Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to

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Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET Hydrolysis Doris Ribitsch,† Antonio Orcal Yebra,† Sabine Zitzenbacher,† Jing Wu,$,& Susanne Nowitsch,† Georg Steinkellner,† Katrin Greimel,† Ales Doliska,‡ Gustav Oberdorfer,†,§ Christian C. Gruber,† Karl Gruber,†,∥ Helmut Schwab,†,⊥ Karin Stana-Kleinschek,‡ Enrique Herrero Acero,*,† and Georg M. Guebitz†,# †

Enzymes and Polymers, Austrian Centre of Industrial Biotechnology ACIB, Petergasse 14, 8010, Graz, Austria Institute for Characterisation and Processing of Polymers, University of Maribor, Smetanova ulica 17, 2000, Maribor, Slovenia § Department of Biochemistry, University of Washington, 3946 West Stevens, Seattle, United States ∥ Institute of Molecular Biosciencies, University of Graz, Humboldtstrasse 50/3, 8010, Graz, Austria ⊥ Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010, Graz, Austria # Institute of Environmental Biotechnology, University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Strasse 20, 3430 Tulln, Austria $ State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China & School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Ave., Wuxi, Jiangsu 214122, China ‡

ABSTRACT: A cutinase from Thermomyces cellullosylitica (Thc_Cut1), hydrolyzing the synthetic polymer polyethylene terephthalate (PET), was fused with two different binding modules to improve sorption and thereby hydrolysis. The binding modules were from cellobiohydrolase I from Hypocrea jecorina (CBM) and from a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis (PBM). Although both binding modules have a hydrophobic nature, it was possible to express the proteins in E. coli. Both fusion enzymes and the native one had comparable kcat values in the range of 311 to 342 s−1 on pNP-butyrate, while the catalytic efficiencies kcat/Km decreased from 0.41 s−1/ μM (native enzyme) to 0.21 and 0.33 s−1/μM for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively. The fusion enzymes were active both on the insoluble PET model substrate bis(benzoyloxyethyl) terephthalate (3PET) and on PET although the hydrolysis pattern was differed when compared to Thc_Cut1. Enhanced adsorption of the fusion enzymes was visible by chemiluminescence after incubation with a 6xHisTag specific horseradish peroxidase (HRP) labeled probe. Increased adsorption to PET by the fusion enzymes was confirmed with Quarz Crystal Microbalance (QCM-D) analysis and indeed resulted in enhanced hydrolysis activity (3.8× for Thc_Cut1+CBM) on PET, as quantified, based on released mono/oligomers.

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INTRODUCTION In recent years, new enzymes have been recognized as powerful tools for hydrolysis of polyethylene terephthalate (PET1−6). Limited enzymatic hydrolysis of the superficial polyester layers, can avoid redeposition of stains onto PET fabrics when cutinases are formulated in detergents;7 in textile processing the dying efficiency can be increased8 and also can increase the bonding of PET to PVC,9 resulting in considerable savings of conventionally used adhesives. On the other hand, complete enzymatic hydrolysis of PET could open up new opportunities in recycling.10,11 However, PET is a non-natural substrate for enzymes and hence biotransformation is rather slow. Consequently, others and we have demonstrated that genetic engineering has a potential to improve enzymes regarding PET © XXXX American Chemical Society

hydrolysis. For example, by enlarging the area around the active site of cutinases from Fusarium solani and Thermobifida fusca, it was possible to increase the hydrolysis rate of PET and oligomers.12,13 Interestingly, apart from the active site area, surface properties of cutinases seem to have a major impact on PET hydrolysis as we have recently shown for two closely related enzymes from Thermobifida fusca.1 Obviously, this should be due to different sorption properties. Received: January 29, 2013 Revised: March 29, 2013

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dx.doi.org/10.1021/bm400140u | Biomacromolecules XXXX, XXX, XXX−XXX

