Polyhydroxyalkanoate Film Formation and Synthase Activity During In

Biomacromolecules 2008, 9, 2811–2818

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Polyhydroxyalkanoate Film Formation and Synthase Activity During In Vitro and In Situ Polymerization on Hydrophobic Surfaces Shun Sato,† Yusuke Ono,† Yukiko Mochiyama,† Easan Sivaniah,‡ Yoshihiro Kikkawa,§ Kumar Sudesh,| Tomohiro Hiraishi,⊥ Yoshiharu Doi,⊥ Hideki Abe,⊥ and Takeharu Tsuge*,† Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8502, Japan, Polymer IRC, School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, United Kingdom, Photonics Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia, and RIKEN Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan Received May 22, 2008; Revised Manuscript Received July 17, 2008

In vitro and in situ enzymatic polymerization of polyhydroxyalkanoate (PHA) on two hydrophobic surfaces, a highly oriented pyrolytic graphite (HOPG) and an alkanethiol self-assembled monolayer (SAM), was studied by atomic force microscopy (AFM) and quartz crystal microbalance (QCM), using purified Ralstonia eutropha PHA synthase (PhaCRe) as a biocatalyst. (R)-Specific enoyl-CoA hydratase was used to prepare R-enantiomer monomers [(R)-3-hydroxyacyl-CoA] with an acyl chain length of 4-6 carbon atoms. PHA homopolymers with different side-chain lengths, poly[(R)-3-hydroxybutyrate] [P(3HB)] and poly[(R)-3-hydroxyvalerate] [P(3HV)] were successfully synthesized from such R-enantiomer monomers on HOPG substrates. After the reaction, the surface morphologies were analyzed by AFM, revealing a nanometer thick PHA film. The same biochemical polymerization process was observed on an alkanethiol (C18) SAM surface fabricated on a gold electrode using QCM. This analysis showed that a complex sequence of PhaCRe adsorption and PHA polymerization has occurred on the hydrophobic surface. On the basis of these observations, the possible mechanisms of the PhaCRe-catalyzed polymerization reaction on the surface of hydrophobic substrates are proposed.

Introduction Polyhydroxyalkanoates (PHAs) are biopolyesters accumulated in bacterial cells as intracellular carbon and energy storages.1-3 Much attention has been paid to PHAs because they are biodegradable, biocompatible, and thermoplastic materials. PHA synthases (PhaC) are a key class of enzymes that play an important role in the biosynthesis of PHA. PhaC polymerizes the (R)-3-hydroxyalkanoate (3HA) moiety of (R)-3HA-CoA with the release of coenzyme A (CoA) and the generation of waterinsoluble inclusions of PHA.4 At present, approximately 60 different PHA synthases have been isolated and characterized.5,6 PHA synthase from Ralstonia eutropha (PhaCRe) is well characterized biochemically and enzymologically, and the expression and purification method for PhaCRe has been developed with the help of gene recombinant techniques.7 These technical developments have allowed the application of soluble PhaCRe for in vitro synthesis of PHA, thus circumventing the limitations associated with bacterial fermentation methods for producing PHAs. The first demonstration of in vitro poly(3-hydroxybutyrate) [P(3HB)] synthesis in aqueous solution was achieved with PhaCRe by Gerngross and Martin.8 Their study revealed that * To whom correspondence should be addressed. Phone: +81-45(924)5420. Fax: +81-45(924)5426. E-mail: [email protected]. † Tokyo Institute of Technology. ‡ University of Leeds. § National Institute of Advanced Industrial Science and Technology (AIST). | Universiti Sains Malaysia. ⊥ RIKEN Institute.

the P(3HB) polymer had significantly higher molecular weight than that synthesized in vivo. This is probably due to a lack of a chain termination step of the PHA polymerization under in vitro conditions, similar to a living polymerization. Taking advantage of the in vitro synthesis, block copolymer biosynthesis, which is difficult to control within a bacterial cell, has been demonstrated by sequential feeding of two kinds of monomer units into an in vitro reactor.9 It is also possible to synthesize PHA with non-naturally occurring compositions and moieties in vitro.10 In further developments, to improve the yield of PHA, some monomer-supplying enzymes have been introduced to construct a byproduct recycling system.11-13 In vitro and in situ PHA synthesis techniques to fabricate micropatterns on solid substrates have recently emerged. Kim et al. reported in situ enzymatic surface-initiated polymerization of PHA and the formation of a PHA film by using immobilized PhaCRe on solid surfaces.14 This research group also fabricated bioactive surfaces on the PHA structures, which were used to support other functional biomolecules such as streptavidin and biotin.15 Such surface modification has biomedical and biotechnological applications. Atomic force microscopy (AFM) is a powerful tool for topological analysis at the nanometer scale. In particular, biological macromolecules such as DNAs and proteins can be imaged in their native state, as the AFM technique does not require any sample pretreatment.16-18 In this paper, we use AFM as a tool to understand the initial stage of PHA granule formation. Previously, individual PHA synthase (PhaCRe) molecules were observed by AFM as globular particles of 2.9 ( 0.4 nm in height and 28 ( 4 nm in width19 on a hydrophobic

