Nonhydrolytic Fragmentation of a Poly[(R)-3

C., Ed.; Elsevier Applied Science: London and New York, 1988;. Vol. .... Kilburn, D. G. Bio/Technology 1991, 9, 1096. ... R.; Warren, J.; Kilburn, D. ...
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Biomacromolecules 2002, 3, 312-317

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Nonhydrolytic Fragmentation of a Poly[(R)-3-hydroxybutyrate] Single Crystal Revealed by Use of a Mutant of Polyhydroxybutyrate Depolymerase Tomohide Murase,†,‡ Yoichi Suzuki,† Yoshiharu Doi,†,§ and Tadahisa Iwata*,† Polymer Chemistry Laboratory, RIKEN Institute, 2-1, Hirosawa, Wako-shi, Saitama 351-0198, Japan, and Department of Innovative and Engineered Materials, Tokyo Institute of Technology, 4259, Nagatsuta, Midori-ku, Yokohama 226-8502, Japan Received August 27, 2001; Revised Manuscript Received November 21, 2001

This paper reports the initial process of the enzymatic degradation of solution-grown lamellar single crystals of bacterial poly[(R)-3-hydroxybutyrate] (P(3HB)) with an extracellular polyhydroxybutyrate (PHB) depolymerase purified from Alcaligenes faecalis T1. We used a hydrolytic-activity-disrupted mutant of the PHB depolymerase in order to avoid the influence of hydrolytic reaction in the system. The effect of addition of the mutant enzyme upon the P(3HB) single crystals was investigated by turbidimetric assay, high-performance liquid chromatography (HPLC), and atomic force microscopy (AFM). Suspension turbidity of the P(3HB) single crystals increased after addition of the mutant enzyme having no hydrolytic activity. No soluble product from the P(3HB) single crystals with the mutant enzyme was detected by HPLC. AFM observation of the P(3HB) single crystals adsorbed on highly ordered pyrolytic graphite revealed that the mutant enzyme yielded a lot of lengthwise crystal fragments from the P(3HB) single crystals. On the basis of these results, we concluded that the mutant enzyme disturbs the molecular packing of the P(3HB) polymer chain around the loose chain packing region in the single crystal, resulting in the fragmentation. Therefore, it is suggested that the enzymatic degradation of P(3HB) single crystals with a wild-type PHB depolymerase progresses via three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chain in the single crystal by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme. Introduction Poly[(R)-3-hydroxybutyrate] (P(3HB)), which is accumulated in a large number of bacteria as intercellular carbon and energy storage material,1-3 is a biodegradable thermoplastic. Bacterial P(3HB) is degraded by many types of extracellular polyhydroxybutyrate (PHB) depolymerases, which are secreted from a number of microorganisms.4 The enzymatic degradation mechanism of P(3HB) material with the PHB depolymerases has been extensively studied from the viewpoint of both enzymatic function of the PHB depolymerase and chemical and solid structures of P(3HB) material.5 Primary structures of several PHB depolymerases have been documented.4,6,7 In all cases, PHB depolymerases have a modular structure that is composed of a catalytic domain, which is responsible for hydrolysis of the P(3HB) polymer chain, and a substrate binding domain, which allows adsorption of the enzyme on the surface of P(3HB) material. The * To whom all correspondence should be addressed. Telephone: +8148-467-9586. Fax: +81-48-462-4667. E-mail: [email protected]. † Polymer Chemistry Laboratory, RIKEN Institute. ‡ Present address: “Nanotechnology” Laboratory, MCC-Group Science & Technology Research Center, Mitsubishi Chemical Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan. § Department of Innovative and Engineered Materials, Tokyo Institute of Technology.

