Biomacromolecules 2004, 5, 1787-1791
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Morphology and Enzymatic Degradation of Oriented Thin Film of Ultrahigh Molecular Weight Poly[(R)-3-hydroxybutyrate] Masahiro Fujita,*,† Yoshitaka Takikawa,‡ Shinya Teramachi,‡ Yoshihiro Aoyagi,§ Tomohiro Hiraishi,† and Yoshiharu Doi†,| Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan, Department of Applied Chemistry, Kogakuin University, Nakanocho 2665-1, Hachioji-shi, Tokyo 192-0015, Japan, Akebono Brake R&D Center, Limited, Higashi 5-4-71, Hanyu-shi, Saitama 348-8501, Japan, and Department of Innovative and Engineered Materials, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan Received February 24, 2004; Revised Manuscript Received May 11, 2004
Thin films of ultrahigh molecular weight poly[(R)-3-hydroxybutyrate] (P(3HB)) were sheared and isothermally crystallized at 100 °C. Transmission electron microscopy and atomic force microscopy (AFM) observations revealed that thick fibrous textures, on which lamellae are overgrown normal to the long axis of the fibril, run parallel to the shearing direction. A selected area electron diffraction pattern taken from the fibrils exhibits a fiber pattern of P(3HB) R-modification, and the crystallographic c-axis (chain axis) of P(3HB) is set parallel to the long axis of the fibril. In situ AFM observations of enzymatic degradation for the thin film were performed with an extracellular P(3HB) depolymerase from Ralstonia pickettii T1 in a buffer solution. The film surface and thickness became rougher and thinner, respectively, with time after adding the enzyme. During the degradation, fine shish-kebab structures appeared gradually. This fact supports that the amorphous region in the film is preferentially degraded rather than the crystalline one by the depolymerase. The in situ AFM observations also revealed that one thick fibril in the original film is composed of three different states, namely, finer fibril (shish), stacked lamellae (kebab) in edge-on state, and the surrounding amorphous phase. Introduction Recently, biodegradable polymer materials have drawn much attention. The materials can be degraded by enzymatic reaction in the natural environment. Poly[(R)-3-hydroxybutyrate] (P(3HB)), produced by various microorganisms as an intracellular carbon and energy reserve, is a biodegradable thermoplastic with a high melting point around 180 °C.1,2 P(3HB) is thus expected to be a new material for a solution to problems concerning the global environment. The P(3HB) material, however, deteriorates due to secondary crystallization and thus exhibits brittleness. The disadvantage in the mechanical strength can be improved using a higher molecular weight polymer than the conventional one. P(3HB) with ultrahigh molecular weight (Mw ∼ 106-107) can be produced by using PHB synthase from a Ralstonia eutropha in vitro system3 or by a recombinant Escherichia coli harboring the R. eutropha biosynthesis phb CAB genes.4 The film and fiber of ultrahigh molecular weight P(3HB) exhibit excellent mechanical strength, compared with those of conventional P(3HB) with medium molecular weight (Mw ∼ 105-106). For example, it was reported recently that the tensile strength * To whom correspondence should be addressed. Phone: +81-48-4679404. Fax: +81-48-462-4667. E-mail:
[email protected]. † Polymer Chemistry Laboratory, RIKEN Institute. ‡ Department of Applied Chemistry, Kogakuin University. § Akebono Brake R&D Center, Ltd. | Department of Innovative and Engineered Materials, Tokyo Institute of Technology.
