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Enzymatic Hydrolysis of Chemosynthesized Atactic Poly(3-hydroxybutyrate) by Poly(3-hydroxyalkanoate) Depolymerase from Acidovorax Sp. TP4 and Ralstonia pickettii T1 Yi Wang,† Yasuhide Inagawa,† Yasushi Osanai,‡ Ken-ichi Kasuya,§ Terumi Saito,| Shuichi Matsumura,‡ Yoshiharu Doi,†,⊥ and Yoshio Inoue*,† Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan, and The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama 351-0198, Japan Received April 29, 2002; Revised Manuscript Received July 4, 2002
The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZral) and AcidoVorax Sp. TP4 (PhaZaci), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZaci depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension 1H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZaci depolymerase at temperatures ranging from 4 to 20 °C, while a-P(3HB) could hardly be hydrolyzed by PhaZral depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase. Bacterial poly([R]-3-hydroxybutyrate) [P(3HB)] with isotactic structure is synthesized and accumulated by a variety of bacteria as a reserve energy source.1,2 A remarkable characteristic of P(3HB) is its biodegradability in various environments. The atactic poly([R, S]-3-hydroxybutyrate) [a-P(3HB)] chemosynthesized by anionic polymerization of β-butyrolactone is an amorphous polymer.3,4 It has been known that the chemosynthetic a-P(3HB) cannot be hydrolyzed in the pure state by the extracellular P(3HB) depolymerases, while when the amorphous a-P(3HB) physically blended with crystalline poly(3-hydroxyalkanoate) (PHA), e.g., bacterial P(3HB)5 and natural poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)],6 the segments of a-P(3HB) could be enzymatically hydrolyzed by P(3HB) depolymerase from Pseudomonas pickettii belonging to the type I5 and that from P. lemoignei belonging to the type III,6-8 respectively. Furthermore, the existence of some other biodegradable crystalline polymers such as poly(-caprolactone) (PCL) and poly(L-lactic acid) (PLLA)9 and in diblock copolymer of a-P(3HB) and crystalline poly(pivalolactone) (PPVL),10 a-P(3HB) was found to be degraded by P(3HB) depolymerase from P. lemoignei. It is noteworthy that PCL, PLLA, †
Tokyo Institute of Technology. Keio University. § Gunma University. | Kanagawa University. ⊥ The Institute of Physical and Chemical Research (RIKEN). ‡
and PPVL are not hydrolyzed by the P(3HB) depolymerases. The phenomenon that a-P(3HB) segments alone were not hydrolyzed but biodegraded in the blends or in the diblock polymer by the P(3HB) depolymerase was called crystallineinduced biodegradation.10 Recently, the enzymatic degradation behavior of a-P(3HB) in binary blends with amorphous polymer with high glass transition temperature, e.g., poly(methyl methacrylate) (PMMA)11,12 and atactic poly(lactide) (a-PLA),13 had also been studied. It was found that the enzymatic degradation of a-P(3HB) can be induced by blending with amorphous polymers which heighten the glass transition temperature of a-P(3HB). The P(3HB) depolymerase from Ralstonia picketti (previously named as Alcaligenes faecalis T1) which belongs to the type I was used in these studies.11-13 On the other hand, many kinds of extracellular P(3HB) depolymerases have been purified from various microorganisms and their enzymatic properties have been characterized.8 All extracellular P(3HB) depolymerases have composite domain structures and consist of a signal peptide segment, a large catalytic domain at the N terminus, a C-terminal substrate-binding domain, and a linking domain between catalytic and binding domains. Analyses of the structure genes of PHA depolymerases and the kinetics of enzymatic hydrolysis of P(3HB) film revealed that the surface hydrolysis of P(3HB) film proceeds through a two-step enzymatic reaction. The first step is the adsorption of enzyme on the surface of P(3HB) film via the binding domain and the
10.1021/bm020052b CCC: $22.00 © 2002 American Chemical Society Published on Web 07/27/2002
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second is the hydrolysis of the polymer chain into watersoluble products by the catalytic domain of the depolymerase.14-16 That is, if there existed another polymer which could provide the stable binding sites for PHA depolymerase, a-P(3HB) could be hydrolyzed by the catalytic domain of the depolymerase. Among the various extracellular PHA depolymerases isolated from a variety of bacteria, the properties of PHA depolymerase purified from R. pickettii T1 (PhaZral) have been studied in great detail.14,17-20 The rate of enzymatic hydrolysis depended on the sequential structures of polyester chain and the crystallinity of the polyester films.19,21-23 Usually, the maximum degradation rates of PHA copolymers were observed at the fraction with the second monomer content of about 20-30 mol % when hydrolyzed by PhaZral depolymerase.17,22 However, it has been found recently for a series of poly(3-hydroxybutyrate-co-3-hydroxypropionate) with different 3-hydroxypropionate (3HP) contents that the decrease of crystallinity leads an