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module (Thr288-Leu323 of CBH I) of 1,4-β-cellobiohydrolase I from Trichoderma reesei (GenBank P62694.135) to the Thc_Cut1 from T. cellulosilytica (GenBank ADV92526.11). Thc_Cut1-PBM was constructed by fusion of the substrate binding molecule (Ala428-Pro488 of PHB) of polyhydroxybutyrate (PHB) depolymerase from Alcaligenes faecalis (PhaZAfa, GenBank AAA21974.120) over the linker region of 1,4beta-cellobiohydrolase I (Pro263-Pro287) from Trichoderma reesei to Thc_Cut1. Both fusion-enzymes carried a C-terminal 6xHisTag for rapid purification by affinity chromatography. Expression and Purification. Enzymes were expressed in E. coli BL21-Gold(DE3) at 20 °C and purified as described by Herrero Acero et al.1 Esterase Activity. Esterase activity was measured using paranitrophenylbutyrate (PNPB) as substrate.36 The final assay concentration was 7.9 mM PNPB. Activity was quantified by following the absorbance increase at 405 nm with a Tecan plate reader. Hydrolysis of the Model Substrate Bis(benzoyloxyethyl) Terephthalate (3PET) and PET Film. 3PET (10 mg) and PET films (1 × 2 cm) were incubated in 2 mL eppendorf tubes in a thermomixer at 50 °C and 100 rpm with 25 mM enzyme in 100 mM K2HPO4/KH2PO4 buffer at pH 7. After time intervals, as indicated below, samples were diluted 1:1 with methanol on ice. Analysis of the released products benzoic acid (BA), terephthalic acid (TA), mono(2hydroxyethyl) terephthalate (MHET), and hydroxyethylbenzoate (HEB) was performed via RP-HPLC, as previously described.1 Dynamic Enzyme Adsorption on PET Films. Enzyme adsorption on PET films was monitored with a quartz crystal microbalance (QCMD) with dissipation from Q-Sense AB, Gothenburg, Sweden. It is based on the change in resonance frequency of a thin AT-cut piezoelectric quartz crystal disc. The quartz crystals (Q-Sense) used had gold plate electrodes and were sputtered with silica on the active surface. The crystal frequency change is in correlation to the adsorbed mass with a Sauerbrey eq 1, which is only valid for rigid, evenly distributed, thin adsorbed layers.

In nature, controlled adsorption and desorption of enzymes during hydrolysis of polymers is achieved by so-called substrate binding modules linked via spacers to the catalytic domain.14 Carbohydrate binding modules of (hemi)cellulases are known for more than a century, but have also discovered for other enzymes such as chitinases.15−18 More recently, substrate binding modules have also been described for enzymes hydrolyzing natural polyesters.19,20 For example, a depolymerase from Pseudomonas stutzeri was only able to hydrolyze waterinsoluble poly[(R)-3-hydroxybutyrate] (P3HB) when carrying a substrate binding module in contrast to mutants without binding domain.21 There are two mechanisms how binding domains enhance hydrolysis of polymeric material,22 on the one hand enzymes with a binding module dramatically increase the amount of active enzyme on the polymer interface.23 On the other hand binding modules can also facilitate the hydrolysis of insoluble substrates by partially disrupting the structure of the polymer and therefore making the targeted bonds more accessible to the catalytic domain.24−26 Genetic engineering was widely used to exchange, modify or add binding modules to (hemi)cellulases improving hydrolysis of lignocellulose.27,28 Particularly interesting is the approach by Zhang et al.29 who fused two different carbohydratebinding modules (CBM) to a cutinase from Thermobifida fusca result in enhanced binding to cotton and consequently improved hydrolysis of waxes contained therein (“ bio-scouring”). Here we have investigated the effect of two different binding domains on polyester (PET) hydrolysis when fused to a cutinase from Thermobifida cellulosilytica (Thc_Cut1). The polyhydroxyalkanoate (PHA) binding module (Thc_Cut1+PBM) from Alcaligenes faecalis was chosen due to similarity of the natural polyester PHA to PET in terms of hydrophobicity. The CBM from Cellobiohydrolase I from Trichoderma reesei was selected as a binding module designed by nature for a more hydrophilic polysaccharide. Although their natural substrates have a complete different chemical structure it is known that both share a common interaction mechanism involving hydrophobic interactions via tryptophan residues30,31 in the case of CBM. In the case of the PBM, the mechanism can also include specific hydrophobic amino acid interactions, which are known to interact with cellulose and PHA via hydrophobic interactions32

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Δm = −

C·Δf n

(1) −1

−2

where C is the mass sensitivity constant (17.7 ng Hz cm for a 5 MHz quartz crystal), n is the overtone number (1, 3, 5, 7, 9, 11, 13), Δm is the change in mass, and Δf is the frequency change. Dissipation occurs when periodically switching the AC voltage on and off over the crystal and the energy from the oscillating crystal dissipates from the system. Dissipation is proportional to film elasticity and viscosity. Dissipation is defined as D=

MATERIALS AND METHODS

E lost 2πEstored

(2)

where Elost is the energy lost during one oscillating cycle, and Estored is the total energy stored in the oscillator.