catalytic domain and the substrate binding domain are connected by a linker peptide. The substrate binding domain confers efficient condensation of the enzyme onto the surface of P(3HB) material. Then, the catalytic domain can readily access the surface of P(3HB) material with help from the flexible linker peptide. Accordingly, the catalytic domain can then hydrolyze the P(3HB) polymer chain efficiently. The structure and properties of PHB depolymerase purified from Alcaligenes faecalis T1 (PhaZAfa) has been extensively investigated. Enzyme activity and hydrolytic function of the PhaZAfa has been studied in detail.8-11 An active center site in the catalytic domain of the PhaZAfa was identified to be 139Ser (serine residue at position 139).12 In addition, adsorption behavior of the PhaZAfa with the substrate binding domain was reported.13-15 Many researchers have investigated the enzymatic degradation mechanism of solution-grown P(3HB) single crystals with PHB depolymerases by using transmission electron microscopy16-19 and atomic force microscopy.20,21 The combination of P(3HB) single crystals and the PhaZAfa is a prominent system for elucidating the enzymatic degradation mechanism at the molecular level,19-21 because both the single crystals and the enzyme are well-characterized. In our recent papers,20,21 we demonstrated that P(3HB) single crystals are converted into lathlike fingers and crystal fragments parallel to the long axis of the crystal during the

10.1021/bm015604p CCC: $22.00 © 2002 American Chemical Society Published on Web 01/10/2002

Enzymatic Degradation of P(3HB)

enzymatic degradation. We interpreted the characteristic degradation texture from the point of the molecular packing state of P(3HB) polymer chain in the single crystal. In this paper, we attempt to obtain more insight on the initial process of enzymatic degradation of solution-grown P(3HB) single crystals by PhaZAfa. To examine the initial process without the influence of hydrolysis in the system, a hydrolytic-activity-disrupted mutant of the PhaZAfa, in which the original 139Ser was replaced by an alanine (Ala), is used. The effect of addition of the mutant enzyme upon the P(3HB) single crystals is investigated by turbidimetric assay, highperformance liquid chromatography, and atomic force microscopy. The results obtained from the mutant enzyme are compared with those obtained from a wild-type PhaZAfa enzyme. Experimental Section Materials. The wild-type and the mutant PhaZAfa enzymes were purified from recombinant Escherichia coli JM 109 carrying plasmids pDP14 or pDP20, respectively,12,22 according to a reported method.23 P(3HB) hydrolysis activities of the wild-type and the mutant enzymes were assayed by suspension turbidity of PHB granules as reported previously.24 A 3.2 µg portion of either the wild type or the mutant enzyme was injected into 2 mL of P(3HB) granule suspension. The turbidity at 660 nm (OD660) was measured spectrophotometrically at 37 °C with stirring. Bovine serum albumin (BSA) was purchased from Sigma Chemical Co. (St. Louis, MO) and was used as received. Bacterial P(3HB) (number-average molecular weight (Mn) and polydispersity (DPI) were 358 000 and 2.8, respectively) was purchased from Aldrich Chemical Co., Ltd. (Milwaukee, WI). The P(3HB) sample used in this study was prepared from the bacterial P(3HB) by alkali hydrolysis as reported previously.18 Single crystals of P(3HB) (Mn and DPI were 45 000 and 1.6, respectively) were grown from dilute solution (0.025 wt %) of 1-octanol under isothermal crystallization conditions. After crystallization, P(3HB) single crystals were collected by centrifugation, washed several times with methanol, and then suspended in methanol. Turbidimetric Assay. The effect of addition of the wild type or the mutant enzyme upon P(3HB) single crystals which were suspended in buffer solution was monitored using a turbidimetric assay reported previously.16,18,19 For the assay, the P(3HB) single crystals suspended in methanol were collected by centrifugation, washed twice with 1 mL of 50 mM Tris-HCl buffer solution (pH 7.5), and resuspended in the same buffer solution (∼0.4 mg/mL). A 1.6 µg portion of either the wild type or the mutant enzyme was injected into 1 mL of the single crystal suspension. The OD660 was measured spectrophotometrically at 37 °C without stirring. Product Analysis. After the turbidimetric assay, soluble products in the buffer solution were analyzed using an LC7A high-performance liquid chromatography (HPLC) system (Shimadzu Corporation, Kyoto, Japan) with a gradient controller and an SPD-10A UV spectrophotometric detector according to the literature.24 The postassay suspension was filtered, and the resulting solution was poured into a dilute

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Figure 1. Hydrolytic activity of PhaZAfa enzyme against a suspension of P(3HB) granules measured at 37 °C: (b) wild-type enzyme; (O) mutant enzyme; (0) buffer only.

