic solutions. Concentrations in leaves, roots, and nutrient solution were quantified by triple quadrupole mass spectrometry, and metabolites in the leaves were identified by Orbitrap high resolution mass spectrometry. This study demonstrates that TWP-derived compounds are readily taken up by lettuce with measured maximum leaf concentrations between ∼0.75 (6PPD) and 20 µg g-1 (HMMM). Although these compounds were metabolized in the plant, we identified several transformation products, most of which proved to be more stable in the lettuce leaves than the parent compounds. Furthermore, continuous leaching from TWP led to a resupply and replenishment of the metabolized compounds in the lettuce leaves. The stability of metabolized TWP-derived compounds with largely unknown toxicities is particularly concerning and is an important new aspect for the impact assessment of TWP in the environment.

Identifying Functional Groups that Determine Rates of Micropollutant Biotransformations Performed by Wastewater Microbial Communities.

The goal of this research was to identify functional groups that determine rates of micropollutant (MP) biotransformations performed by wastewater microbial communities. To meet this goal, we performed a series of incubation experiments seeded with four independent wastewater microbial communities and spiked them with a mixture of 40 structurally diverse MPs. We collected samples over time and used high-resolution mass spectrometry to estimate biotransformation rate constants for each MP in each experiment and to propose structures of 46 biotransformation products. We then developed random forest models to classify the biotransformation rate constants based on the presence of specific functional groups or observed biotransformations. We extracted classification importance metrics from each random forest model and compared them across wastewater microbial communities. Our analysis revealed 30 functional groups that we define as either biotransformation promoters, biotransformation inhibitors, structural features that can be biotransformed based on uncharacterized features of the wastewater microbial community, or structural features that are not rate-determining. Our experimental data and analysis provide novel insights into MP biotransformations that can be used to more accurately predict MP biotransformations or to inform the design of new chemical products that may be more readily biodegradable during wastewater treatment.

Effect of Polymer Properties on the Biodegradation of Polyurethane Microplastics.

The release of fragments from plastic products, that is, secondary microplastics, is a major concern in the context of the global plastic pollution. Currently available (thermoplastic) polyurethanes [(T)PU] are not biodegradable and therefore should be recycled. However, the ester bond in (T)PUs might be sufficiently hydrolysable to enable at least partial biodegradation of polyurethane particles. Here, we investigated biodegradation in compost of different types of (T)PU to gain insights into their fragmentation and biodegradation mechanisms. The studied (T)PUs varied regarding the chemistry of their polymer backbone (aromatic/aliphatic), hard phase content, cross-linking degree, and presence of a hydrolysis-stabilizing additive. We developed and validated an efficient and non-destructive polymer particle extraction process for partially biodegraded (T)PUs based on ultrasonication and density separation. Our results showed that biodegradation rates and extents decreased with increasing cross-linking density and hard-segment content. We found that the presence of a hydrolysis stabilizer reduced (T)PU fragmentation while not affecting the conversion of (T)PU carbon into CO2. We propose a biodegradation mechanism for (T)PUs that includes both mother particle shrinkage by surface erosion and fragmentation. The presented results help to understand structure-degradation relationships of (T)PUs and support recycling strategies.

Towards more Sustainable Peptide- based Antibiotics: Stable in Human Blood, Enzymatically Hydrolyzed in Wastewater?

The emergence and spread of antibiotic resistance is a major societal challenge and new antibiotics are needed to successfully fight bacterial infections. Because the release of antibiotics into wastewater and downstream environments is expected to contribute to the problem of antibiotic resistance, it would be beneficial to consider the environmental fate of antibiotics in the development of novel antibiotics. In this article, we discuss the possibility of designing peptide-based antibiotics that are stable during treatment (e.g. in human blood), but rapidly inactivated through hydrolysis by peptidases after their secretion into wastewater. In the first part, we review studies on the biotransformation of peptide-based antibiotics during biological wastewater treatment and on the specificity of dissolved extracellular peptidases derived from wastewater. In the second part, we present first results of our endeavour to identify peptide bonds that are stable in human blood plasma and susceptible to hydrolysis by the industrially produced peptidase Subtilisin A.

Exploring the Specificity of Extracellular Wastewater Peptidases to Improve the Design of Sustainable Peptide-Based Antibiotics.

New antimicrobial peptides are emerging as promising alternatives to conventional antibiotics because of their specificity for target pathogens and their potential to be rapidly hydrolyzed (i.e., inactivated) by extracellular peptidases during biological wastewater treatment, thereby limiting the emergence and propagation of antibiotic resistance in the environment. However, little is known about the specificity of extracellular peptidases derived from wastewater microbial communities, which is a major impediment for the design of sustainable peptide-based antibiotics that can be hydrolyzed by wastewater peptidases. We used a set of natural peptides to explore the specificity of dissolved extracellular wastewater peptidases. We found that enzyme-catalyzed hydrolysis occurred at specific sites and that a subset of these hydrolyses was conserved across enzyme pools derived from three independent wastewater microbial communities. An analysis of the amino-acid residues flanking the hydrolyzed bonds revealed a set of residue motifs that were linked to enzyme-catalyzed hydrolysis and are therefore candidates for incorporation into new and sustainable peptide-based antibiotics.