Chemicals and Reagents. The model substrate bis(benzoyloxyethyl)terephthalate (3PET) was synthesized according to the method described by Heumann et al.33 and purity was confirmed via NMR and HPLC-MS. All the other chemicals were of analytical grade and purchased from Sigma-Aldrich. Polyethylene terephthalate (PET) films were completely amorphous and purchased from Goodfellow (U.K.). General Recombinant DNA Techniques. All DNA manipulations described in this work were performed by standard methods (Sambrook et al.34). Digestion of DNA with restriction endonucleases (New England Biolabs, U.S.A.), dephosphorylation with alkaline phosphatase (Roche, Germany), and ligation with T4 DNA-ligase (Fermentas, Germany) were performed in accordance to the manufacturer’s instructions. Plasmid Mini Kit from Qiagen (Germany) was used to prepare plasmid DNA. Plasmids and DNA fragments were purified by Qiagen DNA purification kits (Qiagen, Germany). Vector pET26b(+) (Novagen) was used for expression of fusion proteins in E. coli BL21Gold(DE3) (Stratagene). Fusion of Binding Modules to Thc_Cut1. The genes coding for Thc_Cut1-CBM and Thc_Cut1-PBM were synthesized by Mr.Gene GmbH (Germany). Thc_Cut1-CBM was constructed by fusion of the linker region (Pro263-Pro287 of CBH I) and carbohydrate-binding

Δti

E lost C·Δt D= n 2πEstored

(3)

In our particular case, crystals were spin-coated with a 20% solution of PET in 1,1,2,2-tetrachloroethane (Fluka, 86960), as described by Indest et al.37 The solution was spin-coated at 2000 rpm for 60 with a SCC spin coater from LOT-Oriel, Germany. Solvent was evaporated overnight at 30 °C. Initially, 100 mM K2HPO4/KH2PO4 buffer, pH 7, was pumped into the system at a flow of 0.1 mL/min until a stable baseline was reached regarding frequency and dissipation. Thereafter, the buffer was substituted by the corresponding enzyme solution (2.5 mM) for a further 3 min. The flow was then stopped in order to allow equilibration. After 30 min, again buffer was flushed through the system in order to study the desorption dynamics. Enzyme Adsorption on PET. Enzyme adsorption on PET film was monitored by specific binding of horseradish peroxidase (HRP) labeled probe to the 6xHisTag of the enzymes using HisProbe-HRP and SuperSignal West HisProbe Kit (Thermo Scientific). In a 2 mL Eppendorf tube, PET film (0.3 × 0.7 mm) was incubated with 600 μL of B


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Figure 1. Fusion of binding molecules to Thc_Cut1 (blue). Carbohydrate-binding module of 1,4-beta-cellobiohydrolase I from Hypocrea jecorina (CBM; green). Substrate binding domain of polyhydroxybutyrate depolymerase from Alcaligenes faecalis (PBM; red). Linker region of 1,4-betacellobiohydrolase I from T. reesei (yellow). enzyme solution (20 μM) for 2 h at 30 °C. The PET film was washed twice by dipping in TBS buffer at room temperature, in order to remove nonadsorbed enzyme. After incubation with HisProbe-HRP conjugate (1:2500 dilution in TBS buffer) at room temperature and shaking for 40 min, the film was washed again (3× TBS buffer, room temperature). The PET film was incubated with 600 μL of SuperSignal West Pico Substrate Working Solution for 5 min. Chemiluminscence was detected by the G:BOX Chemi from Syngene, and quantified with a Colorlite sph 850 (Colorlite:Inovative color measurements; GERMANY).38 Molecular Modeling. To build the fusion model of Thc_Cut+PBM a model of Thc_Cut1 from Thermofibifida cellulosilytica DSM44535 was used which was generated in a previous work1 applying the Streptomyces exfoliatus lipase (PDB Code: 1JFR)39 as a template. As a suitable template for the PBM part is not available a template free modeling of PBM was performed using the molecular modeling suite Rosetta40 following a previously described ab initio structure prediction protocol.41 A total of 2000 structures were calculated and clustered according to their root-mean-square-deviations (rmsd), with an rmsd cutoff of 3 Å. Over all structural clusters, a stringent energy cutoff was used to select the ten lowest energy structures. These structures were visually inspected using PyMOL.42,42,43 The final model was chosen by chemical intuition, and fused with the linker to the model of Thc_Cut1 relaxation of the fusion model was carried out by molecular dynamics simulation of 3 ns employing GROMACS 4.5.542,42 and the OPLS-aa force field in explicit solvent (TIP3P) with standard parameters.