and Young’s modulus of the ultrahigh molecular weight P(3HB) fiber have reached 1.3 and 18 GPa, respectively, owing to development of a new drawing process.5 Generally, it is known that the mechanical properties of polymers strongly depend on their crystalline morphology in the materials. Orienting polymer chains brings about anisotropy of mechanical properties because the strength along the polymer chain is greater than the intermolecular forces. For example, the aligning of polymer chains is achieved under a shear field during processing of materials such as extrusion, injection, and fiber spinning. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) have revealed that subjecting the polymers to a strain field leads to the formation of so-called row-nucleated or shish-kebab structure as the resulting crystalline morphology.6-18 Recently, in situ X-ray scattering and diffraction in synchrotron radiation and rheo-optical measurements have enabled one to provide the initial mechanism of the crystallization induced by shearing.19-22 It is widely accepted that the orientation of higher molecular weight polymers results in the formation of bundles of parallel chain segments or filament-like entities (shish), followed by a subsequent crystallization of the folded-chain lamellae (kebab) perpendicularly from them, and that an oriented morphology such as the shish-kebab structure is finally developed. A simple structural model of the shish is described as a fully extended chain crystal.9 In practice, however, various structural models
10.1021/bm049886f CCC: $27.50 © 2004 American Chemical Society Published on Web 06/29/2004
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and formation mechanisms of the shish have been proposed (for example, whether the shish consists of continuously extended crystals or not), although it probably depends on the sample preparation method.6-22 For biodegradable polymers including P(3HB), to disclose the details of the oriented structure such as shish-kebab is of significance to control the biodegradation rate as well as the physical properties, because the biodegradability is strongly affected by the crystal size and chain packing state.23 In a previous paper, the shish-kebab-like structures in the P(3HB) oriented material after enzymatic degradation by an extracellular P(3HB) depolymerase from Ralstonia pickettii T1 were visualized at the micrometer scale by scanning electron microscopy (SEM), due to preferential erosion of the amorphous regions in the material.24 To reveal the relation between the crystalline morphology induced by shearing and the biodegradability of ultrahigh molecular weight P(3HB), it is important to analyze these details at the nanometer scale. In the present study, uniaxially oriented thin films of the ultrahigh molecular weight P(3HB) with about 100 nm in thickness as a model sample of the oriented material16,18 were prepared and then analyzed with TEM and AFM. Recently, AFM has been able to visually provide the enzymatic degradation behavior of biodegradable polymers in real time.25,26 In this study, we have used an in situ AFM technique to follow the enzymatic degradation of the oriented thin film by an extracellular P(3HB) depolymerase from R. pickettii T1. From the enzymatic biodegradation view, the structural details of the shish-kebab will be discussed. Experimental Section Materials. Ultrahigh molecular weight P(3HB) was synthesized from glucose by a recombinant E. coli XL-1 blue (pSYL105) harboring R. eutropha PHB biosynthesis phbCAB genes, according to the same procedure reported in a previous work.4 The weight-average molecular weight (Mw) and polydispersity (Mw/Mn) were 4.2 × 106 and 1.5, respectively, determined by gel permeation chromatography (Shimadzu 10A GPC system). An extracellular P(3HB) depolymerase from R. pickettii T1 was purified by a method similar to that reported previously.27 Preparation of the Oriented Thin Film. P(3HB) was dissolved in chloroform to give a 0.5% w/v solution. A drop of the solution was sandwiched between two glass plates on a hot stage thermostated at about 60 °C. A supercooled thin film was uniaxially sheared just after evaporation of chloroform and then annealed (crystallized) in an oven at 100 °C for 8-16 h by displacing one of the glass plates.16,18 Transmission Electron Microscopy. For TEM observations, the oriented thin films of P(3HB) were reinforced with carbon. To calibrate the camera length in selected area electron diffraction (SAED) experiments, the samples were shadowed under a vacuum with Au before carbon coating. The shadowing direction is perpendicular to the shearing direction of the film. TEM was performed with a JEOL JEM 2000 FXII operated at 120 kV. Bright-field images and SAED patterns were recorded on Kodak 4489 and Mitsubishi MEM films, respectively.
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Figure 1. A bright-field TEM image of the oriented thin film of ultrahigh molecular weight P(3HB), sheared and subsequently annealed at 100 °C for 16 h. The arrow indicates the shearing direction. The regions with darker contrast correspond to the crystalline phase in the film: fibrous appearance parallel to the shearing direction and lamellar morphology with edge-on manner grown perpendicularly to the shearing direction were observed.