Photochemical Transformation of Poly(butylene adipate- co-terephthalate) and Its Effects on Enzymatic Hydrolyzability.

Biodegradable polyesters are being increasingly used to replace conventional, nondegradable polymers in agricultural applications such as plastic film for mulching. For many of these applications, poly(butylene adipate- co-terephthalate) (PBAT) is a promising biodegradable material. However, PBAT is also susceptible to photochemical transformations. To better understand how photochemistry affects the biodegradability of PBAT, we irradiated blown, nonstabilized, transparent PBAT films and studied their enzymatic hydrolysis, which is considered the rate-limiting step in polyester biodegradation. In parallel, we characterized the irradiated PBAT films by dynamic mechanical thermal analysis. The rate of enzymatic PBAT hydrolysis decreased when the density of light-induced cross-links within PBAT exceeded a certain threshold. Mass-spectrometric analysis of the enzymatic hydrolysis products of irradiated PBAT films provided evidence for radical-based cross-linking of two terephthalate units that resulted in the formation of benzophenone-like molecules. In a proof-of-principle experiment, we demonstrated that the addition of photostabilizers to PBAT films mitigated the negative effect of UV irradiation on the enzymatic hydrolyzability of PBAT. This work advances the understanding of light-induced changes on the enzyme-mediated hydrolysis of aliphatic-aromatic polyesters and will therefore have important implications for the development of biodegradable plastics.

Sustainable Polyester Elastomers from Lactones: Synthesis, Properties, and Enzymatic Hydrolyzability.

Chemically cross-linked elastomers are an important class of polymeric materials with excellent temperature and solvent resistance. However, nearly all elastomers are petroleum-derived and persist in the environment or in landfills long after they are discarded; this work strives to address these issues by demonstrating the synthesis of renewable, enzymatically hydrolyzable, and mechanically competitive polyester elastomers. The elastomers described were synthesized using a novel bis(ß-lactone) cross-linker and star-shaped, hydroxyl-terminated poly(γ-methyl-ε-caprolactone). Using model compounds, we determined that the bis(ß-lactone) cross-linker undergoes acyl bond cleavage to afford ß-hydroxyesters at the junctions. The mechanical properties of the cross-linked materials were tunable and competitive with a commodity rubber band. Furthermore, the elastomers demonstrated high thermal stability and a low glass transition (-50 °C), indicating a wide range of use temperatures. The polyester networks were also subjected to enzymatic hydrolysis experiments to investigate the potential for these materials to biodegrade in natural environments. We found that they readily hydrolyzed at neutral pH and environmentally relevant temperatures (2-40 °C); complete hydrolysis was achieved in all cases at temperature-dependent rates. The results presented in this work exemplify the development of high performance yet sustainable alternatives to conventional elastomers.

Uukuniemi Virus as a Tick-Borne Virus Model.

UNLABELLED: In the last decade, novel tick-borne pathogenic phleboviruses in the family Bunyaviridae, all closely related to Uukuniemi virus (UUKV), have emerged on different continents. To reproduce the tick-mammal switch in vitro, we first established a reverse genetics system to rescue UUKV with a genome close to that of the authentic virus isolated from the Ixodes ricinus tick reservoir. The IRE/CTVM19 and IRE/CTVM20 cell lines, both derived from I. ricinus, were susceptible to the virus rescued from plasmid DNAs and supported production of the virus over many weeks, indicating that infection was persistent. The glycoprotein GC was mainly highly mannosylated on tick cell-derived viral progeny. The second envelope viral protein, GN, carried mostly N-glycans not recognized by the classical glycosidases peptide-N-glycosidase F (PNGase F) and endoglycosidase H (Endo H). Treatment with ß-mercaptoethanol did not impact the apparent molecular weight of GN On viruses originating from mammalian BHK-21 cells, GN glycosylations were exclusively sensitive to PNGase F, and the electrophoretic mobility of the protein was substantially slower after the reduction of disulfide bonds. Furthermore, the amount of viral nucleoprotein per focus forming unit differed markedly whether viruses were produced in tick or BHK-21 cells, suggesting a higher infectivity for tick cell-derived viruses. Together, our results indicate that UUKV particles derived from vector tick cells have glycosylation and structural specificities that may influence the initial infection in mammalian hosts. This study also highlights the importance of working with viruses originating from arthropod vector cells in investigations of the cell biology of arbovirus transmission and entry into mammalian hosts. IMPORTANCE: Tick-borne phleboviruses represent a growing threat to humans globally. Although ticks are important vectors of infectious emerging diseases, previous studies have mainly involved virus stocks produced in mammalian cells. This limitation tends to minimize the importance of host alternation in virus transmission to humans and initial infection at the molecular level. With this study, we have developed an in vitro tick cell-based model that allows production of the tick-borne Uukuniemi virus to high titers. Using this system, we found that virions derived from tick cells have specific structural properties and N-glycans that may enhance virus infectivity for mammalian cells. By shedding light on molecular aspects of tick-derived viral particles, our data illustrate the importance of considering the host switch in studying early virus-mammalian receptor/cell interactions. The information gained here lays the basis for future research on not only tick-borne phleboviruses but also all viruses and other pathogens transmitted by ticks.