Figure 2. SDS-PAGE analysis (4−12%) of fusion proteins expressed in E. coli BL21-Gold(DE3) and purified by 6xHisTag. Lane 1: soluble cell fraction of Thc_Cut1+PBM; lane 2: purified Thc_Cut1+PBM; lane 3: insoluble cell fraction of Thc_Cut1+PBM; lane 4: soluble cell fraction of Thc_Cut1+CBM; lane 5: purified Thc_Cut1+CBM; lane 6: insoluble cell fraction of Thc_Cut1+CBM; lane 7: standard Novex Sharp Protein Standard (Invitrogen).

substrate were determined for the fusion proteins and compared to the cutinase without binding module (Table 1). The kcat values Table 1. Kinetic Parameters of Cutinase from T. cellulosilytica Fused to Carbohydrate-Binding Module of 1,4-βCellobiohydrolase I from H. jecorina (CBM) and the Substrate Binding Domain of Polyhydroxybutyrate Depolymerase from A. faecalis (PBM) on p-Nitrophenyl Butyrate

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RESULTS Cloning, Expression, and Purification of Fusion Proteins. Two binding modules, differing in their affinity toward hydrophilic or hydrophobic substrates, were fused to Thc_Cut1 for modulating the adsorption of the cutinase onto polyester (Figure 1). First, the noncatalytic carbohydratebinding module (CBM, 3.7 kDa) of the multidomain fungal 1,4-β-cellobiohydrolase I (CBH I) from Hypocrea jecorina (formerly Trichoderma reesei44) was fused to the C-terminus of Thc_Cut1 (Thc_Cut1+CBM). Like in CBH I, the CBM was fused over a linker region (2.3 kDa) to the catalytic domain. The CBH I- linker region was also applied for fusion of the substrate-binding domain (PBM, 6.3 kDa) of the polyhydroxybutyrate depolymerase from A. faecalis (PhaZAfa) to Thc_Cut1 (Thc_Cut1+PBM, Figure 1). The synthetic genes were cloned into pET26b(+) lacking the pelB leader peptide. For rapid purification, both proteins were tagged with (His)6 at the C-terminus. Expression of fusion enzymes was performed in E. coli BL21-Gold(DE3) at 20 °C and 0.05 mM IPTG to minimize formation of inclusion bodies. As shown in Figure 2, Thc_Cut1+PBM was expressed in higher yields in its soluble form than Thc_Cut1+CBM. Both fusion proteins were purified by the C-terminal (His)6 affinity Tag and displayed protein bands which corresponded well to the calculated molecular weights of 38 kDa (Thc_Cut1+PBM) and 35 kDa (Thc_Cut1+CBM). Kinetic Characterization on Soluble Substrates. The kinetic parameters with p-nitrophenyl butyrate PNPB as

Thc_Cut1 Thc_Cut1+PBM Thc_Cut1+CBM

Km (μM)

kcat (s−1)

kcat/Km (s−1/ μM)

800 ± 20 1500 ± 90 1050 ± 110

325 311 342

0.41 0.21 0.33

for the three enzymes were in same range, while the Km values of the fusing proteins were clearly higher with Thc_Cut1+PBM showing a Km value twice as high when compared to Thc_Cut1. Hydrolysis of the Model Substrate 3PET and PET Films. In a next step, the cutinases fused to binding modules were compared to the native enzymes in terms of hydrolysis of the water insoluble PET model oligomer 3PET and of PET. In both cases a time study was performed in order to show the evolution of the released products benzoic acid (BA), terephthalic acid (TA), mono(2-hydroxyethyl) terephthalate (MHET), and hydroxyethylbenzoate (HEB; Figure 3). Both fused enzymes showed a lower activity with the insoluble PET. For the new designed proteins, the distribution of the released products differed from the native enzyme. Particularly the fused proteins seemed to have difficulties in hydrolyzing the soluble released esters HEB and MHET as shown by the higher levels of such compounds compared with Thc_Cut1. Time profile of the PET released products were obtained for the native enzyme and the fused ones (Figure 4). All the three C


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Figure 3. Time profile of soluble hydrolysis products of 3PET by Thc_Cut1, Thc_Cut1+CBM, and Thc_Cut1+PBM measured at 50 °C and pH 7: TA, terephthalic acid; MHET, mono(2-hydroxyethyl) terephthalate; BA, benzoic acid; HEB, hydroxyethylbenzoate.