HCl solution (pH 2.5). A 50 µL aliquot of the sample was injected into the LiChrospher RP-8 column. The gradient from distilled water (adjusted to pH 2.5 with HCl solution) to acetonitrile was carried out for 40 min with a pump speed of 1.0 mL/min at 40 °C. The products were detected at 210 nm. Atomic Force Microscopy Measurement. Morphological changes of the P(3HB) single crystals after incubation with the wild-type or the mutant enzyme were observed by atomic force microscopy (AFM). For the AFM measurement, the P(3HB) single crystals were deposited on the surface of highly ordered pyrolytic graphite (HOPG; Nihon Veeco K.K., Japan) as reported previously.20,21 The deposition of the P(3HB) single crystals onto HOPG was carried out by immersing freshly cleaved HOPG into a suspension of P(3HB) single crystals in methanol. A 1.6 µg portion of either the wild type or the mutant enzyme was injected into 1 mL of 50 mM Tris-HCl buffer solution in a transparent plastic cuvette containing P(3HB) single crystals on HOPG. The cuvette was incubated at 37 °C without stirring. After 120 min, the sample was removed, rinsed several times with distilled water and methanol, and dried in air. AFM measurements were performed with an SPA400/SPI3800N atomic force microscope (Seiko Instruments Inc., Japan) operating in the dynamic force microscope mode. A 20 µm scanner (maximum scan range ca. 24 µm) and a rectangular silicon cantilever (Si-DF20, Seiko Instruments Inc.; 200 µm in length, resonant frequency of ca. 134 kHz, stiffness of ca. 16 N/m) was applied in all experiments. All measurements were carried out in ambient conditions at room temperature. The resulting images were flattened and plane-fit using Seiko Instruments software. Results Characterization of Wild-Type and Mutant PhaZAfa Enzymes. The wild-type and the mutant PhaZAfa enzymes were purified to electrophoretic homogeneity. Apparent molecular weights of both the wild-type and the mutant enzymes were ca. 47 kDa. No difference in secondary structure profile between the wild-type and the mutant enzymes was observed by circular dichroism spectra. Figure 1 shows hydrolytic activity of the wild-type and the mutant

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Figure 2. Time-dependent changes in turbidity of a suspension of P(3HB) single crystals measured at 37 °C: (b) wild-type enzyme; (O) mutant enzyme; (+) BSA; (0) buffer only.

enzymes against P(3HB) granules (about 0.5 µm in diameter) at 37 °C. Upon addition of the enzyme, the turbidity rapidly decreased with the wild-type enzyme, whereas it remained unchanged with the mutant enzyme. The turbidity was also unchanged in the absence of enzyme during the measurement (control experiment). Thus, the mutant enzyme had no hydrolytic activity against the P(3HB) granules. Incubation of P(3HB) Single Crystals with Wild-Type or Mutant of PhaZAfa. Figure 2 shows turbidimetric profiles for the suspension of P(3HB) single crystals at 37 °C with the wild-type enzyme, with the mutant enzyme, and in the absence of enzyme. The turbidity drastically decreased after addition of the wild-type enzyme. It indicated that the wildtype enzyme degraded and hydrolyzed the suspended P(3HB) single crystals into soluble products. Interestingly, the turbidity slightly increased after addition of the mutant enzyme. It appeared that the mutant enzyme produced insoluble and fine substances from the suspended P(3HB) single crystals. Turbidity remained unchanged for 120 min in the absence of enzyme. Even if the same amount of bovine serum albumin (BSA) was added instead of the wild-type or the mutant enzyme, turbidity remained unchanged for 120 min. Furthermore, the turbidity was unchanged after addition of heat-inactivated PhaZafa enzyme (data not shown). All these results indicated that the mutant enzyme brought about the increase in turbidity during the measurement. After the turbidimetric assay, soluble products in the buffer solution were analyzed by HPLC. Figure 3 shows typical HPLC curves of the soluble products with the wild-type enzyme, with the mutant enzyme, and in the absence of enzyme. Two characteristic peaks were observed with the wild-type enzyme. The peak components at retention times of 12.0 and 16.5 min were assigned to dimeric and trimeric esters of (R)-3-hydroxybutanoic acid, respectively.24 The results indicated that the wild-type enzyme hydrolyzed P(3HB) polymer chains in the single crystals, yielding dimers and trimers as soluble products.8-11 On the other hand, no noticeable peak was detected with the mutant enzyme or in the absence of enzyme. Therefore, it was confirmed that the wild-type enzyme hydrolyzed P(3HB) polymer chains in the single crystals whereas the mutant enzyme did not hydrolyze P(3HB) polymer chains in the single crystals during the turbidimetric assay.