Atomic Force Microscopy. The surface morphologies of P(3HB) oriented thin films were observed by SPI3800N/ SPA400 (Seiko Instruments Inc.) AFM with dynamic force mode at room temperature. A rectangular Si tip with a spring constant of 20 N/m and a length of 100 µm was used. In situ AFM observations of the enzymatic degradation behavior of P(3HB) oriented thin film were performed at room temperature in a small vessel containing 1.2 mL of 10 mM phosphate buffer solution (pH 7.0) with enzymes: Five microliters of a 40 ng/µL solution of P(3HB) depolymerase from R. pickettii T1 was added to the buffer solution, and AFM images were then obtained with contact mode. A triangular Si3N4 tip mounted on a 200 µm cantilever with a spring constant of 0.02 N/m was applied. The contact force between the sample surface and the cantilever tip was controlled to minimize damage to the sample. Results and Discussion Morphology of the Oriented Thin Film of Ultrahigh Molecular Weight P(3HB). Figure 1 shows a bright-field TEM image of the oriented thin film of the ultrahigh molecular weight P(3HB), crystallized at 100 °C for 16 h. This image was taken at a rather large amount of underfocus in the bright-field imaging mode (defocus contrast method).28 The arrow in this figure indicates the shearing direction, which was, for example, determined from the shadowing direction in the TEM image of the specimens coated with Au at a particular direction. It is recognized that most of the lamellae, which are oriented normal to the shearing direction, are grown in an edge-on manner. Some fibrils of 100-200
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Figure 2. (a) SAED pattern of the oriented thin film of ultrahigh molecular weight P(3HB) and (b) schematic illustration of part a. Concentric rings in this figure are from Au that was deposited to calibrate the camera length in SAED. The c*-axis is parallel to the shearing direction.
Figure 3. AFM height images of the oriented thin film of ultrahigh molecular weight P(3HB), taken in atmosphere at room temperature: (a) lower- and (b) higher-sheared regions, respectively. The arrow in each figure indicates the shearing direction.
Table 1. Lattice Spacings for Reflections of the P(3HB) Oriented Thin Film Observed in the SAED Patterna hkl
dobs, nm
dcalc, nm
hkl
dobs, nm
dcalc, nm
020 A 040 110 011 021
0.657 0.438 0.329 0.526 0.544 0.451
0.660
031 101 111 002 202 232
0.354 0.415 0.395 0.298 0.208 0.188
0.354 0.414 0.395 0.298 0.207 0.187
0.330 0.528 0.543 0.442
a d obs, lattice spacing observed in this study; dcalc, lattice spacing calculated on the basis of the P(3HB) R-form (ref 29).
nm in width running parallel to the shearing direction are also clearly observed in the same field. As can be seen, the fibrils are passing through several lamellae like shish-kebab structures, which consist of a central fibril (shish) surrounded by lamellae (kebab). On the other hand, the lamellae are regularly oriented even in the area where definitive fibrils cannot be recognized. It is supposed that the lamellae overgrow from the considerably thin fibril or the row nuclei.20 Figure 2 shows a SAED pattern taken from the shishkebab-like structures in Figure 1 and a schematic illustration. All reflections in Figure 2a, except for one pair of reflections labeled A, could be well indexed with lattice dimensions of the P(3HB) R-form (P212121, orthorhombic a ) 0.576 nm, b ) 1.320 nm, c (chain axis) ) 0.596 nm) reported previously.29 The observed lattice spacings are listed in Table 1. In Figure 2, the 002 reflections are recognized on the meridian (c*-axis), which is set parallel to the shearing direction. In addition, the hk0 reflections such as the 020 and 110 reflections on the equator and the hk1 and hk2 reflections on the lth ()1, 2) layer lines are also clearly observed. The SAED pattern shown in Figure 2 represents a fiber diagram of the P(3HB) R-form; namely, the c-axes of crystallites or molecular chain axes are found to orient in the shearing direction. The reflections appearing in this diffractogram are slightly arced due to incomplete orientation of the crystallites in the direction of shear. The reflection labeled A on the equator in Figure 2 cannot be ordinarily indexed as 030 using the lattice dimension of the R-form because the structure factor of odd-numbered 0k0 reflections must be null according to the space group of the P(3HB) R-form, P212121. If the specimen is thick enough to
Figure 4. A series of AFM height images for the enzymatic degradation of the oriented thin film of ultrahigh molecular weight P(3HB) by depolymerase from R. pickettii T1. These images were taken at room temperature in a phosphate buffer solution (pH 7.0) before degradation (a) and 60 min (b), 90 min (c), and 120 min (d) after adding the enzymes. The arrow in part a indicates the shearing direction.