Figure 4. Time profile of soluble hydrolysis products of PET by Thc_Cut1, Thc_Cut1+CBM, Thc_Cut1+PBM measured at 50 °C and pH 7: TA, terephthalic acid; MHET, mono(2-hydroxyethyl) terephthalate.

Figure 5. QCM analysis of adsorption of the native cutinase (Thc-Cut1) and fusion proteins with the substrate-binding module (PBM) and the carbohydrate-binding module (CBM) onto PET films. Frequency changes after adsorption of Thc_Cut1, Thc_Cut1+CBM, and Thc_Cut1+PBM onto PET films. Right picture, Thc_Cut1+PBM model.

enzymes released enzymes released TA and MHET, while no bishydroxyethyl terephtalate (BHET) was detected. In this case both fused enzymes showed higher PET hydrolysis rates.

However, the ratio between the hydrolysis products and the total amount was clearly different for Thc_Cut1 when compared to both Thc_Cut1+CBM and Thc_Cut1+PBM. In the case of D


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concentration of CBH I in the vicinity of the natural polar substrate cellulose. The domain is highly stable, with two internal disulfide bonds stabilizing the secondary structures of the overall fold.47 The CBM is fused over a linker region to the CBH I. This 27 residue linker peptide is described to be heavily Oglycosylated with mannose residues at the serines (total of 2) and threonines (total of 7) in H. jecorina.44 The substrate-binding domain of the PHA depolymerase from A. faecalis (PBM) is connected over a fibronectin type 3 domain to the catalytic domain and has an affinity not only to various apolar polyesters (polyhydroxyalkanoates, PHAs) but also for polystyrene.48 Since the function of the fibronectin type 3 domain is not fully understood we decided to use again the linker region of CBH I to fused the PBM to the cutinase. Quite expectedly, on small soluble substrates (i.e., PNPB) the catalytic efficiency (kcat/Km) of the enzymes carrying binding modules with 0.21 s−1/ μM for Thc_Cut1+PBM and 0.33 s−1/ μM for Thc_Cut1+CBD were somewhat lower than measured for the native enzyme (0.41 s−1/μM). Nevertheless, the kcat/Km values are in a range with those previously reported for Thc_Cut11 and for two cutinases fused with binding modules from C. fimi and cellulase Cel6A.49 In the latter study, a maximum decrease in the catalytic efficiency of 15% was observed for the fusion enzymes. The use of a different and longer linker between enzyme and binding modules could decrease potential sterical hindrance in diffusion of the substrate and reaction products to/from the active site. In agreement with these results higher activities toward soluble substrates were measured when binding domains were deleted from Trichoderma reesei β-mannanase.50 In contrast, binding modules of chitinases from Bacillus circulans WL-12 and Streptomyces olivaceoviridis did not have an effect on their activity on small soluble substrates as demonstrated with deletion of the binding domains.51,52 The hydrolysis of 3PET, a water insoluble oligomeric model substrate for PET, revealed interesting mechanistic information based on a different product pattern seen with the fusion enzymes and the native cutinase. Thc_Cut1+CBM released lower total amounts (−21%) of hydrolysis products when compared with the native enzyme and with Thc_Cut1+PBM (−8%). Comparing the pattern of the released products a clear difference was seen for the three enzymes. The native enzyme releases initially comparable amounts of all possible hydrolysis products whereas at longer incubation time the amounts of BA and TA increased most likely because exotype hydrolysis of HEB and MHET. For fusion enzymes the initial trend was similar with all the released products present in comparable quantities whereas the final distribution after longer incubation times was different. The ratios HEB/BA and MHET/TA were significantly higher after hydrolysis of 3PET with the fused proteins compared with the native cutinase. Previously, different specificities of an esterase from Bacillus subtilis,36 a cutinase from Humicola insolens,53 different Thermobifida sp. cutinases1,53 and lipases from T. lanuginosus54were seen on this same model substrate. Bacillus subtilis esterase and bacterial cutinases1,53 released mainly BA and TA while MHET was detected in significant lower amounts and no BHET has been reported in any of them. An opposite hydrolysis pattern was found for the cutinases from T. fusca and F. solani, where BHET appeared as an intermediate.54 Interestingly, Bacillus36 esterase and Humicola insolens53 cutinase released preferentially BA in a exowise mechanism. Contrary to the Bacillus36 esterase being unable to