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Figure 3. HPLC chromatograms of soluble products from suspension of P(3HB) single crystals after turbidimetric assay with PhaZAfa enzyme at 37 °C for 120 min: (a) wild-type enzyme; (b) mutant enzyme; (c) buffer only. The two peaks in (a) at retention times of 12.0 and 16.5 min were assigned to dimeric and trimeric esters of (R)-3-hydroxybutanoic acid, respectively.

To clarify the reason of an increase in turbidity caused by the mutant enzyme, AFM observation was performed on P(3HB) single crystals before and after incubation with the wild-type enzyme, with the mutant enzyme, and in the absence of enzyme. P(3HB) single crystals adsorbed on HOPG were used for the AFM observation because morphological change of a P(3HB) single crystal after the enzymatic degradation could be clearly observed without an influence of physical disruption.20,21 Figure 4a shows an AFM image of P(3HB) single crystals on HOPG before the incubation. The P(3HB) single crystals had multilamellar lath-shaped morphology, and the thickness of the monolamellar part of the single crystals was ∼5 nm. The surface and edges of the P(3HB) single crystals were smooth. It was reported that P(3HB) single crystals have a chain-folding surface, and the long axes of the crystals correspond to both crystallographic a-axis and the average chain folding direction.25,26 The dimensions of the P(3HB) single crystals were 0.3-2 µm in width and 5-15 µm in length by optical microscopy. Figure 4b shows an AFM image of P(3HB) single crystals on HOPG after incubation with the wild-type enzyme at 37 °C for 120 min. The P(3HB) single crystals were degraded by the wild-type enzyme, forming lengthwise crystal fragments separated by cracks.20,21 The width of each fragment was about 50 nm. The lamellar thickness remained unchanged during the enzymatic degradation. No morphological change of P(3HB) single crystals was observed after incubation in the absence of the wild-type enzyme at 37 °C for 120 min. Thus, it was confirmed that the morphological changes in Figure 4b were caused by the action of the wildtype enzyme. The characteristic degradation texture of the P(3HB) single crystals can be interpreted as predominant degradation of inherent straight degradation pathways parallel to the long axis of the crystal.20 The straight degradation pathway probably corresponds to loose chain packing regions in the P(3HB) single crystals. We speculated that the straight degradation pathway was recorded in the single crystals as a history line in the crystal growth process in solution.20,21 Figure 5 shows AFM images of P(3HB) single crystals on HOPG after incubation with the mutant enzyme at 37 °C

Enzymatic Degradation of P(3HB)

Figure 4. AFM images of P(3HB) single crystals adsorbed on a surface of HOPG (a) before and (b) after enzymatic degradation with wild-type PhaZAfa enzyme at 37 °C for 120 min. The images show (a) multilamellar P(3HB) single crystals which have lath-shaped morphology and (b) long and narrow crystal fragments parallel to the long axis of the crystal formed during the enzymatic degradation with the wild-type enzyme. The arrow in each image indicates the long axis of the crystal, which corresponds to both the crystallographic a-axis and the average chain-folding direction.