cause dynamical scattering, this reflection may appear at the lattice point. Another possibility is that the reflection labeled A may be attributed to the β-form of P(3HB),30 because it is known that the β-form of P(3HB) occurs under high stress.5,30 Therefore, the R- and β-forms may coexist in the present highly oriented film, though the lattice spacing obtained in this study (0.438 nm shown in Table 1) is slightly less than the calculated one reported previously30 and the present reflection is not broad but sharp. The thickness and surface structure of the P(3HB) oriented thin film were examined using conventional AFM at room temperature. The overall film thickness was estimated as about 100 nm on the basis of the substrate surface exposed at some places. Figure 3 shows typical AFM height images of the P(3HB) thin films, which were annealed at 100 °C for 16 h after shearing. The arrow in each figure indicates the shearing direction. Similar to the TEM results as shown in Figure 1, fibrous structures of 100-200 nm in width parallel to the shearing direction and edge-on lamellae
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Figure 5. Surface roughness of the P(3HB) oriented thin film during enzymatic degradation. All data were evaluated for a selected square area (2.0 × 2.0 µm2) in Figure 4. Ra (0) means the averaged absolute value of the deviation of the selected surface from a flat surface with the mean height of the selected one, and S/S0 (O) is the ratio of selected surface area to ideal flat surface area.
overgrown on the fibrils normal to the shearing direction are recognized even on the film surface. It is inferred that an increase in shearing stress leads to an increase in the population density of formation of fibrils so that growth of the lamellae perpendicularly to the shearing direction is fairly limited. Compared with Figure 3a, the fibril structures are more dominant and the length of lamellae is shorter in Figure 3b. This fact means that the area observed in Figure 3b was more highly strained than that in Figure 3a during shearing. In both figures, the film surface is not so flat. Probably, transportation of polymer chains during annealing and/or secondary crystallization at room temperature, viz., incorporation of the amorphous chains into the crystalline phase, leads to the formation of hollows observed in some places of the film surface.
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Enzymatic Degradation of P(3HB) Oriented Thin Film by P(3HB) Depolymerase from R. pickettii T1. In this study, in situ observations of enzymatic degradation of the P(3HB) oriented thin film by AFM were carried out at room temperature in phosphate buffer solution (pH 7.0). The extracellular P(3HB) depolymerase from R. pickettii T1 was used. A series of AFM height images of morphological change in the surface structure of P(3HB) thin film with time after adding the enzyme are presented in Figure 4. The arrow indicates the shearing direction of the film, that is, the molecular chain axis of P(3HB). Before adding the enzyme (Figure 4a), the surface topography of the film observed in the buffer solution is similar to that in Figure 3b. Obviously, the appearance reflects the existence of many fibrils of 100200 nm in width, which run parallel to the shearing direction, and it is deduced that this region was considerably strained because the fibrous texture is dominant as shown in Figure 3b. After the enzyme is added, the film surface seems to become gradually rougher as shown in Figure 4b-d with elapsed time (in practice, it was confirmed that the surface morphology does not change by the buffer solution used here at all). To support this observation, the mean roughness Ra for a surface and the ratio of surface area (S/S0) as surface roughness parameters were estimated by using a surface analysis program in SPI3800N (Seiko Instruments Inc.) for the image data in a rectangular area of 2 µm × 2 µm obtained at the same location in each figure. If the film is degraded homogeneously from its surface, the surface roughness (the surface topography) remains unchanged while the overall thickness decreases monotonically. As demonstrated in Figure 5, however, both roughness and surface area parameters increase with time. These results indicate that the film surface became surely rough by enzymatic degradation. It is accepted that the degradation rate of P(3HB) material by the P(3HB) depolymerase from R. pickettii T1 depends on the chain packing state, and the amorphous regions in the material are eroded faster by the enzyme than the crystalline regions.23 Thus, the roughening of the film surface with time
Figure 6. Enlarged images of Figure 4: (a) before degradation and (b) 90 min after adding the enzyme. These images are the deflection images.