10.1021/bm800566s CCC: $40.75  2008 American Chemical Society Published on Web 09/05/2008

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highly oriented pyrolytic graphite (HOPG) substrate. Also, the in vitro polymerization reaction on the surface of HOPG substrates was carried out at a low concentration of monomers in order to observe the initial stages of the reaction. As a result, the clusters of PhaCRe molecules could be imaged. Therefore, AFM studies will provide insight into the behavior of PHA synthase and its product during in vitro PHA polymerization. Because it is difficult to follow a rapid reaction with AFM, an additional analytical approach is needed to gain a better understanding of reaction dynamics. The quartz crystal microbalance (QCM) technique provides real-time analyses of mass uptake and viscosity change on a QCM electrode.20-22 In many cases, QCM has been used for the analysis of protein adsorption and desorption at the surface of an electrode.23,24 For PHA thin films, the enzymatic degradation behavior has been studied by QCM.25 However, QCM has not been applied to in vitro PHA polymerization for real-time analyses thus far. The aim of this study is to understand the behavior of PHA and PHA synthases during the in vitro polymerization on hydrophobic surfaces. For this purpose, AFM and QCM were applied to follow the polymerization reaction catalyzed by PhaCRe on two hydrophobic surfaces, HOPG and alkanethiol (C18) self-assembled monolayer (SAM). The combination of AFM and QCM provides complementary data on the biopolymerization reaction that occurs on hydrophobic surfaces. On the basis of our observations, possible mechanisms of this reaction are discussed.

Experimental Section Expression and Purification of Enzymes. (R)-Specific enoyl-CoA hydratase from Aeromonas caViae (PhaJAc) and histidine-tagged PHA synthase (PhaCRe) from Ralstonia eutropha were used in this study. These enzymes were expressed in recombinant Escherichia coli BL21(DE3) strains (Novagen, CA), as described previously.26,27 PhaJAc and His-tagged PhaCRe were purified from soluble protein fractions of E. coli with a HiLoad Q-Sepharose HP 16/10 column (Amersham Biosciences, NJ) and HisTrap HP column (Amersham Biosciences), respectively. The hydratase activity of PhaJAc was assayed by the hydration of trans-2-crotonyl-CoA.28 The PhaCRe activity was assayed by measuring the absorption change at 236 nm, which corresponds to the cleavage of the thioester bond of (R)-3-hydroxybutyryl-CoA [(R)3HB-CoA].29 The enzyme purity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to the standard procedure,30 and protein concentration was determined by the method of Bradford by using a Protein Assay Kit (Bio-Rad, CA) and bovine serum albumin (BSA) as the standard. Synthesis of CoA Derivatives. trans-2-Enoyl-CoAs with acyl chain lengths of 4-6 carbon atoms, the substrates of PhaJAc, were synthesized from lithium salts of CoA (Wako Chemicals, Japan) and the corresponding trans-2-alkenoic acids (Tokyo Kasei, Japan), based on mixedanhydride methods.31 The purifications of trans-2-enoyl-CoAs were performed with Sep-Pak C18 columns (Waters, MA).32 Purified trans2-enoyl-CoA was converted into (R)-3HA-CoA by an enzymatic hydration reaction catalyzed by 10 nM of PhaJAc (3.4 U) in 3 mL of 50 mM phosphate buffer solution (pH 8.0) for 10 min at 30 °C. After this reaction, PhaJAc proteins were removed by ultrafiltration with a Vivaspin 500 (Vivascience, Germany), and the resultant (R)-3HA-CoA solutions were used for the in vitro polymerization reaction. HPLC Analysis. The CoA derivatives generated by hydration and polymerization reactions were analyzed by high performance liquid chromatography (HPLC) using a Shimadzu 10A LC-VP system and a SPD-10A UV-vis detector.33 Samples, 50 µL, after enzymes removal, were injected into the Luna C18(2) column (Shimadzu, Japan) at 40 °C equilibrated with buffer A (200 mM sodium phosphate buffer, pH 5.0) containing 5% buffer B (200 mM sodium phosphate buffer and