the native enzyme, TA was the predominant hydrolysis product, while the enzymes containing binding domains released considerable amounts of MHET. Clearly, attachment of the CBM to cutinase had a positive effect on hydrolysis as the total amount of hydrolysis products at all the time points measured. After 72 h, Thc_Cut1+CBM released a total of 1.7 mol/mol of enzyme compared with 1.2 mol/mol released in the case of Thc_Cut1. This effect was even more pronounced when the PBM was fused to Thc_Cut1, resulting in an overall amount of hydrolysis products of 4.5 mol/mol enzyme. Adsorption Profiles via QCM. In QCM analysis, adsorption of the enzymes onto the PET-coated quartz crystals caused a frequency decrease with varying extents. The adsorption of Thc_Cut1 decreased frequency for around 18 Hz compared to rinsing with buffer. Thc_Cut1+CBM and Thc_Cut1+PBM adsorbed in a higher amount as indicated by frequency changes for more than 35 Hz for Thc_Cut1+PBM and for 25 Hz for Thc_Cu1+CBM (Figure 5). Because dissipation changes were close to zero and did not change more than 1 × 10−6/10 Hz, the Sauerbrey equation was used to calculate the adsorbed amount of proteins to PET surface. The calculated adsorbed mass of Thc_Cut1 was around 330 ng/cm−2, around 500 ng/cm2 of Thc_Cut1+PBM were adsorbed and Thc_Cut1+PBM adsorbed to the highest extent (600 ng/cm2). The adsorption for three enzymes was initially very fast, within a few seconds, followed by a slower increase. During the equilibrium phase no significant changes were detected. Upon rinsing with buffer an increase in frequency, implying mass desorption, was measured. It includes both hydrolyzed PET molecules and desorbing enzyme, and therefore, the interpretation of this part of the graph is not possible. Detection of Adsorbed Protein. To visualize adsorption of the various cutinases on PET, the fusion proteins were incubated with PET-film. After various washing steps, the adsorbed protein was detected by chemiluminescence using a nickel (Ni2+)activated derivative of horseradish peroxidase (HRP) that enables direct, IMAC (immobilized metal affinity chromatography)-based detection of His-tagged proteins. As shown in Table 2, the strongest signal was obtained from Thc_Cut1+PBM, while both Thc_Cut1+CBM and Thc_Cut1 showed rather weak signals. Table 2. Chemilumiscence Differences after Reaction of the Adsorbed Enzyme with Nickel (Ni 2+)-Activated Derivative of Horsereadish Peroxidase Thc_Cut1

Thc_Cut1+CBM

Thc_Cut11+PBM

16.6

19.7

38.0

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DISCUSSION For efficient biotransformation of water insoluble polymeric substrates, nature has equipped enzymes with substrate-binding modules which are well studied for enzymes converting carbohydrates45 and to a lesser extend for poly(3-hydroxyalkanoate) depolymerases.46 Here, we have taken advantage of this concept by designing enzymes for the hydrolysis of synthetic polyesters. Therefore, a cutinase known for PET hydrolysis (Thc_Cut1)1 was fused to a binding module (Thc_Cut1+PBM) of a polyhydroxyalkanoate depolymerase from Alcaligenes faecalis and to a CBM from cellobiohydrolase I from Trichoderma reesei (Thc_Cut1+CBM). The CBM, which belongs to family 1 of carbohydrate-binding modules, is predicted to increase the local E