for 120 min. In Figure 5a, a lot of long and narrow crystal fragments were observed at the surface. In the absence of the mutant enzyme, the morphology of P(3HB) single crystals on HOPG remained unchanged under identical incubation conditions. Therefore, it was concluded that the crystal fragments were produced from the P(3HB) single crystals due to the action of the mutant enzyme. The width of the crystal fragments was 50-200 nm, and the thickness was ∼5 nm. Sometimes these crystal fragments could not be clearly imaged by AFM, because they might weakly adsorb on the surface and be moved easily by the scanning cantilever. Figure 5b shows the ends of P(3HB) single crystals. The edges of the P(3HB) single crystals became milled, and cracks parallel to the long axes of the crystal were created. In other words, the ends of the P(3HB) single crystals were also fragmented by the mutant enzyme. The interval between cracks, that is, the width of the fragment, was ∼50 nm. The thickness of the single crystals was ∼5 nm. It was suggested that the long and narrow crystal fragments in Figure 5a were

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Figure 5. AFM images of P(3HB) single crystals adsorbed on a surface of HOPG after incubation with the mutant PhaZAfa enzyme at 37 °C for 120 min. The images show (a) the formation of many crystal fragments from P(3HB) single crystals and (b) ends of P(3HB) single crystals having cracks parallel to the long axes of the crystals. The arrow in (b) indicates the long axis of the crystal.

cut off from the P(3HB) single crystals by the formation of the cracks parallel to the long axis of the crystal, which were produced by the mutant enzyme. Discussion The fragmentation of P(3HB) single crystals with the mutant enzyme in Figure 5 is likely to lead to an increase in the turbidity of the P(3HB) single crystal suspension with the mutant enzyme in Figure 2. When crystal fragments are released from P(3HB) single crystals by the action of the mutant enzyme, a larger number of smaller single crystals are dispersed into the suspension, resulting in the increase in turbidity of the suspension. Thus, we have concluded that the mutant enzyme adsorbed on the surface of the single crystals fragments P(3HB) single crystals. It should be emphasized that the fragmentation is not related to enzymatic hydrolysis of P(3HB) polymer chain in this system. The width of the crystal fragments formed at the end of the P(3HB) single crystals during incubation with the mutant enzyme (Figure 5b) is very close to that formed during the enzymatic degradation with the wild-type enzyme (Figure

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Figure 6. Schematic representation of nonhydrolytic fragmentation of P(3HB) single crystal by the mutant PhaZAfa enzyme (top view).

4b). Because P(3HB) polymer chains pack loosely around the straight degradation pathway,20,21 it is suggested that the mutant enzyme that adsorbs on the surface of P(3HB) single crystals efficiently disturbs molecular packing of the P(3HB) polymer chain around the straight degradation pathway in the single crystals. The disturbance could weaken van der Waals interaction between the P(3HB) polymer chains in the single crystals, leading to the fragmentation of the single crystals or the formation of cracks. Figure 6 shows a schematic representation of the nonhydrolytic fragmentation of a P(3HB) single crystal by the adsorbed mutant enzyme. The gray line parallel to the long axis of the crystal represents the straight degradation pathway in the single crystals. The adsorbed mutant enzyme efficiently disturbs the molecular packing of the P(3HB) polymer chain around the straight degradation pathway, yielding a lengthwise narrow crystal fragment and cracks parallel to the long axis of the crystal. The difference between the wild-type and the mutant enzyme is only the amino acid substitution from 139Ser to Ala at the active center site in the catalytic domain. Therefore, we have concluded that the adsorbed wild-type enzyme also disturbs molecular packing of P(3HB) polymer chains in the single crystals on the initial process of the enzymatic degradation. It is widely accepted that the enzymatic degradation of P(3HB) single crystals by PHB depolymerase involves two steps: (1) adsorption of the enzyme onto a surface of the single crystal; and (2) hydrolysis of the P(3HB) polymer chain by the adsorbed enzyme.27 However, according to the finding in this study, the enzymatic degradation of P(3HB) single crystals by the wild-type PhaZAfa enzyme involves three steps: (1) adsorption of the enzyme onto the surface of the single crystal; (2) disturbance of the molecular packing of P(3HB) polymer chains around the straight degradation pathway in the single crystals by the adsorbed enzyme; and (3) hydrolysis of the disturbed polymer chain by the adsorbed enzyme. The disturbance between the enzyme molecule and the P(3HB) polymer chains in the single crystals can be speculated in terms of the modular structure of PhaZAfa