Sato et al. 20% acetonitrile, pH 5.0). The elution of CoA derivatives were performed by a gradient of buffer B concentration as follows: 0-5 min, 5% B; 5-15 min, linear gradient of 5-20% B; 15-30 min, linear gradient of 20-50% B; 30-40 min, linear gradient of 50-100% B; 40-50 min, 100% B. Effluents was detected at 254 nm due to UV absorption by the adenyl moiety of CoA. Lithium salts of CoA, crotonyl-CoA (Sigma, MO) and (SR)-3HB-CoA (Sigma) were used to make calibration curves of CoA, enoyl-CoA, and 3HA-CoA, respectively. In Vitro Polymerization in Aqueous Solution. The in vitro polymerization reaction in aqueous solution was carried out in 25 mL of 20 mM potassium phosphate buffer (pH 7.0) containing 2 mM crotonyl-CoA, 20 nM PhaJAc, and 200 nM PhaCRe (17.5 U) at 30 °C. After 18 h of the reaction, 40 µg/mL of proteinase K (Roche Diagnosis, Germany) and 2 mM of CaCl2 were added to the reaction solution and incubated overnight at 30 °C. After centrifugation, the precipitations were lyophilized. White precipitations were dissolved in chloroform at room temperature for 72 h. The chloroform solution was filtered (PVDF membranes, 0.45 µm pore) and used for molecular weight analysis by gel permeation chromatography (GPC) at 40 °C, using a Shimadzu 10A GPC system and a 10A refractive index detector with Shodex K-806 M and K-802 columns. Chloroform was used as the eluent at a flow rate of 0.8 mL/min and the molecular weights determined against a calibration curve made from polystyrene standards. In Vitro Polymerization on HOPG Substrate. In vitro and in situ polymerization of PHA using PhaCRe and (R)-3HA-CoAs were performed on freshly cleaved HOPG substrates (ZYH quality, Veeco, NY) incubated at 30 °C on hot plate.19 First, 5 µL of PhaCRe solution (10 pM) was added as a droplet on the HOPG substrate and incubated for 5 min. A total of 5 µL of (R)-3HB-CoA monomer solution with a concentration of 0.01-100 µM was then added to the enzyme droplet, of which total volume was 10 µL, to initiate polymerization reaction. The final concentrations of PhaCRe and (R)-3HA-CoA were thus half that of the initially added solutions. After incubation for 5 min, the droplet was removed by pipet and the substrate was carefully washed with Milli-Q water several times, dried in a desiccator overnight, and then its surface observed by AFM. AFM Observation. The morphology of enzymes and polyesters complexes was observed by dynamic force mode (tapping mode) AFM (SPI3800/SPA400, SII Nanotechnology, Japan) in air at room temperature. Long silicon cantilevers, 400 µm, with average spring constants of 2.6 N/m were used for the AFM observation under light tapping mode (set-point value ) 0.8-0.9 of free oscillation amplitude of the tip). The scan rate ranged from 0.5 to 1.0 Hz. Enzymatic Degradation of PHA. Enzymatic degradation of the thin films of PHA on the surface of HOPG substrate was carried out using PHB depolymerase from Ralstonia pickettii T1, which was purified as previously reported and stocked in laboratory.34 An aliquot of 10 µL of 100 mM potassium phosphate buffer solution (pH 7.4) containing 10 µg/mL of PHB depolymerase was added to the HOPG substrate surface after in vitro polymerization for 10 min at room temperature. The reaction solution was removed by pipet and the surface washed with Milli-Q water. After drying, AFM observation was performed in the same manner as described above. Fabrication of SAM. QCM crystals, AT-cut quartz crystals with a fundamental resonance frequency of 9 MHz, were purchased from SEIKO EG&G (Japan) with bare gold electrodes on both sides (area size: 0.196 cm2 × 2). The gold electrodes were washed with 1.2 N NaOH and then 1.2 N HCl and rinsed with Milli-Q water. The gold electrode were then incubated in concentrated HCl for 1 min, rinsed with Milli-Q water,35 and finally immersed in 10 mM octadecanethiol solution (ethanol/THF ) 4/1 (v/v)) for 24 h to form a SAM on the surface.36 The resultant SAM were washed with Milli-Q water and incubated in ethanol with sonication for 5 min. For QCM measurements, the SAM-coated electrodes were dried in air prior to use. QCM Measurement. QCM measurements were performed on QCM934 (SEIKO EG&G) with a QA-CL5 well-type cell at room temperature. A total of 180 µL of 50 mM Tris-HCl buffer (pH 7.5)

PHA Film Formation and Synthase Activity Scheme 1. Enzymatic Reaction of the In Vitro Polymerization for PHA Employing (R)-Specific Enoyl-CoA Hydratase (PhaJAc) and PHA Synthase (PhaCRe)

was added to the dry QCM cell and system was stabilized (no further frequency change). The changes of resonant frequency (∆F) and resonant resistance (∆R) were then monitored as 2 µL of PhaCRe solution (1 µM) and 20 µL of (R)-3HB-CoA solution (860 µM) were added to the QCM cell. The total volume in the cell was 202 µL and the final concentrations of PhaCRe and (R)-3HB-CoA were 9.9 and 85 nM, respectively. The sequence in which the enzyme and monomer were added was an experimental parameter. After QCM measurements, SAM-coated electrode surfaces were washed with Milli-Q water, dried in air, and observed by tapping-mode AFM. Contact Angle Measurement. The wettability of the sample surface was estimated by static contact angle measurements with water, using a Phenix Alpha contact angle system (SEO. Co., Ltd., Korea). The sample surfaces were dried in a desiccator overnight prior to measurement.