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hydrolase MHET, high amounts of it were detected for T. halotolerans55 and Humicola insolens.53 In a next step, hydrolysis of PET was studied. Indeed, attachment of binding modules had a positive effect on enzymatic hydrolysis of PET. Thc_Cut1+PBM released significantly higher amounts of both terephthalic acid (TA) and mono-(2-hydroxyethyl) terephthalate (MHET) from PET than Thc_Cut1. Specifically, after 72 h of incubation, Thc_Cut1+PBM yielded 2.9 mols of TA and 1.6 mols of MHET per mole of enzyme, while the native enzyme only released 1 mol of TA and 0.14 mols of MHET, per mole of enzyme. Interestingly, the cutinase fused to the carbohydratebinding module (Thc_Cut1+CBM) released similar amounts of TA like Thc_Cut1+PBM while the amount of MHET released was only 0.74 mols per mole enzyme, but still almost four times more than Thc_Cut1. In line with many known examples in nature were binding domains that enhance the hydrolysis of natural insoluble substrates.56−59 For both fusion enzymes, the ratio of TA/MHET decreased after prolonged incubation. This indicates that some of the resulting MHET may be hydrolyzed, however, to a much lower extend than seen for the native cutinase. The same trend was seen during hydrolysis of the model substrate, 3PET, were for both fusion enzymes the ratios of released TA/MHET and BA/HEB were lower than for Thc_Cut1. This nicely demonstrates the value of the 3PET model substrate to predict quite accurately the performance of enzymes regarding PET hydrolysis. The effect ultimately expected for industrial applications from limited enzymatic surface hydrolysis of PET is an increase in hydrophilicity. This has been quantified with methods like water contact angle assessment (WCA), XPS analysis60 or derivatization of hydroxyl groups,4 which, however all can suffer from some inaccuracy if complete enzyme removal from the polymer surface is not achieved.3,23 To investigate the effect of the binding modules attached to cutinases on PET hydrolysis more in detail, we carried out dynamic adsorption experiments of the fusion enzymes at the same concentrations as the hydrolysis experiments that were done on amorphous PET. Recognition of water insoluble polymers by binding domains has been done previously,15,17,26 while Quartz Crystal Microbalance (QCM) is a powerful technique to study adsorption and hydrolysis phenomena in a dynamic approach.61−63 The amounts of protein adsorbed to PET were 730 ng/cm2 (18.95 μmol/cm 2 ) and 550 ng/cm 2 (15.16 μmol/cm 2 ) for Thc_Cut1+PBM and Thc_Cut1+CBM, respectively, while for the native enzyme only, 440 ng/cm2 (14.27 μmol/cm2) was calculated. The amounts of enzyme adsorbed were comparable with the review by Nakanishi64 in which, depending on the substrate and the protein values, between 630 and 60 ng/cm2 were described. In addition to QCM analysis, the amount of enzyme adsorbed on PET films was determined based on specific binding of the His-tags to a peroxidase-based probe. When incubated at the same molar concentration (20 μM) with the PET film, the same trend was seen like with QCM analysis. The chemiluminescence measured according to the CIE Lab concept and quantified as ΔE was highest for Thc_Cut1+PBM, 38.0 followed by Thc_Cut1+CBM, 19.7 and for Thc_Cut1 16.6, which confirm the strongest adsorption of Thc_Cut1+PBM. Adsorption measurements have shown a good correlation with enzyme activity on PET, being Thc_Cut1+PBM the enzyme adsorbing in a highest extent and releasing 3.75 fold more hydrolysis products than the native enzyme. Thc_Cut1+CBM

showed an intermediate behavior between Thc_Cut1 and Thc_Cut1+PBM both in activity and adsorption. It is well-known for a variety of natural polymers of varying complexities15,16,29,61,62,65 that binding modules enhance enzyme adsorption and, consequently, biotransformation. Consequently, this study demonstrates for the synthetic polymer PET that likewise higher amounts of enzymes recruited on the polymer surface by binding modules can enhance hydrolysis. Quite expectedly, this effect was more pronounced when the cutinase was fused to PBM. This is the binding module of enzymes hydrolyzing PHA, a microbial polyester that is rather hydrophobic like PET.66 In contrast, CBMs are designed by nature for hydrophilic cellulose and were thus less effective in enhancing binding of the cutinase to polyester. Nevertheless, attachment of CBMs to cutinases has previously been demonstrated to increase enzyme binding to cellulosic materials.23,29

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CONCLUSIONS We have successfully fused two different binding domains from to a cutinase from Thermobifida cellulosilytica already known to show PET hydrolytic activity. Both fused proteins were cloned and expressed in E. coli. The new enzymes adsorb in a much higher extent to PET films as we proven via QCM-D and enzyme detection by fluorimetric methods. Overall, we observed that the activity of both fused proteins toward PET was increased compared with the native enzyme as shown by the higher amount of released products. On the other hand, the hydrolysis of soluble substrates PNPB, MHET, and HEB was diminished, most likely due to steric reasons.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +43 316 876 9342. Fax: +43 316 876 9343. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol and ZIT - Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.

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