Morphology and Degradation of P(3HB) Thin Film
shown in Figures 4 and 5 is attributed to preferential erosion of the amorphous region. The surface appearance shown in Figure 4c reflects the crystalline entities that emerged during degradation. Enlarged images are shown in parts a and b of Figure 6, which are deflection images corresponding to rectangular regions in Figure 4a (before degradation) and Figure 4c (after 90 min), respectively. These figures demonstrate that one fibril in the original film (Figure 6a) is composed of some thinner filaments (shish) and many edge-on lamellar crystals (kebab), and these surfaces are surrounded by amorphous phase. In Figure 6b, the apparent thickness and lateral length of the edge-on crystal could be estimated as about 50 nm or less and 100-200 nm, respectively. The former value is far more than one lamellar thickness of P(3HB), ca. 5-10 nm. This is probably because the lamellae are closely stacked and/or tilted, and accordingly the width of the crystal was considerably more than the true lamellar thickness. On the other hand, the latter value is comparable with that of fibril width before degradation. The apparent width of a thin filament of several micrometers in length also could be estimated as about 50 nm or less in Figure 6b, suggesting that one thick fibril includes several thinner filaments. With further degradation by the enzyme, both the fine filament and the lamellae were gradually degraded and as a result the glass surface was exposed in major part (Figure 4d). Interestingly, Figure 4d indicates that most of the filaments connecting adjacent lamellae disappear prior to the disappearance of the lamellae by enzymatic degradation. This is presumably because the crystal size normal to the chain axis may affect the degradation rate. Accordingly, the disappearance of filament is faster than that of lamellar crystal. The enzymatic degradation progresses preferentially from the defective regions within the crystal. Even if the appearance of a filament is well-defined, there will exist many structural defects in the shish along its long axis,14,15 and the chains in the filament are not likely to extended fully.21,22 The starting of erosion by the enzyme will be preferentially from such defective sites in the filament. Conclusions The TEM and AFM results demonstrate that the uniaxially oriented thin film of ultrahigh molecular weight P(3HB) can be prepared by shearing and subsequent annealing (crystallization) and the chain axis (crystallographic c-axis) is parallel to the shearing direction. In the oriented thin film with about 100 nm thickness, fibrils of 100-200 nm in width running parallel to the shearing direction and rodlike structure, that is, folded-chain lamellar crystals with edge-on manner overgrown on the fibrils normal to the shearing direction, were recognized. In situ AFM observations of enzymatic degradation of the P(3HB) oriented thin film by P(3HB) depolymerase from R. pickettii T1 were successful. The degradation behavior has indicated that the surface topography become rough gradually and the film thickness decreases, suggesting that the amorphous phase is more preferentially eroded than the crystalline one so that the fine shish-kebab structure is observed clearly during enzymatic degradation. In addition, it is clarified that the fibrils of 100-