Results Monomer Preparation through R-Hydration Reaction. Scheme 1 was designed for in vitro synthesis of PHA, using two enzymes R-hydratase (PhaJAc) and PHA synthase (PhaCRe). The (R)-3HA-CoA monomers were prepared through a Rspecific hydration reaction catalyzed by PhaJAc. Taking into consideration the substrate specificities of PhaJAc and PhaCRe, we synthesized and used enoyl-CoAs with acyl chain length of 4-6 carbon atoms: crotonyl-CoA (C4), pentenoyl-CoA (C5), and hexenoyl-CoA (C6). The samples after hydration reaction by PhaJAc were analyzed by HPLC and the typical elution profiles are shown in Figure 1. In all cases, the dominant peaks after the hydration reactions corresponded to 3HA-CoAs, suggesting that the enzymatic conversions were successful. The equilibrium constants (Keq ) [3HA-CoA]/[enoyl-CoA]) and conversion ratios of the hydration reactions were determined from the HPLC analysis, as listed in Table 1. For crotonyl-

Figure 1. HPLC elution profiles of hydration reaction products of trans2-enoyl-CoA catalyzed by PhaJAc. Crotonyl-CoA (C4), pentenoyl-CoA (C5), and hexenoyl-CoA (C6) were used as substrates.


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CoA, Keq of the hydration reaction was as high as 9.77, and the conversion ratio to (R)-3HB-CoA was approximately 0.9. However, with increasing acyl chain length of substrates, the equilibrium constants and the conversion ratios decreased. Keq and conversion ratio for hydration reaction of hexenoyl-CoA were approximately 4 and 0.8, respectively. Both Keq and the conversion ratio were not functions of the initial enoyl-CoA concentration in the concentration ranges used in our experiments. To elucidate the feasibility of using the reactants after R-hydration reaction, 10 nM PhaCRe enzyme (35 mU) was added to the filtrates of reactants to initiate in vitro polymerization. Consumption of 3HA-CoAs and the polymerization-induced release of CoA were measured by HPLC, and the conversion ratios from 3HA-CoA to PHA were calculated as listed in Table 1. P(3HB) and poly(3-hydroxyvarerate) [P(3HV)] were successfully synthesized with conversion ratios of 0.69 and 0.61, respectively, in aqueous solution. Unfortunately, PhaCRe showed no polymerization activity toward 3HHx-CoA in vitro. The P(3HB) synthesized in aqueous solution was subjected to GPC analysis that revealed that the final P(3HB) was of high molecular weight (The number average molecular weight (Mn) and polydispersity (Mw/Mn) were 4.02 × 106 and 1.42, respectively). AFM Imaging of PhaCRe or (R)-3HB-CoA Adsorbed on HOPG. Freshly cleaved HOPG provided a smooth surface with the average vertical roughness of approximately 0.3 nm. PhaCRe from solutions at different concentrations (1-10 nM) was deposited on the surfaces and visualized by AFM (see Figure 2). The globular objects observed were approximately 1.3 nm in height and 29 nm in width, which is very similar in size to previous measurements of PhaCRe molecules.19 At a PhaCRe concentration of 10 nM, large aggregates of PhaCRe molecules were observed on HOPG surface, while individual particles of PhaCRe are clearly visualized from 1000-fold diluted solution (10 pM). From the molecular sizes, PhaCRe must initially adsorb on HOPG as a monomeric subunit. These low concentrations are better suited for experimental clarity, and subsequent experiments reported here involved a PhaCRe concentration of 10 pM. On the other hand, no surface structure was observed when the monomer alone was added (100 µM (R)-3HB-CoA), as in Figure 2E, indicating that monomers did not adsorb onto hydrophobic surface due to high hydrophilicity of CoA moiety. Morphology Analysis of the Substrate Surface after Polymerization on HOPG. When C4 and C5 monomers prepared by the R-hydration reaction are used, in vitro and in situ polymerization of PHA on hydrophobic HOPG surfaces was conducted. After drying the surfaces, AFM observation was carried out in air. Figure 3 shows the morphology of HOPG surfaces after in vitro and in situ polymerization using (R)-3HB-CoA at different concentrations (0.01-100 µM of initial crotonyl-CoA). At an (R)-3HB-CoA concentration of approximately 0.01 µM, organization of PhaCRe molecules and fibrils elongated from small aggregates substances were imaged by AFM (see images B and H). As (R)-3HB-CoA concentration increased, the globular substances connect to form a network structure. At approximately 10 µM of (R)-3HB-CoA, the surface of the substrate was covered by a thin film with some pits (see Figure 3; images E, K). The thickness of the thin film was 1 nm from the crosssection analysis. This thin film is P(3HB) polymerized on the HOPG surface. As the (R)-3HB-CoA concentration increased further, the number of pits in the thin film declined and the

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Table 1. Hydration and Polymerization Reactions for In Vitro Polymerization of PHAs hydration reaction of enoyl-CoAsa concentration (µM) acyl chain length of enoyl-CoA C4 C5 C6

after hydration initial enoyl-CoA

enoyl-CoA (A)

3HA-CoA (B)

Keq (B/A)

conversion ratio B/(A + B)