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enzyme.28,29 The catalytic domain of the mutant enzyme may retain its binding ability toward the P(3HB) polymer chain, although the catalytic domain loses hydrolytic activity. Such nonproductive complex formation between the catalytic domain and P(3HB) polymer chain that is exposed near the surface of the single crystal may bring about the disturbance of the molecular packing of the P(3HB) polymer chain in the single crystal. On the other hand, Nojiri et al. speculated that the linker peptide of the PhaZAfa may have a role in disrupting the crystalline structure on the surface of crystalline P(3HB) when the linker peptide is located next to the catalytic domain.30 In the field of enzymatic degradation of cellulose by a cellulase, several researchers reported that a binding domain of cellulase enhances the physical disruption of cellulose fibers and releases small particles from cotton fibers.31-33 The cellulose binding domain functions for adsorption of the cellulase on the substrate surface. By analogy, the substrate binding domain of the PhaZAfa may cause the disturbance of molecular packing of P(3HB) polymer chains in the single crystal due to adsorption. It would be of interest to address which part of the PhaZAfa plays a key role for the disturbance of molecular packing of P(3HB) polymer chain in the single crystal and how it works. Conclusions In this paper, we discussed the initial process of the enzymatic degradation of a solution-grown P(3HB) single crystal with an extracellular PHB depolymerase purified from A. faecalis T1 (PhaZAfa). To avoid the influence of hydrolytic reaction in the system, a hydrolytic-activity-disrupted mutant of the PhaZAfa enzyme was used in this study. On the basis of the results including direct observation by AFM, we have concluded that the mutant enzyme fragments the P(3HB) single crystals. The mutant enzyme adsorbed on the surface of a P(3HB) single crystal may disturb molecular packing of P(3HB) polymer chain around a straight degradation pathway corresponding to a loose chain packing region in the single crystal. The disturbance would be involved in the initial process of the enzymatic degradation of P(3HB) single crystal by the wild-type PhaZAfa enzyme. The enzymatic degradation of P(3HB) single crystal by PhaZAfa progresses via three steps: (1) adsorption, (2) disturbance, and (3) hydrolysis. Acknowledgment. We are indebted to Professor T. Saito (Kanagawa University, Japan) for kind gifts of plasmids pDP14 and pDP20 used in this work. We are grateful to Dr. S. Taguchi for countless discussions and useful suggestions. T.M. is a recipient of Special Postdoctoral Researchers Program of RIKEN Institute. This work was supported by the grant for Ecomolecular Science Research, RIKEN Institute Japan and a Grant-in-Aid for Scientific Research on a Priority Area (Sustainable Biodegradable Plastics, No. 11217216 (2001)) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. References and Notes (1) Doi, Y. Microbial Polyesters; VCH Publishers: New York, 1990.

