Citation: Yoshiji Hantani, Hiroshi Imamura, Tsubasa Yamamoto, Akane Senga, Yuri Yamagami, Minoru Kato, Fusako Kawai, Masayuki Oda. Functional characterizations of polyethylene terephthalate-degrading cutinase-like enzyme Cut190 mutants using bis(2-hydroxyethyl) terephthalate as the model substrate[J]. AIMS Biophysics, 2018, 5(4): 290-302. doi: 10.3934/biophy.2018.4.290
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Polyethylene terephthalate (PET) has been widely used in our everyday lives, as it possesses great strength, stiffness, ductility, transparency, and lightness. As PET is practically non-degradable, post-consumer PET is a major cause of environmental concern. Chemical recyclings involving glycolysis, hydrolysis, and aminolysis are some of the potential treatments, but they generally require high temperatures and often generate additional environmental pollutants. Enzymatic methods are being investigated, as they are less energy consuming and environmentally friendly; several PET-degrading enzymes have also been reported [1,2]. However, most of the enzymes can only degrade the surface morphology. Significant decay of the polymer structure, especially the inner block leading to significant weight loss, is only detected at temperatures higher than the glass transition temperature of PET, which is between 60-65 °C in aqueous solutions [3,4,5]. Therefore, there is a need to develop enzymes with higher degradation activity and thermal stability, as well as eco-friendly chemical PET-degrading methods for industrial applications.
We have structurally and functionally characterized Cut190, a cutinase-like enzyme from Saccharomonospora viridis AHK190, and also obtained its mutants with higher degradation activity and thermal stability [4,5,6,7,8,9]. A unique feature of Cut190 is that its function and stability are regulated by Ca2+ binding [4,5,6,7,8]. The mutant, Cut190_S226P/R228S (Cut190*), has higher activity and thermal stability than the wild-type of Cut190 [4]. We used Cut190* as the template to obtain mutants with high activity, and successfully obtained the mutant, Cut190*Q138A/D250C-E296C, which exhibited the degradation rate of 26.6% towards amorphous PET (PET-GF) films [5].
The functional analysis of PET-degrading enzymes is generally performed using substrates such as poly(butylene succinate-co-adipate) (PBSA) and p-nitrophenyl butyrate (pNPB), although their structures are different from that of PET. Bis(2-hydroxyethyl) terephthalate (BHET) is produced as an intermediate of enzymatic degradation of PET, and the structure of BHET is similar to that of PET compared to PBSA and pNPB (
In this study, we analyzed the effects of Ca2+, substrate, and enzyme concentrations on the enzymatic activity of Cut190* on BHET under various conditions including pH and temperature to find the optimum condition, and compared them with the results for PBSA and pNPB reported previously [4]. We also analyzed the effect of pressure on the enzyme activity. Enzyme activity enhancement by high pressure has been reported [10,11,12,13]. Quartinello et al. recently reported that pressure of 4 MPa along with a high temperature of 250 °C enhanced PET degradation non-enzymatically [14]. We therefore expected synergistic effects of enzyme activity under high pressure and analyzed the effects of pressure on BHET hydrolysis by enzymes, Cut190* and Cut190*Q138A/D250C-E296C, whose degradation rates towards amorphous PET films were quantitatively evaluated [4,5]. We further analyzed the structural stability of Cut190* under high pressure using Fourier transform infrared (FTIR) measurements.
TPA and BHET were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively. MHET was synthesized, as described previously [5,15]. Acetonitrile (HPLC grade) was purchased from Kanto Chemical Co., INC. (Tokyo, Japan). The enzymes, Cut190* and Cut190*Q138A/D250C-E296C, were overexpressed in Escherichia coli, and purified as described previously [5].
For enzyme activity assays, the model substrate of PET, BHET, was used. Because of low solubility, BHET was dissolved in dimethyl sulfoxide (DMSO). The assays were carried out in 50 mM Tris-HCl (pH 7.5) containing 1% (v/v) DMSO, 5 mM CaCl2, and 1 mM BHET, which was slightly modified for the purpose of respective experiments. Reactions were initiated by addition of the enzyme (final concentration, 1 µM). Assays except high-pressure experiments were performed in a final volume of 75 µL, and incubated at 37 °C for 2 h. The reaction was stopped by the addition of 75 µL of 1% (v/v) trifluoroacetic acid. Products were analyzed using HPLC as described below.