increase in the hydrolysis rate by P(3HB) depolymerase PhaZaci isolated from AcidoVorax Sp. TP4 independent of the 3HP unit content. That is, PhaZaci hydrolyzed PHA films with a low degree of crystallinity at a much high rate than PhaZral.24 The sequence of PhaZaci has been studied and defined as type II PHA depolymerase,25 while PhaZral defined as type I by differences in the linker domain structure or in the position of the lipase box in the catalytic domain.8 Considering this property of PhaZaci PHA depolymerase, it is quite reasonable to expect that PhaZaci could degrade amorphous a-P(3HB) in the pure state. In this work, the enzymatic hydrolysis behavior of a-P(3HB) by PhaZaci is studied and the results will be compared with those by PhaZral. Bacterial P(3HB) [b-P(3HB)] were biosynthesized from sucrose by one-stage fermentation with Alcaligenes latus (ATCC 29713) at 30 °C in a fermentor.26 Atactic poly([R,S]3-hydroxybutyrate) [a-P(3HB)] was synthesized through anionic polymerization of racemic β-butyrolactone in bulk with potassium oleate/18-crown-6 ether complex as an initiator.27 The resultant polymer was precipitated twice into methanol from the chloroform solution and dried under vacuum for a week before use. The weight-averaged molecular weight (Mw) and the molecular weight distribution (Mw/Mn) of a-PHB were 5.0 × 104 and 1.1, respectively, while those of b-P(3HB) were 4.5 × 105 and 1.6, respectively. The enzymatic hydrolysis experiments of a-P(3HB) were performed as the films adhering to the Teflon film. The films were aged at room temperature for at least 4 weeks to remove completely the solvent. Two kinds of extracellular PHA depolymerase were used. The extracellular PHA depolymerase coded as PhaZral was purified from R. pickettii T1 according to the method of Shirakura et al.20 The extracellular PHA depolymerase coded as PhaZaci was purified from the cultivation medium of Aci. sp. TP4 according to the method of Kobayashi et al.25 Considering that the optimum conditions of P(3HB) degradation by the PhaZaci depolymerase25 were different from those by PhaZral depolymerase,20 the degradation experiment by the PhaZaci depolymerase was carried out in 50 mM Tris
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Figure 1. Effect of experimental temperature on the enzymatic degradation rate of b-P(3HB) and a-P(3HB).
buffer (pH 8.0), while that by PhaZral has been carried out in 0.1 M potassium phosphate buffer (pH 7.5).17 The activity of PHA depolymerase was measured by the decrease in turbidity of a P(3HB) suspension in aqueous buffer solutions.25 The aqueous solution of PhaZaci depolymerase prepared here had a P(3HB) degradation activity of 50 U/mg, while the degradation activity of PhaZral was 200 u/mg. One unit of enzyme activity is defined as the decrease of the absorbance (at 660 nm) by 0.001 units/min under the assay conditions.19 The polyester film of initial weight about 10 mg and initial dimension 10 × 10 × 0.1 mm was placed in the small bottle containing the given amount of enzyme and 1.0 mL of buffer solution. For each polymer sample, three films were used and the average value of their weight losses was reported as the result. Control tests, carried out for all samples in buffer solution free from the enzyme, showed no appreciable weight losses under the same condition. The degradation products were filtered with a microfilter, then dissolved in deuterated water and lyophilized twice, and then dissolved again in deuterated water (99.96% D2O, Merck Co., USA) before 1H NMR analysis. The surfaces of the films after degradation were observed with a scanning electron microscope (JEOL JSM-5200) after gold coating of the films with an ion coater. The 1H-1H COSY spectra were collected at room temperature using the π/2-t1-π/2-t2 pulse, with 2000 Hz spectral width and 256 data points for t1 axis and 512 data points for t2 axis. The results of the enzymatic degradation of b-P(3HB) and a-P(3HB) by PHA depolymerases isolated from R. picketti T1 (PhaZral) and Aci. Sp. TP4 (PhaZaci) were shown in Figure 1 as degradation rates plotted against the hydrolysis temperature. The enzymatic degradations of a-P(3HB) by PhaZral and PhaZaci and b-P(3HB) by PhaZaci were performed for 10 days, while that of b-P(3HB) by PhaZral was performed for 1 day. As the a-P(3HB) film was supported by the Teflon film, only one side of the a-P(3HB) film was exposed to the enzyme solution. Both surfaces of the b-P(3HB) were exposed to the enzyme solution. Thus, for a comparison, the
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Figure 3. 1D 1H NMR and 2D 1H-1H COSY NMR spectra of watersoluble degradation products from a-P(3HB) after 10 days of hydrolysis by 2 µg of PhaZaci in Tris buffer (pH ) 8.0). Figure 2. The SEM photographs of the a-P(3HB) films after the enzymatic hydrosis at 4 °C for 10 days. The film surface exposed to aqueous solution of enzyme (a and c) and the film surface contacted with Teflon substrate(b) in 50 mM Tris buffer solution (pH 8.0) including 2 µg of PhaZaci (a and b) and in 0.1 M phosphate buffer solution (pH 7.5) including 2 µg of PhaZral (c).