Enzymatic Degradation of P(3HB) (2) Holmes, P. A. In DeVelopments in Crystalline Polymers; Bassett, D. C., Ed.; Elsevier Applied Science: London and New York, 1988; Vol. 2, pp 1-65. (3) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. (4) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451. (5) Sudesh, K.; Abe, H.; Doi, Y. Prog. Polym. Sci. 2000, 25, 1503. (6) Kita, K.; Mashiba, S.; Nagata, M.; Ishimaru, K.; Okamoto, K.; Yanase, H.; Kato, N. Biochim. Biophys. Acta 1996, 1352, 113. (7) Kasuya, K.; Inoue, Y.; Tanaka, T.; Akehata, T.; Iwata, T.; Fukui, T.; Doi, Y. Appl. EnViron. Microbiol. 1997, 63, 4844. (8) Tanio, T.; Fukui, T.; Shirakura, Y.; Saito, T.; Tomita, K.; Kaiho, T.; Masamune, S. Eur. J. Biochem. 1982, 124, 71. (9) Shirakura, Y.; Fukui, T.; Saito, T.; Okamoto, Y.; Narikawa, T.; Koide, K.; Tomita, K.; Takemasa, T.; Masamune, S. Biochim. Biophys. Acta 1986, 880, 46. (10) Bachmann, B. M.; Seebach, D. Macromolecules 1999, 32, 1777. (11) Scherer, T. M.; Fuller, R. C.; Goodwin, S.; Lenz, R. W. Biomacromolecules 2000, 1, 577. (12) Shinohe, T.; Nojiri, M.; Saito, T.; Stanislawski, T.; Jendrossek, D. FEMS Microbiol. Lett. 1996, 141, 103. (13) Shinomiya, M.; Iwata, T.; Doi, Y. Int. J. Biol. Macromol. 1998, 22, 129. (14) Kasuya, K.; Ohura, T.; Masuda, K.; Doi, Y. Int. J. Biol. Macromol. 1999, 24, 329. (15) Yamashita, K.; Aoyagi, Y.; Abe, H.; Doi, Y. Biomacromolecules 2001, 2, 25. (16) Hocking, P. J.; Marchessault, R. H.; Timmins, M. R.; Lenz, R. W.; Fuller, R. C. Macromolecules 1996, 29, 2472. (17) Nobes, G. A. R.; Marchessault, R. H.; Chanzy, H.; Briese, B. H.; Jendrossek, D. Macromolecules 1996, 29, 8330.

Biomacromolecules, Vol. 3, No. 2, 2002 317 (18) Iwata, T.; Doi, Y.; Kasuya, K.; Inoue, Y. Macromolecules 1997, 30, 833. (19) Iwata, T.; Doi, Y.; Tanaka, T.; Akehata, T.; Shiromo, M.; Teramachi, S. Macromolecules 1997, 30, 5290. (20) Murase, T.; Iwata, T.; Doi, Y. Macromolecules 2001, 34, 5848. (21) Murase, T.; Iwata, T.; Doi, Y. Macromol. Biosci. 2001, 1, 275. (22) 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. (23) Suzuki, Y.; Taguchi, S.; Saito, T.; Toshima, K.; Matsumura, S.; Doi, Y. Biomacromolecules 2001, 2, 541. (24) Hiraishi, T.; Ohura, T.; Ito, S.; Kasuya, K.; Doi, Y. Biomacromolecules 2000, 1, 320. (25) Birley, C.; Briddon, J.; Sykes, K. E.; Barker, P. A.; Organ, S. J.; Barham, P. J. J. Mater. Sci. 1995, 30, 633. (26) Iwata, T.; Doi, Y.; Kokubu, F.; Teramachi, S. Macromolecules 1999, 32, 8325. (27) Mukai, K.; Yamada, K.; Doi, Y. Int. J. Biol. Macromol. 1993, 15, 361. (28) Fukui, T.; Narikawa, T.; Miwa, K.; Shirakura, Y.; Saito, T.; Tomita, K. Biochim. Biophys. Acta 1988, 952, 164. (29) Saito, T.; Iwata, A.; Watanabe, T. J. EnViron. Polym. Degrad. 1993, 1, 99. (30) Nojiri, M.; Saito, T. J. Bacteriol. 1997, 179, 6965. (31) Din, N.; Gilkes, N. R.; Tekant, B.; Miller, R. C. J.; Warren, R. A. J.; Kilburn, D. G. Bio/Technology 1991, 9, 1096. (32) Henrissat, B. Cellulose 1994, 1, 169. (33) Tomme, P.; Driver, D. P.; Amandoron, E. A.; Miller, R. C. J.; Antony, R.; Warren, J.; Kilburn, D. G. J. Bacteriol. 1995, 177, 4356.

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