High-Throughput Analysis of Enzymatic Hydrolysis of Biodegradable Polyesters by Monitoring Cohydrolysis of a Polyester-Embedded Fluorogenic Probe.

Biodegradable polyesters have the potential to replace nondegradable, persistent polymers in numerous applications and thereby alleviate plastic accumulation in the environment. Herein, we present an analytical approach to study enzymatic hydrolysis of polyesters, the key step in their overall biodegradation process. The approach is based on embedding fluorescein dilaurate (FDL), a fluorogenic ester substrate, into the polyester matrix and on monitoring the enzymatic cohydrolysis of FDL to fluorescein during enzymatic hydrolysis of the polyester. We validated the approach against established techniques using FDL-containing poly(butylene adipate) films and Fusarium solani cutinase (FsC). Implemented on a microplate reader platform, the FDL-based approach enabled sensitive and high-throughput analysis of the enzymatic hydrolysis of eight aliphatic polyesters by two fungal esterases (FsC and Rhizopus oryzae lipase) at different temperatures. While hydrolysis rates for both enzymes increased with decreasing differences between the polyester melting temperatures and the experimental temperatures, this trend was more pronounced for the lipase than the cutinase. These trends in rates could be ascribed to a combination of temperature-dependent polyester chain flexibility and accessibility of the enzyme active site. The work highlights the capability of the FDL-based approach to be utilized in both screening and mechanistic studies of enzymatic polyester hydrolysis.

Enzymatic Hydrolysis of Polyester Thin Films at the Nanoscale: Effects of Polyester Structure and Enzyme Active-Site Accessibility.

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.

Enzymatic Hydrolysis of Polyester Thin Films: Real-Time Analysis of Film Mass Changes and Dissipation Dynamics.

Cleavage of ester bonds by extracellular microbial hydrolases is considered a key step during the breakdown of biodegradable polyester materials in natural and engineered systems. Here we present a novel analytical approach for simultaneous detection of changes in the masses and rigidities of polyester thin films during enzymatic hydrolysis using a Quartz Crystal Microbalance with Dissipation monitoring (QCM-D). In experiments with poly(butylene succinate) (PBS) and the lipase of Rhizopus oryzae (RoL), we detected complete hydrolysis of PBS thin films at pH 5 and 40 °C that proceeded through soft and water-rich film intermediates. Increasing the temperature from 20 to 40 °C resulted in a larger increase of the enzymatic hydrolysis rate of PBS than of nonpolymeric dibutyl adipate. This finding was ascribed to elevated accessibility of ester bonds to the catalytic site of RoL due to increasing polyester chain mobility. When the pH of the solution was changed from 5 to 7, initial hydrolysis rates were little affected, while a softer film intermediate that lead to incomplete film hydrolysis was formed. Hydrolysis dynamics of PBS, poly(butylene adipate), poly(lactic acid), and poly(ethylene terephthalate) in assays with RoL showed distinct differences that we attribute to differences in the polyester structure.

Host cell entry of respiratory syncytial virus involves macropinocytosis followed by proteolytic activation of the F protein.

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na⁺/H⁺ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.

Biomimetic Approach to Enhance Enzymatic Hydrolysis of the Synthetic Polyester Poly(1,4-butylene adipate): Fusing Binding Modules to Esterases.