value of weight loss of a-P(3HB) film shown in Figure 1 was twice as much of the experimentally observed weight loss. The hydrolyses rates of b-P(3HB) degraded by PhaZral at the temperature range from 4 to 20 °C were much higher than those rates obtained by PhaZaci. However, the a-P(3HB) films were hardly degraded by PhaZral at the same temperature. The degradation rate of the b-P(3HB) films by PhaZral depolymerase increased with the testing temperature from 4 to 20 °C, while they were degraded by PhaZaci at a similar rate of about 0.07 mg per day (mg day-1) at 15 and 20 °C. Moreover, using PhaZaci depolymerase, a-P(3HB) film was degraded at a rate of about 0.13 mg day-1 at 4 °C, and the rate decreased a little to about 0.10 mg day-1 when the temperature increased to 15 and 20 °C. When the temperature increased to 25 °C, it was hardly for PhaZral depolymerase to degrade the a-P(3HB) films (data not shown). It is noted that in the temperature range from 4 to 20 °C, PhaZaci depolymerase degraded a-P(3HB) faster than b-P(3HB). The surfaces of the a-P(3HB) films were observed under scanning electron microscopy (SEM) (Figure 2) after incubated in buffer solution at 4 °C for 10 days contained PhaZaci or PhaZral depolymerase. It is obvious that the hydrolyzed surface of a-P(3HB) incubated in PhaZaci depolymerase solution became porous. The largest hole had a diameter of about 40-50 µm. On the other hand, no change was observed on the surface contacted with Teflon substrate, and it was considered to be practically the same as that before degrada-
Table 1. The Adsorption of PHA Depolymerase onto the Polyester Films for 15 min with a Initial Enzyme Concentration of 2.0 µg enzyme
temp/°C
a-P(3HB) /µg
b-P(3HB)/µg
PhaZaci PhaZaci PhaZaci PhaZral
4.0 15.0 20.0 4.0
0.32 ( 0.04 0.08 ( 0.02 0.06 ( 0.02 0
0.16 ( 0.02 0.09 ( 0.02 0.08 ( 0.02 0.56 ( 0.01
tion. On the hydrolyzed surface of a-P(3HB) incubated in PhaZral solution, no trace of being eroded was observed. The water-soluble products were investigated by NMR analyses furthermore. Figure 3 shows the 1D 1H NMR and 2D 1H-1H COSY spectra of water-soluble degradation products from a-P(3HB) degraded by PhaZaci at 4 °C for 10 days. In the spectra, the cross-peaks of methine (4.1 ppm) with methylene (2.4 ppm) and methyl (1.2 ppm) of the 3HB monomer, and those of methine (5.2 ppm) with methylene (2.5 ppm) and methyl (1.3 ppm) were observed as previously reported.24 The signals at about 5.3 (1) and 2.7 (2) ppm corresponded to the middle methine and methylene of 3HB trimer were observed in the 1D 1H NMR spectrum. Furthermore, the cross-peak of the mentioned signals was also observed in the 2D 1H-1H COSY spectrum, thus confirmed that trimer was one of the products produced from a-P(3HB) hydrolyzed by PhaZaci. The existence of monomer, dimer, and trimer in the water-soluble products was also confirmed by high-performance liquid chromatography (data not shown). The amount of enzyme bound onto the polyester films was reported in Table 1. At 4 °C, a similar amount of PhaZaci depolymerase bound to a-P(3HB) as well as to b-P(3HB) films. Because the a-P(3HB) film was supported by the Teflon film, for a comparison, the amount of depolymerase
Communications