Products were monitored at a wavelength of 254 nm on an Agilent 1200 series HPLC (Agilent technologies, USA). Sample (5 µL) was loaded onto a reversed phase column RP-C18 (Agilent ZORBAX Eclipse Plus C18 (particle size, 1.8 µm; pore size 95 Å; length, 50 mm; inside diameter, 2.1 mm)) and eluted using a mobile phase (acetonitrile/water/formic acid (15/85/0.1, v/v/v)) at a flow rate of 0.6 mL min−1. The amounts of MHET and TPA produced in the reaction mixture were calculated from respective standard curves.
High-pressure experiments were performed using high-pressure hand pump and 25 mL high-pressure reactor (Syn Corporation Ltd, Kyoto, Japan) under the condition as follows: 1 µM enzyme and 1 mM BHET in 50 mM Tris-HCl (pH 7.5) containing 1% (v/v) DMSO and 5 or 25 mM CaCl2 were loaded into Tygon tube (Tygon®2375, Saint-Gobain Performance Plastics, Tokyo, Japan) and pressurized to keep at 50, 100, 200, 300 and 400 MPa, respectively, at 37 °C for 2.5 h. After 2.5 h, reactors were decompressed. Non-pressure treated samples were also examined as a controlled experiment. Trifluoroacetic acid (1% (v/v), 50 µL) was added to 50 µL of the reaction mixtures to stop the enzyme reaction, and the product was analyzed using HPLC as described above.
FTIR measurement was used to monitor secondary structures of proteins under high pressure [16,17]. The solution of 0.29 mM Cut190* in 50 mM Tris-DCl with pD of 8.5 (adding 0.4 to the pH meter reading) was loaded into a diamond anvil cell with a gasket with a thickness of 100 µm. Barium sulfate was added to the sample as an internal pressure calibrant [18]. The cell was connected to a circulating water bath to control the sample temperature at 23.6 ± 0.1 °C. IR spectra were recorded on a FTIR spectrometer (Jasco FT/IR-6600) equipped with a MCT liquid nitrogen-cooled detector. Prior to measurement, pressure was applied to the sample at the average rate of ∼0.2 GPa h−1 (up to 1.2 GPa) for ∼6 h to complete hydrogen-deuterium exchange of the amide protons, which was confirmed by monitoring the intensity of the amide II band. To obtain a spectrum with a resolution of 2 cm−1, 512 interferograms were collected. Spectral analyses were performed using GRAMS Research for System2000 FTIR version 3.01B (Galactic Software) and IGOR Pro version 6.22A (WaveMetrics, Portland, OR).
Enzymatic hydrolysis of BHET was evaluated monitoring hydrolysis products by reversed-phase HPLC. TPA and MHET were identified based on references molecules (
Due to the low solubility of BHET, DMSO was used as the solvent. Hence, the effects of DMSO on BHET hydrolysis by Cut190* were analyzed (
Effects of Ca2+, substrate, and enzyme concentrations on Cut190* activity towards BHET were investigated. The enzyme activity was observed even in the absence of Ca2+ and increased in a Ca2+ concentration-dependent manner up to 25 mM (Figure 1A). There was little difference in the amount of product when CaCl2 concentration was between 25 and 125 mM. The concentration of Ca2+ used in the following experiments was 5 mM or 25 mM, which was used in the previous studies on activities of Cut190 and its mutants towards PBSA or pNPB [4]. These concentrations were selected so that we could compare our results with the Cut190* activity towards PBSA and pNPB reported previously [4].
Figure 1. Effect of Ca2+ (A), substrate (B), and enzyme (C) concentrations on Cut190* activity towards BHET. Black and grey bars were MHET amounts in the reaction mixtures with and without enzyme, respectively. Grey circles were MHET amounts subtracted without enzyme from with enzyme to indicate net enzyme activities. Data are shown with the standard deviation of triplicate measurements.Product amount increased as the substrate concentration increased (Figure 1B). Because substrate concentration above 4 mM could not be used due to low solubility, we could not detect the maximum velocity (Vmax). Therefore, we could not determine the Michaelis constant (Km) of BHET. Substrate concentration above 4 mM could not be used due to low solubility. The amount of product increased until the enzyme concentration reached 2 µM, but slightly decreased at 4 µM compared to 2 µM of the enzyme (Figure 1C).