Mimicking a concept of nature for the hydrolysis of biopolymers, the Thermobifida cellulosilytica cutinase 1 (Thc_Cut1) was fused to a polymer binding module (PBM) to enhance the hydrolysis of the polyester poly(1,4-butylene adipate) (PBA). Namely, the binding module of a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (Thc_Cut1_PBM) was attached to the cutinase via two different linker sequences varying in length. In order to investigate the adsorption behavior, catalytically inactive mutants both of Thc_Cut1 and Thc_Cut1_PBM were successfully constructed by site-directed mutagenesis of serine 131 to alanine. Quartz crystal microbalance with dissipation monitoring (QCM-D) analysis revealed that the initial mass increase during enzyme adsorption was larger for the inactive enzymes linked with the PBM as compared to the enzyme without the PBM. The hydrolysis rates of PBA were significantly enhanced when incubated with the active, engineered Thc_Cut1_PBM as compared to the native Thc_Cut1. Thc_Cut1_PBM completely hydrolyzed PBA thin films on QCM-D sensors within approximately 40 min, whereas twice as much time was required for the complete hydrolysis by the native Thc_Cut1.

Biotransformation of antibiotics: Exploring the activity of extracellular and intracellular enzymes derived from wastewater microbial communities.

Evaluating the activity of extracellular and intracellular enzymes derived from wastewater microbial communities is essential to improve our fundamental understanding of micropollutant removal during wastewater treatment. To study biotransformations with respect to enzyme biogeography, we developed a method to separate soluble extracellular, extracellular polymeric substance (EPS)-bound, and intracellular enzymes from wastewater microbial communities and assessed the protease and peptidase activity of the resulting enzyme pools. We also evaluated the biotransformation of six antibiotics (amoxicillin, ampicillin, clindamycin, daptomycin, linezolid, and vancomycin) in each enzyme pool because we expect that the kinetics, pathways, and biogeography of antibiotic biotransformations influence the selection of antibiotic resistance within wastewater microbial communities and in downstream environments. Our results demonstrated that biotransformation rate constants varied among the tested antibiotics, and that the observed rank order was consistent across three wastewater treatment plants. Importantly, many of the observed biotransformations eliminated the functional groups associated with antibiotic activity. Furthermore, we found that ß-lactam hydrolysis and daptomycin hydrolysis were catalyzed by enzymes extracted from the EPS, while none of the tested antibiotics were biotransformed by soluble extracellular enzymes. Finally, our results demonstrated that the number of enzyme-catalyzed antibiotic transformations was larger for intracellular than for extracellular enzymes. Together, this study provides novel insights on the kinetics, pathways, and biogeography of antibiotic biotransformations performed by wastewater microbial communities and can be used to inform pathway prediction or the development of biodegradable chemicals.

Biodegradation of synthetic polymers in soils: Tracking carbon into CO2 and microbial biomass.

Plastic materials are widely used in agricultural applications to achieve food security for the growing world population. The use of biodegradable instead of nonbiodegradable polymers in single-use agricultural applications, including plastic mulching, promises to reduce plastic accumulation in the environment. We present a novel approach that allows tracking of carbon from biodegradable polymers into CO2 and microbial biomass. The approach is based on 13C-labeled polymers and on isotope-specific analytical methods, including nanoscale secondary ion mass spectrometry (NanoSIMS). Our results unequivocally demonstrate the biodegradability of poly(butylene adipate-co-terephthalate) (PBAT), an important polyester used in agriculture, in soil. Carbon from each monomer unit of PBAT was used by soil microorganisms, including filamentous fungi, to gain energy and to form biomass. This work advances both our conceptual understanding of polymer biodegradation and the methodological capabilities to assess this process in natural and engineered environments.

Polyol Structure Influences Enzymatic Hydrolysis of Bio-Based 2,5-Furandicarboxylic Acid (FDCA) Polyesters.

Polyesters of 2,5-furandicarboxylic acid (FDCA) have gained attention as they can be regarded as the bio-based alternatives to the petroleum-based polyesters of terephthalic acid. However, only little is known about the biodegradation and enzymatic hydrolysis of FDCA-based polyesters. This work aims to investigate the influence of different polyols on enzymatic hydrolysis of FDCA-based polyesters. A series of polyesters containing various polyols are synthesized and analyzed regarding susceptibility to enzymatic hydrolysis by cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1). FDCA-based polyesters' number average molecular weight (Mn ) ranged from 9360-35 800 g mol-1 according to gel permeation chromatography (GPC) analysis. Differential scanning calorimetry (DSC) analyses show decreasing glass transition temperature (Tg ) with increasing diol chain length. Crystallinity of all polyesters is below 1% except for polyesters containing 1,6-hexanediol, 1,8-octanediol, and 1,12-dodecanediol for which calculated crystallinities are 27, 37, and 30%, respectively. Thc_Cut1 hydrolyzes all tested polyesters with preference for polyesters containing 1,5-pentanediol and 1,9-nonanediol (57.7 ± 7.5 and 52.8 ± 4.0% released FDCA). Enzyme activity increases when the linear diol 1,3-propanediol is replaced by the branched analog 1,2-propanediol or ethoxy units are introduced into the polyester chain. The results will contribute to expand the knowledge of microbial biodegradation of FDCA-based polyesters.

Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica.

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.