600 40 700 80 5200 750

55.7 4.4 101 11.9 948 177

544 36.3 582 65.0 4236 546

9.77 8.29 5.76 5.48 4.47 3.09

0.91 0.89 0.85 0.85 0.82 0.76

c

polymerization reactionb

overall

monomer conversion ratio (C)

reaction yield C × B/(A + B)

0.69 0.61 NDd -

0.63 0.52 NDd -

a Hydration reaction was catalyzed by 10 nM of PhaJAc (3.4 U) in 3 mL of 50 mM phosphate buffer (pH 8.0) at 30 °C for 10 min. b Polymerization reactions were performed in 1 mL of 20 mM potassium phosphate buffer (pH 7.0) containing 10 nM of PhaCRe (35 mU) and 100 µM of 3HA-CoA. The monomer conversion ratio, defined by the molar ratio of released CoA (mol) to initial 3HA-CoA (mol), was determined by HPLC analysis. c Keq, equilibrium constant. d ND, not detectable.

Figure 2. AFM height images of PhaCRe molecules or (R)-3HB-CoA solution dispersed on HOPG surface: (A) no PhaCRe, (B) 10 pM, (C) 1 nM, (D) 10 nM of PhaCRe, and (E) 100 µM of (R)-3HB-CoA. The color contrast for the height images represents a total range of 2 nm.

Figure 3. AFM height (A-F) and phase (G-L) images of PhaCRe and synthesized P(3HB) on HOPG surfaces. In vitro polymerization was performed with 10 pM PhaCRe and (A, G) 0 µM, (B, H) 0.01 µM, (C, I) 0.1 µM, (D, J) 1 µM, (E, K) 10 µM, (F, L) 100 µM C4 monomer for 5 min at 30 °C, respectively. The C4 monomer solution dominantly contained (R)-3HB-CoA, as shown in Figure 1. Cross sectional data along the black lines in images (A-F) are shown in (M-R), respectively. The white bar inside the images represents 100 nm.

film thickened. In the latter images, PhaCRe molecules were hardly distinguished. After polymerization with approximately 100 µM (R)-3HBCoA on the HOPG surface, the residual reaction mixture was subjected to HPLC analysis to measure free CoA concentration, which is important to understand the reaction progress. From HPLC analysis, the generation of free CoA was indeed detected. However, unlike polymerization in aqueous solution, the conversion ratio of (R)-3HB-CoA was as low as 0.05 on HOPG surface. When (R)-hydroxyvareryl-CoA [(R)-3HV-CoA] was used as a monomer, the surface morphologies of HOPG after polymerization were observed as shown in Figure 4. Similar to the case of (R)-3HB-CoA, the structure observed on HOPG surface varied from PhaCRe aggregates to a P(3HV) thin film, depending on the concentration of monomer. The thickness of the P(3HV) film produced by 100 µM monomer of initial pentenoyl-CoA was approximately 1 nm, which is similar in size to P(3HB) thin film. Interestingly, globular substances could be seen in

phase mode images (see Figure 4K) as very small black particles. These substances are probably PhaCRe molecules trapped within polymerized P(3HV). Because P(3HV) is more hydrophobic than P(3HB), it may facilitate detection of phase contrast between polyesters and proteins. Degradation of PHA Thin Film. We suggest that the HOPG surface, after in vitro polymerization of PHA, is covered by a PHA film. To confirm this finding, we carried out an enzymatic degradation experiment by using P(3HB) depolymerase from Ralstonia pickettii T1. The surface morphology before degradation is shown in Figure 5A. After P(3HB) depolymerase treatment for 10 min, surface morphologies of the HOPG was visualized by AFM, as shown in Figure 5C, indicating drastic degradation of the PHA film. As control experiments, treatment with either Milli-Q water or proteinase K (a typical serine protease) was also performed in the same manner as P(3HB) depolymerase, but no significant changes in surface morphology was observed. These results strongly suggest that the initial thin film was P(3HB).

PHA Film Formation and Synthase Activity


Figure 4. AFM height (A-E) and phase (F-K) images of PhaCRe and synthesized P(3HV) on HOPG surfaces. In vitro polymerization was performed with 10 pM PhaCRe and (A, F) 0.01 µM, (B, G) 0.1 µM, (C, H) 1 µM, (D, I) 10 µM, and (E, J) 100 µM C5 monomer for 5 min at 30 °C, respectively. The C5 monomer solution mainly contained (R)-3HV-CoA, as shown in Figure 1. Panel K inset in panel J shows a higher enlarged image of dotted square (50 × 50 nm2) in panel J. Cross-sectional data along the black lines in images (A-E) are shown in (L-P), respectively. The white bar inside the images represents 100 nm.