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200 nm in width which are observed before the degradation consist of finer filaments and edge-on lamellar crystals. The finer filaments pass through many edge-on lamellae. The degradation rate of the filament was relatively fast. Most of the filaments connecting adjacent lamellae disappear prior to the disappearance of the lamellae by enzymatic degradation. This fact implies that the existence of defective sites in the filament, in addition to the effect of crystal size, causes the fast degradation rate of the filament. Acknowledgment. This research was supported by grants for Ecomolecular Science Research from RIKEN Institute and for SORST (Solution Oriented Research for Science and Technology) from the Japan Science and Technology Agency (JST). References and Notes (1) Doi, Y. Microbial Polyesters; VCH: New York, 1990. (2) Sudesh, K.; Abe, H.; Doi, Y. Prog. Polym. Sci. 2000, 25, 15031555. (3) Gerngross, T. U.; Martin, D. P. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6279-6283. (4) Kusaka, S.; Abe, H.; Lee, S. Y.; Doi, Y. Appl. Microbiol. Biotechnol. 1997, 47, 140-143. (5) Aoyagi, Y.; Doi, Y.; Iwata, T. Polym. Degrad. Stab. 2003, 79, 209216. (6) Pennings, A. J.; Kiel, A. M. Kolloid. Z. Z. Polym. 1965, 205, 160162. (7) Keller, A.; Machin, M. J. J. Macromol. Sci., Phys. 1967, B1, 41-91. (8) Wikjord, A. J.; Manley, R. St. J. J. Macromol. Sci., Phys. 1968, B2, 501-537. (9) Pennings, A. J.; van der Mark, J. M. A. A.; Kiel, A. M. Kolloid. Z. Z. Polym. 1969, 237, 336-358. (10) Hill, M. J.; Keller, A. J. Macromol. Sci., Phys. 1969, B3, 153-169. (11) Petermann, J.; Gleiter, H. J. Polym. Sci., Polym. Lett. 1977, 15, 649654. (12) Petermann, J.; Miles, M. J. Polym. Sci., Polym. Phys. 1979, 17, 55-62. (13) Hobbs, J. K.; Humphris, A. D. L.; Miles, M. J. Macromolecules 2001, 34, 5508-5519. (14) Liu, T. X.; Lieberwirth, I.; Petermann, J. Macromol. Chem. Phys. 2001, 202, 2921-2925. (15) Liu, T. X.; Tjiu, W. C.; Petermann, J. J. Cryst. Growth 2002, 243, 218-223. (16) Tsuji, M.; Kohjiya, S. Prog. Polym. Sci. 1995, 20, 259-308. (17) Puiggali, J.; Ikada, Y.; Tsuji, H.; Cartier, L.; Okihara, T.; Lotz, B. Polymer 2000, 41, 8921-8930. (18) Yoshioka, T.; Tsuji, M.; Kawahara, Y.; Kohjiya, S. Polymer 2003, 44, 7997-8003. (19) Kumaraswamy, G.; Issaian, A. M.; Kornfield, J. A. Macromolecules 1999, 32, 7537-7547. (20) Somani, R. H.; Hsiao, B. S.; Nogales, A.; Srinivas, S.; Tsou, A. H.; Sics, I.; Balta-Calleja, F. J.; Ezaquerra, T. A. Macromolecules 2000, 33, 9385-9394. (21) Seki, M.; Thurman, D. W.; Oberhauser, J. P.; Kornfield, J. A. Macromolecules 2002, 35, 2583-2594. (22) Somani, R. H.; Yang, L.; Hsiao, B. S.; Agarwal, P. K.; Fruitwala, H. A.; Tsou, A. H. Macromolecules 2002, 35, 9096-9104. (23) Abe, H.; Doi, Y.; Aoki, H.; Akehata, T. Macromolecules 1998, 31, 1791-1797. (24) Kusaka, S.; Iwata, T.; Doi, Y. Int. J. Biol. Macromol. 1999, 25, 87-94. (25) Rossini, C. J.; Arceo, J. F.; McCarney, E. R.; Augustine, B. H. Macromol. Symp. 2001, 167, 139-151. (26) Kikkawa, Y.; Murase, T.; Abe, H.; Iwata, T.; Inoue, Y.; Doi, Y. Macromol. Biosci. 2002, 2, 189-194. (27) Saito, T.; Suzuki, K.; Yamamoto, J.; Fukui, T.; Miwa, K.; Tomita, K.; Nakanishi, S.; Odani, S.; Suzuki, J.; Ishikawa, K. J. Bacteriol. 1989, 171, 184-189. (28) Petermann, J.; Gleiter, H. Philos. Mag. 1975, 31, 929-934. (29) Yokouchi, M.; Chatani, H.; Tadokoro, H.; Teranishi, K.; Tani, H. Polymer 1973, 14, 267-272. (30) Orts, W. J.; Marchessault, R. H.; Bluhm, T. L.; Hamer, G. K. Macromolecules 1990, 23, 5368-5370.
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