Effects of temperature and pH on BHET hydrolysis by Cut190* were investigated. The enzyme activity increased as the temperature increased up to 60 °C (Figure 2A). The enzyme activity also slightly increased with increase in pH up to 8.5 (Figure 2B). Under the condition of 1 mM BHET in 1% DMSO, 50 mM Tris-HCl (pH 7.5), 5 or 25 mM CaCl2 at 37 °C, the time-course experiments showed that the progress curve was linear until 8 or 4 h, respectively (Figures 3A,B and
Figure 2. Effect of temperature (A) and pH (B) on Cut190* activity towards BHET. Black and grey bars were MHET amounts in the reaction mixtures with and without enzyme, respectively. Grey circles were MHET amounts subtracted without enzyme from with enzyme to indicate net enzyme activities. Data are shown with the standard deviation of triplicate measurements.
Figure 3. Time course of BHET hydrolysis by Cut190* (A, B) and Cut190*Q138A/D250C-E296C (C, D) in the presence of 5 mM Ca2+. Black squares and grey diamonds in A and C were MHET amounts in the reaction mixtures with and without enzyme, respectively. Grey circles in B and D were MHET amounts subtracted without enzyme from with enzyme to indicate net enzyme activities. Data are shown with the standard deviation of triplicate measurements.Activities of Cut190* and Cut190*Q138A/D250C-E296C in the presence of 5 or 25 mM CaCl2 were measured under different pressures (up to 400 MPa). The results showed that the amounts of product depended on pressure, and the highest values were obtained at 400 MPa (Figure 4A). Hydrolysis of BHET was observed not only in the presence of enzyme, but also in the absence of enzyme. In order to compare the net enzyme activities, the MHET amount without the enzyme was subtracted from that obtained with enzyme. The results revealed that increase in enzyme activity due to pressurization was not observed. The enzyme activity in the presence of 5 mM Ca2+ was kept under high pressure of 400 MPa (Figure 4A,C). On the other hand, the enzyme activity at 25 mM Ca2+ tended to decrease under high pressure (Figure 4B,D).
Figure 4. Effect of pressure on Cut190* activity towards BHET (A, B) and Cut190*Q138A/D250C-E296C (C, D) in the presence of 5 mM (A, C) or 25 mM Ca2+ (B, D). Black and grey bars were MHET amounts in the reaction mixtures with and without enzyme, respectively. Grey circles were MHET amounts subtracted without enzyme from with enzyme to indicate net enzyme activities. All data are shown with the standard deviation of triplicate measurements.Effects of pressure on the secondary structure of Cut190* were analyzed using FTIR experiments. At relatively low pressures, the FTIR absorbance spectra of Cut190* showed a prominent peak at ∼1638 cm−1 with shoulders at ∼1652 cm−1 and ∼1674 cm−1 (Figure 5A). Resolution enhancement by a second derivative analysis [19] indicated that there were peaks at 1629 cm−1, 1638 cm−1, 1654 cm−1, and 1674 cm−1 in the spectra at relatively low pressures (Figure 5B) (note that a second derivative spectrum gives a negative peak). According to the empirical and theoretical rules in literature [20,21,22,23], it is probable that the peaks at 1629 cm−1/1638 cm−1, 1654 cm−1, and 1674 cm−1 stem from β-sheets, α-helices, and β-sheets (or turns), respectively. This assignment is in line with the secondary structures in the crystal structure of the enzyme [6,9]. The second derivative spectra remained almost unchanged in the region of pressure < 300 MPa, suggesting that the secondary structures were preserved under these pressures. Indeed, pressure dependence of the second derivative spectral intensity at 1638 cm−1, i.e., the β-sheets depicts that the onset of structural change was at ∼400 MPa (Figure 5C). Figure 5C also suggested that the midpoint of the unfolding is ∼730 MPa. Reaching a plateau of the unfolding curve required a pressure over 1000 MPa. The spectrum after decompression was almost identical to those of the native structure at relatively low pressures (Figure 5B), indicating that the structural change induced by pressure was reversible.