Figure 5. AFM height images of the postpolymerization HOPG sample surface before and after enzymatic degradation of P(3HB) thin film: (A) before enzymatic treatment, (B) treatment with Milli-Q water, (C) treatment with PHA depolymerase from Ralstonia pickettii T1, and (D) treatment with proteinase K. All treatments were conducted at room temperature. The color contrast for the height images represents a total range of 2 nm. Table 2. Static Contact Angles (θ) with Water on the Surface of the Substratesa substrates

contact angle (°)

HOPG SAM-coated electrode before polymerization after incubation with PhaCRe after polymerization (case 1)b after polymerization (case 2)b

74 ( 3 97 ( 3 67 ( 3 60 ( 9 79 ( 2

a Values are the averages and standard deviations of three measurements. Cases 1 and 2 represent the experimental sequence of QCM analysis. In case 1, first PhaCRe solution and then (R)-3HB-CoA solution were added to a well-type QCM cell, whereas the order of addition was reversed in case 2. b

QCM Analysis. To gain a better understanding of the dynamics of the in vitro polymerization process that occurs on the hydrophobic surface, real-time monitoring of the reaction was performed with QCM. Because HOPG substrate is not available as a QCM electrode, a SAM of alkanethiol (C18) on the gold-coated electrode was used. The hydrophobicity of the two surfaces were estimated by water contact angles as follows: HOPG, 74 ( 3°; SAM-coated electrode, 97 ( 3° (see Table 2). The SAM surface was more hydrophobic than HOPG surface. Two QCM experiments were conducted. In case 1, first PhaCRe solution and then (R)-3HB-CoA solution were added to a well-type QCM cell at time points of (A) and (B) in Figure 6, respectively. In case 2, the order of addition was reversed at time points of (A) and (C) in Figure 6, respectively. Figure 6 shows the QCM data for these two cases. In case 1, there is a reduction in frequency after addition of PhaCRe, due

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Figure 6. Time-dependent changes of resonant frequency (∆F) and resonant resistance (∆R) of QCM during in vitro and in situ polymerization of P(3HB) on alkanethiol (C18) SAM. The solid and dotted lines represent cases 1 and 2, respectively. In case 1, first PhaCRe solution and then (R)-3HB-CoA solution were added to a well-type QCM cell at time points of (A) and (B), respectively. In case 2, the order of addition was reversed at time points of (A) and (C), respectively.

to adsorption of PhaCRe on the SAM surface. The frequency change before and after PhaCRe addition was 347 Hz, which corresponds to a mass change of 123 ng (as estimated by the Sauerbrey equation37,38), suggesting that over 90% of PhaCRe that was added to the cells was adsorbed on the SAM surface. Subsequently, (R)-3HB-CoA solution was added to the QCM cell. After a 10 min induction period, a steady decrease of frequency was observed over 40 min. This frequency change (528 Hz) is attributable to the synthesis of P(3HB) on the SAM surface. An estimated 188 ng of P(3HB) is produced (assuming no initially attached PhaCRe desorbed from the surface). That this process corresponds to P(3HB) synthesis is corroborated by the change of resonant resistance monitored by QCM. Increases in resonant resistance are associated with a more viscous material at the electrode surface. PhaCRe adsorption did not significantly affect resonant resistance (10-20 min), as seen from Figure 6. However, after the polymerization reaction was initiated (20 min), the resonant resistance increased due to the damped response of the PHA film to the oscillating QCM crystal. In case 2, first (R)-3HB-CoA solution and then PhaCRe solution was added to the QCM cell. The addition of (R)-3HBCoA had no effect on the frequency or the resonant resistance, suggesting there was no interaction between (R)-3HB-CoA and SAM surface. Significant changes in frequency and resonant resistance were only observed once PhaCRe was added. The rapid decrease in frequency was due to both synthesized P(3HB) and PhaCRe adsorption. The frequency change, 957 Hz, corresponds to the mass uptake of 340 ng. Because resonant resistance was not influenced by adsorption of PhaCRe, as demonstrated above, the observed resistance change represents P(3HB) synthesis on a SAM surface. The slight difference in total mass change between case 1 (311 ng) and case 2 (340 ng) may result from enzyme activity, where not all of the PhaCRe (in case 1) could retain activity once absorbed. AFM Imaging of QCM SAM Surfaces. The SAM surfaces, before and after QCM measurements, were observed by AFM in air, as shown in Figure 7. The SAM-coated surfaces contained hemispherical structures, typical of deposited gold films, with the average surface roughness of 1.2 nm. After PhaCRe molecules were deposited on the SAM surface, a slight change

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between cases 1 and 2 could be related to the surface coverage of the SAM with PHA. The surface coverage by P(3HB) were 91 and 56%, calculated from AFM height images (1 × 1 µm2 area) in cases 1 and 2, respectively.