adsorbed onto the a-P(3HB) film shown in Table 1 was twice as much as experimentally observed. The amount of PhaZaci depolymerase bound to a-P(3HB) was more than that bound to b-P(3HB). At the temperature of 15 and 20 °C, which is 10-15 °C higher than the glass transition, a-P(3HB) could also be bound by PhaZaci depolymerase. In the case of PhaZral depolymerase, it could hardly bind to the surface of a-P(3HB) even at the temperature near the glass transition, while it could bind to the surface of b-P(3HB) better than PhaZaci depolymerase. The same experiments had been performed at the same temperature on Teflon films as controls using two depolymerases; no enzyme adsorption was observed. As revealed in the previous section, a-P(3HB) could be enzymatically hydrolyzed by PhaZaci depolymerase in the temperature range from 4 to 20 °C, while it could not be evidently degraded by PhaZral depolymerase. In the previous study,24 we have found that the profile of the degradation rates of PhaZaci depolymerase on P(3HBco-3HP) films vs 3HP content was different from that of PhaZral depolymerase. PhaZaci could degrade P(3HB-co-3HP) films with low crystallinities with higher rate than PhaZral. This indicated that the adsorption of PhaZaci depolymerase to the PHA films with low crystallinities is easier than other depolymerase. In this report, we studied the enzymatic degradation behavior of a-P(3HB) by PHA depolymerase from Aci. TP4 and R. pickettii T1 at temperatures ranging from 4 to 20 °C, which are near the glass transition temperature (Tg) of a-P(3HB) (ca. 6 °C). The direct enzymatic degradation of a-P(3HB) by PhaZaci was confirmed to undergo in the temperature range from 4 to 20 °C. However, at the same temperature, a-P(3HB) could not be evidently degraded by PhaZral depolymerase. Furthermore, the adsorption of PhaZaci onto the surface of a-P(3HB) at the same temperature range was confirmed. In other words, at the temperature near the Tg of the amorphous a-P(3HB) film, the PHA depolymerase, PhaZaci, is able to bind to the surface of a-P(3HB) and degrade it. Thus, in addition to the previous study,24 it is known that the enzymatic properties of PhaZaci depolymerase belonging to type II are different from those of PhaZral depolymerase belonging to type I. The binding ability of PhaZaci depolymerase onto the amorphous PHA or PHA films with low crystallinities is better than that of PhaZral depolymerase. From the adsorption results (Table 1), the amount of PhaZaci depolymerase bound to the polyester films decreased with the increase of the temperature. On the other hand, studies on the kinetics of P(3HB) depolymerase from R. pickettii14 and P. stutzeri28 have revealed that the rate of enzymatic hydrolysis increased with the increase of reaction temperature. Thus, the tendency of the degradation rates shown in Figure 1 could be explained as the effects of the balance between the decrease of adsorption and the increase of enzymatic hydrolysis with the increase of degradation temperature, at least within the temperature range investigated here. Analyses of the water-soluble degradation products indicated that degradation of a-P(3HB) by PhaZaci PHA depolymerase could produce 3HB monomer, dimer, and
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trimer, while from the water-soluble degradation products of bacterial b-P(3HB), only monomer and dimer could be observed.24 It had been assumed that attack to a-P(3HB) by PHA depolymerase could occur only at the ester linkages between (R) repeating units.5,6,29 The existence of trimer could be considered due to the presence of undegradable (S) units.30 According to the result reported on the active site of PHA depolymerase by Bachmann and Seebach,29 the central two subsides between which enzymatic degradation occurs must be occupied by (R) 3HB units, whereas the terminal ones may also be (S) units. It has also been reported that the P(3HB) depolymerases hydrolyzed P(3HB) through endoexo modes.19,21,23 The trimer containing two continuous (R) units will be further degraded into (R-S) dimer and (R) monomer. Thus, the trimer in the