Figure 5. Effect of pressure on the structure of Cut190*. (A) Pressure dependence of FTIR spectra in the amide I' region of Cut190*. The spectra at higher pressures are shown as greyer colors. (B) Their second-derivative spectra. A dotted line indicates the spectrum after decompression. (C) Pressure dependence of the second derivative spectral intensity at 1638 cm−1. A sigmoidal curve obtained by fitting the observed data (closed circle) is shown as the dotted line (for guiding the eyes).Due to high activity and thermal stability of Cut190*, it can be used in the industrial applications to hydrolyze PET. We previously analyzed the catalytic activity of Cut190* towards PBSA, pNPB and PET [4,7]; our results indicate that the mutant, Cut190*Q138A/D250C-E296C, exhibited higher activity and stability as compared to Cut190* [5]. In this study, we used BHET (the intermediate product of PET degradation (
In addition to Cut190*, the catalytic activity of Cut190*Q138A/D250C-E296C towards BHET was evaluated. The kcat values could not be determined due to the low solubility of BHET [29,30]. An analogue of BHET with high solubility will make it possible to determine the enzyme kinetics. The ratio of the substrate conversion indicated that the mutation Q138A together with the introduction of disulfide bond between the residues 250 and 296 could increase the activity, similar to the findings of studies on PBSA and PET-GF films [5]. Using the two mutants, we also analyzed the effect of pressure on the catalytic activity. With increasing pressure up to 400 MPa, the conversion rate of BHET into MHET gradually increased up to approximately 20% (Figure 4). Quartinello et al. recently reported that pressure (4 MPa) at high temperature (250 °C) is effective in PET degradation [14]. To our knowledge, this is the first report of BHET degradation analysis at high pressures (∼400 MPa), showing that high pressure even at low temperature (37 °C) is effective in BHET degradation. Under all the conditions of high pressures analyzed, the activities of enzymes were remained, and the BHET conversion rate in the presence of enzyme was higher than that in the absence of enzyme. The FTIR experiments under high pressure indicated that Cut190* molecules with the native structure were still dominantly populated at 400 MPa (Figure 5). In the structural stability analysis against pressure in terms of the secondary structural change using FTIR, the loss of the β-sheets was used as an indicator of the folding-unfolding equilibrium (Figure 5C). This analysis indicated that most of the enzyme molecules were populated in the native structure below ∼400 MPa. Thus, the loss of activity by pressure-induced unfolding would be negligible in the region of pressure. The midpoint of the Cut190* unfolding was determined to be ∼730 MPa (Figure 5C), which is comparable to those of other globular proteins reported previously [31,32]. The reversibility of pressure-induced unfolding is contrary to the irreversibility of thermal denaturation [8]. There were minor spectral changes such as a red shift of the peak at ∼1654 cm−1 even between 4 MPa and 300 MPa. This could be because high-pressure FTIR spectral changes involve an elastic effect including a change in strength of hydrogen bond in helices in addition to effect of a conformational change [16]. Under the conditions of high pressure where the enzyme activity is increased [10,11,12], high-pressure FTIR will be a powerful tool to evaluate the structure - activity relationship (SAR) of the enzyme. Taken together, as pressure increases, the structural flexibility of Cut190* might be unchanged or the increase might be smaller than the increase in the non-enzymatic degradation rate of BHET.
In conclusion, we showed that BHET could be used as a substitute for PBSA, pNPB, and PET for the analysis of Cut190* activity. The investigation for Ca2+ effect on the enzyme activity suggested that small size of substrate could bind to not only “open” form of Cut190* but also the “closed” form. We also found out the additive effects of enzyme and pressure (up to 400 MPa). The present finding that hydrolysis of BHET even in the absence of enzyme increased with increase in pressure suggests the possibility of PET degradation by high pressure.
The authors thank Drs. Akihiro Maeno and Kazuyuki Akasaka for technical support of high-pressure experiments. This work was supported by Institute for Fermentation, Osaka (IFO).
All authors declare no conflict of interest in this paper.
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Figures(5)
Yoshiji Hantani, Hiroshi Imamura, Tsubasa Yamamoto, Akane Senga, Yuri Yamagami, Minoru Kato, Fusako Kawai, Masayuki Oda. Functional characterizations of polyethylene terephthalate-degrading cutinase-like enzyme Cut190 mutants using bis(2-hydroxyethyl) terephthalate as the model substrate[J]. AIMS Biophysics, 2018, 5(4): 290-302. doi: 10.3934/biophy.2018.4.290
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