Discussion

Figure 7. AFM height images of the surface of quartz crystals modified with alkanethiol (C18) SAM before and after in vitro polymerization of P(3HB): (A) before polymerization, (B) after depositing PhaCRe molecules on SAM, (C) after in vitro polymerizations in case 1, (D) after in vitro polymerizations in case 2. In case 1, first PhaCRe solution and then (R)-3HB-CoA solution were added to a well-type QCM cell at time points of (A) and (B) in Figure 6, respectively. In case 2, the order of addition was reversed at time points of (A) and (C) in Figure 6, respectively. QCM data during in vitro polymerization in cases 1 and 2 are shown in Figure 6. Cross-sectional profiles along the black line in images (C) and (D) are shown in (E) and (F), respectively.

in morphology of SAM surface was imaged probably due to coverage of the surface by PhaCRe. The different morphologies of the SAM surfaces after QCM measurements were imaged between cases 1 and 2, depending on the order of addition of monomer and enzyme. In case 1, SAM surface was covered by P(3HB) thin film with the thickness of up to 6 nm and average surface roughness of 0.9 nm. This morphology was very similar to that observed on the HOPG surface. In case 2, large globular objects and their network structure on SAM surface were observed by AFM. These heights were as high as approximately 80 nm. The globular objects are most probably P(3HB) granules generated in aqueous solution and deposited on the SAM surface because such objects were only observed when both monomer and catalyst were present in solution. Thus, in case 2, different modes of polymerization proceed on the hydrophobic surface and within the aqueous solution, generating P(3HB) network structure and granules, respectively. Contact Angle of Surfaces. After P(3HB) polymerization, the water contact angles of SAM surfaces were measured, as listed in Table 2. The surface, covered with a nearly complete P(3HB) film (case 1), had a contact angle of 60 ( 9°, whereas the surface with a heterogeneous network of film and P(3HB) granules (case 2) had a contact angle of 79 ( 2°. Both surfaces were more hydrophilic than that of untreated SAM (97 ( 3°). This hydrophilic property can result from P(3HB) polymer and PhaCRe molecules localized on the surface. In fact, the surface incubated with PhaCRe solution only had a contact angle of 67 ( 3°. However, the significant difference in contact angle

PHA synthases only catalyze the reaction of R-enantiomers of 3HA-CoA as monomers. In this study, (R)-3HA-CoAs with different acyl chain length were prepared by using R-hydratase, one of the monomer-supplying enzymes in bacterial PHA biosynthesis. HPLC analysis of the R-hydration reaction catalyzed by PhaJAc revealed that the equilibrium direction of the reaction is mainly toward 3HA-CoA, with an equilibrium constant of 9 for C4 substrates, and conversion ratios of over 0.7 even for C6 substrates (Table 1). Therefore, this enzyme is a very good monomer-supplier for in vitro PHA synthesis. PHA synthases and PhaJs with various substrate specificities have been found so far. By proper combination of PHA synthases and PhaJs, PHA with novel monomer components and nonnaturally occurring compositions can be synthesized. The PhaCRe subunit is a 64 kDa protein, and is considered to become active once it is associated to other subunits in a dimeric or oligomeric form.4,19 Because PhaCRe has been purified as soluble enzyme from recombinant E. coli, the protein surface is principally hydrophilic. This idea is also supported in our previous study19 where we showed that PhaCRe clusters had a flatter profile when adsorbed on mica surface (hydrophilic surface) than on the HOPG surface (hydrophobic surface). However, as demonstrated by the AFM observation, unreacted PhaCRe molecules are capable of adsorbing onto the HOPG surface. Careful observation by AFM revealed that the size of the unreacted PhaCRe enzyme on HOPG surface is about 14 nm, after appropriate AFM tip deconvolution. This corresponds to a spherical object of diameter about 8 nm if the PhaCRe molecule is assumed to be globular.19 Because this is comparable to the hydrodynamic diameter of PhaCRe, which is estimated as 6.5-8.0 nm based on its molecular weight,19 the unreacted PhaCRe enzyme observed on the HOPG substrate is identical as a single subunit of PhaCRe. Thus, we assume that a region of the PhaCRe subunit surface is locally hydrophobic and would interact with hydrophobic HOPG surface. This region would also play an important role in dimerization or aggregation of PhaCRe molecules by inherent hydrophobic interaction. In fact, clusters of unreacted PhaCRe were rarely observed on the HOPG surface by AFM, suggesting that clustered PhaCRe formed a hydrophilic surface by altering its conformation and burying the hydrophobic region inside. This may have led to a reduced ability for the enzyme to bind to the HOPG substrate. In contrast, after initiation of polymerization reaction, PhaCRe molecules were observed in an aggregate form on the HOPG substrate. From this observation, we infer that clustering of PhaCRe molecules are prompted by the presence of (R)-3HACoA monomers. In addition, fibrils elongated from clustered PhaCRe were imaged by AFM (Figures 3B and 4A). In our previous study, the possibility that these fibrils may be a PHA polymer chain was discussed.19 However, these fibril-like objects could be a novel AFM imaging artifact rather than PHA polymer chains. Because the PhaCRe molecules are thought to be weakly adsorbed on the HOPG surfaces compared with single subunits of PhaCRe, they might be easily pushed aside from their original positions by the scanning motion of the AFM cantilever tip. Thus, the cumulative image of the moving PhaCRe molecules

PHA Film Formation and Synthase Activity


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Figure 8. Proposed model in which PHA thin film and PHA granule are formed on a hydrophobic surface.