water-soluble products should be most of (R-S-R) and seldom (S-S-R) stereoisomer. From the results, we can draw the following conclusions: (1) The enzymatic degradation of the chemosynthesized amorphous a-P(3HB) film by PhaZaci PHA depolymerase was directly confirmed. (2) The degradation condition of the enzymatic hydrolysis of a-P(3HB) by PhaZaci PHA depolymerase is that the hydrolysis temperature should be near Tg, that is, 4-20 °C. (3) The binding properties of PhaZaci PHA depolymerase onto the PHA film is different from those of PhaZral PHA depolymerase. (4) The watersoluble degradation products with higher molecular weight could be obtained from the degradation of a-P(3HB) by PhaZaci PHA depolymerase than from that of the b-P(3HB) films. References and Notes (1) Doi, Y. Microbial Polyesters; VCH: New York, 1990. (2) Inoue, Y.; Yoshie, N. Prog. Polym. Sci. 1992, 17, 571. (3) Jedlinski, Z.; Kurcok, P.; Kowalczuk, M.; Kasperczyk, J. Makromol. Chem. 1986, 187, 1651. (4) Jedlinski, Z.; Kowalczuk, M.; Kurcok, P.; Brzoskowska, L.; Franek, J. Makromol. Chem. 1987, 188, 1575. (5) Abe, H.; Matsubara, I.; Doi, Y. Macromolecules 1995, 28, 844. (6) Scandola, M.; Focarete, M. L.; Adamus, G.; Sikorska, W.; Baranowska, I.; Swierczek, S.; Gnatowiski, M.; Kowalczuk, M. Jedlinski, Z. Macromolecules 1997, 30, 2568. (7) Tomasi, G.; Scandola, M. Macromolecules 1996, 29, 507. (8) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451. (9) Focarete, M. L.; Ceccorulli, G.; Scandola, M.; Kowalczuk, M. Macromolecules 1998, 31, 8485. (10) Scandola, M.; Focarete, M. L.; Gazzano, M.; Matuszowicz. A.; Sikorska, W.; Adamus, G.; Kurcok, P.; Kowalczuk, M.; Jedlinski, Z. Macromolecules 1997, 30, 7743. (11) He, Y.; Shuai, X.; Cao. A.; Kasuya, K.; Doi, Y.; Inoue, Y. Macromol. Rapid Commun. 2000, 21, 1277. (12) He, Y.; Shuai, X.; Cao. A.; Kasuya, K.; Doi, Y.; Inoue, Y. Polym. Degrad. Stab. 2001, 73, 193. (13) He, Y.; Shuai, X.; Kasuya, K.; Doi, Y.; Inoue, Y. Biomacromolecules 2001, 2, 1045. (14) Kasuya, K.; Inoue, Y.; Yamada, K.; Doi, Y. Polym. Degrad. Stab. 1995, 48, 167. (15) Kasuya, K.; Inoue, Y.; Doi, Y. Int. J. Biol. Macromol. 1996, 19, 35. (16) Scandola, M.; Focarete, ML.; Frisoni, G. Macromolecules 1998, 31, 1, 3846. (17) Cao, A.; Arai, Y.; Yoshie, N.; Kasuya, K.; Doi, Y.; Inoue, Y. Polymer 1999, 40, 6821. (18) Abe, H.; Doi, Y.; Aoki, H.; Akehata, T.; Hori, Y.; Yamaguchi, A. Macromolecules 1995, 28, 7630.
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(19) Scherer, T. M.; Fuller, R. C.; Goodwin, S.; Lenz, R. W. Biomacromolecules 2000, 1, 577. (20) Shirakura, Y.; Fukui, T.; Saito, T.; Okamoto, Y.; Norikawa, T.; Koide, K.; Tomita, K.; Takemasa, T.; Masamune, S. Biochim. Biophys. Acta 1986, 880, 46. (21) Iwata, T.; Doi, Y.; Tanaka, T.; Akehata, T.; Shiromo, M.; Teramachi, S. Macromolecules 1997, 30, 5290. (22) Abe, H.; Doi, Y. Int. J. Biol. Macromol. 1999, 25, 185. (23) Kasuya, K.; Ohura, T.; Masuda, K.; Doi, Y. Int. J. Biol. Macromol. 1999, 24, 329. (24) Wang, Y.; Inagawa, Y.; Saito, T.; Kasuya, K.; Doi, Y.; Inoue, Y. Biomacromolecules, in press.
Communications (25) Kobayashi, T.; Sugiyama, A.; Kawase, Y.; Saito, T.; Mergaert, J.; Swings J. J. EnViron. Polym. Degrad. 1999, 7, 9. (26) Wang, Y.; Ichikawa, M.; Cao, A.; Yoshie, N.; Inoue, Y. Macromol. Chem. Phys. 1999, 200, 1047. (27) Kurcok, P.; Kowalczuk, M.; Hennek, K.; Jedlinski, Z. Macromolecules 1992, 25, 2017. (28) Uefuji, M.; Kasuya, K.; Doi, Y. Polym. Degrad. Stab. 1997, 58, 275. (29) Bachmann, B. M.; Seebach, D. Macromolecules 1999, 32, 1777. (30) Abe, H.; Matsubara, I.; Doi, Y.; Hori, Y.; Yamaguchi, A. Macromolecules 1994, 27, 50.
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