leaves behind a trace that appears, erroneously, to be a PHA polymer chain. In fact, the orientation of this fibrous image is always perpendicular to the AFM scan direction. Thus, the dimeric or clustered PhaCRe may be easily moved during scanning by AFM cantilever tip. On the other hand, the subsequent growth of the hydrophobic PHA polymer chain would improve the adhesion of PhaCRe to HOPG surface. The QCM analysis provided insight into the process of P(3HB) polymerization on hydrophobic surfaces. Adsorbed PhaCRe showed very slow change in QCM frequency immediately after initiation of the polymerization reaction (Figure 6, case 1). This result supports the idea that adsorbed PhaCRe molecules are single subunits and do not have full catalytic activity, as demonstrated by our AFM studies. Subsequently, the QCM frequency change increased with incubation time in behavior related to the activation of PhaCRe and PHA polymerization. In addition, by monitoring the resonant resistance change, it was possible to distinguish the process of P(3HB) polymerization on the SAM surface from simple PhaCRe adsorption. PHA film formation on hydrophobic surfaces was observed when the monomer concentration in the reaction mixture was higher than 10 µM. The morphologic appearance observed in this study is quite different from previous reports on in vitro PHA polymerization. Kim et al., in studies of in situ P(3HB) polymerization by immobilized PhaCRe on silicon wafers, reported globular P(3HB) of 0.6 µm in diameter and 200 nm in height on the surface by AFM.14 They also investigated this reaction by surface plasmon resonance in real-time and revealed that initial concentration of PhaCRe affected morphology of P(3HB) films.39 Tajima et al. showed aggregated globular P(3HB) on a PhaCRe-immobilized resin by scanning electron microscope observation.40 Hiraishi et al. investigated P(3HB) polymerization by ex situ AFM studies, and the development of P(3HB) granules in aqueous solution.41 However, the PHA film formation observed here has never been reported. Such film formation may be aided by the mobility of PhaCRe molecules and the relative hydrophobicity of the substrate. On the basis of these results, we propose a model in which PHA films and PHA granules are formed on hydrophobic surfaces (Figure 8). First, single PhaCRe subunits are adsorbed to hydrophobic surfaces due to hydrophobic interactions; this

leads to inactivation of PhaCRe. As (R)-3HA-CoA monomers approach the adsorbed PhaCRe, clustering of PhaCRe molecules are prompted. The polymeric PhaCRe gains catalytic activity and starts PHA polymerization near the substrate surface. The PHA chain intramolecularly aggregates and immediately adsorbs onto the surface via hydrophobic interactions. However, PhaCRe probably remains in the aqueous solution by repulsive forces between the hydrophilic PhaCRe and the hydrophobic surface. All subsequently produced PHA growing from the PhaCRe unit continues to preferentially adsorb onto the hydrophobic surface. As a result, the hydrophobic surface is covered with product PHA to form a thin film. According to the AFM observation of P(3HV), PhaCRe appeared to be buried in the PHA film. In addition, HPLC analysis revealed that only 5% of monomer was converted to P(3HB) during the in vitro reaction on HOPG substrate. Taking these results together, the polymerization reaction is terminated earlier than that in aqueous solution because PhaCRe is trapped with product PHA and buried into the resultant thin film. In contrast, PhaCRe that has reacted with monomers in aqueous solution far from the hydrophobic surface generate small PHA granule, without interference from the hydrophobic surface. These small granules aggregate to form larger granules that finally adhere to the substrate surface by hydrophobic interactions. PhaCRe molecules close to the hydrophobic surface also generated small PHA granules in aqueous solution; however, PHA granules, once generated, would quickly adhere to hydrophobic surface because of their hydrophobic interaction. If PhaCRe molecules on such PHA granules were still active, they would continue the polymerization reaction by moving not only over PHA granule but also over the HOPG surface, resulting in the formation of a network structure.

Conclusions The behavior of PHA and PhaCRe molecules during in vitro and in situ polymerization on the surface of two hydrophobic substrates, HOPG and alkanethiol SAM, was studied by AFM and QCM. From AFM analysis, we found for the first time that in vitro polymerization on hydrophobic surfaces forms PHA thin films with 1-6 nm of thickness. QCM analysis provided insight into the reaction processes involved in the film formation: first, PhaCRe molecules adsorbed on the hydrophobic surface.

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The adsorbed PhaCRe molecules would recover their catalytic activity by the presence of (R)-3HA-CoA monomer and then formed PHA thin film if the enzymes locate near the hydrophobic surface. According to this model, localization of PHA synthase near a hydrophobic substrate surface is important to give thin film formation. Acknowledgment. This work was supported by KAKENHI 19681008 (to T.T.) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. E.S acknowledges the Royal Society (London) for a short visit grant that allowed participation in this project. S.S. is a recipient of the Japan Society for the Promotion of Science Research Fellowship. Supporting Information Available. Adsorbed mass changes of PhaCRe onto SAM surface. This material is available free of charge via the Internet at http://pubs.acs.org.

References and Notes (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15)

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Polyhydroxyalkanoate Film Formation and Synthase Activity During In

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