Enzymatic Degradation of Atactic Poly(R,S

The phase structure and biodegradability were investigated for amorphous blends of chemosynthetic fully amorphous atactic poly(R,S-3-hydroxybutyrate) ...
1 downloads 0 Views 89KB Size
Download PDF
Biomacromolecules 2001, 2, 1045-1051

1045

Enzymatic Degradation of Atactic Poly(R,S-3-hydroxybutyrate) Induced by Amorphous Polymers and the Enzymatic Degradation Temperature Window of an Amorphous Polymer System Yong He,† Xintao Shuai,† Ken-ichi Kasuya,‡ Yoshiharu Doi,§ and Yoshio Inoue*,† Department of Biomolecular Engineering, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, Japan, Department of Biological and Chemical Engineering, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan, and Polymer Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama 351-0198, Japan Received May 9, 2001; Revised Manuscript Received June 15, 2001

The phase structure and biodegradability were investigated for amorphous blends of chemosynthetic fully amorphous atactic poly(R,S-3-hydroxybutyrate) (a-PHB) with atactic poly(methyl methacrylate) (PMMA) and atactic poly(R,S-lactide) (a-PLA). The differential scanning calorimetry thermal analysis indicated that a-PHB/PMMA blends were partially miscible while a-PHB/a-PLA blends were miscible in the studied composition range. The biodegradations of the blends were carried out in phosphate buffer solution in the presence of bacterial poly(R-3-hydroxybutyrate) extracellular depolymerases purified from Alcaligenes faecalis T1 and P. stutzeri. Although a-PHB in the pure state was not degraded by these depolymerase, it was degraded by blending with PMMA and a-PLA. The results demonstrated that the enzymatic degradation of a-PHB can be induced by amorphous polymers such as PMMA and a-PLA. Also, the biodegradation rate of a-PHB in the blends decreased drastically when the degradation temperature is too much away from the polymer glass transition temperatures. On the basis of these results, a temperature window of the enzymatic degradation was first proposed for the blend and the essence of induced degradation was discussed. Introduction Optically active poly(R-3-hydroxybutyrate) (PHB) with isotactic structure is synthesized and accumulated by a variety of bacteria as a reserve energy source.1-4 PHB isolated from the bacteria is a highly crystalline polymer with a melting point and a glass transition temperature of about 180 and 5 °C, respectively.2,3 A remarkable characteristic of PHB is its biodegradability in various environments. A number of microorganisms in the environment excrete extracellular PHB depolymerases to hydrolyze the solid PHB into water-soluble monomer and oligomers. Several extracellular PHB depolymerases have been purified from Pseudomonas lemoignei,5 Pseudomonas pickettii,6 Pseudomonas stutzeri,7 Alcaligenes faecalis T1,8 Comamonas testosteroni,9 and Comamonas acidoVorans.10 Atactic poly(R,S-3-hydroxybutyrate) (a-PHB) obtained from the anionic polymerization of racemic β-butyrolactone is a fully amorphous polymer with a glass transition temperature of about 5 °C.11,12 a-PHB cannot be hydrolyzed in the pure state by the PHB depolymerases, while the segments of a-PHB undergo enzymatic attack when a-PHB is in physical blends with or it constitutes a diblock copolymer with crystalline poly(hydroxyalkanoic acid) (PHA); regardless of the partner, PHA can be or cannot be degraded †

Tokyo Institute of Technology. Gunma University. § The Institute of Physical and Chemical Research (RIKEN). ‡

by the PHB depolymerases. Abe et al. observed that a-PHB was hydrolyzed by PHB depolymerase in the presence of natural crystalline PHB.13 Scandola et al. reported that a-PHB was biodegraded in binary blends with natural crystalline poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV),14 chemosynthesized crystalline poly(-caprolactone) (PCL), and poly(S-lactic acid) (PLA)15 and in diblock copolymer with crystalline poly(pivalolactone) (PPVL).16 It is noteworthy that PCL, PLA, and PPVL are not hydrolyzed by the PHB depolymerases but they are biodegradable polyesters as PHB and PHBV. The phenomenon that a-PHB segments are not hydrolyzed in the pure state but are hydrolyzed in the crystalline blends or the crystalline diblock polymer by the PHB depolymerase is very interesting and was called crystalline-induced biodegradation by Scandola et al.16 Analyses of the structural genes of the PHB depolymerases have revealed that the enzymes are organized with two domains and a linker region.17,18 One of the domains, the substrate binding domain, plays the role in its binding to the PHB surface. The other domain is the catalytic domain, which contains the catalytic triad Ser-His-Asp.19 The serine residue is a part of the lipase box pentapeptide Gly-X-SerX-Gly,20 which has been found in all known serine hydrolases.21-23 In addition, the two domains are connected by a fibronectin type III or threonin-rich linker.24 On the other hand, the kinetics of heterogeneous hydrolysis of PHB film, in the presence of the extracellular PHB depolymerase, have been extensively studied.25-27 The results demonstrated

10.1021/bm010087w CCC: $20.00 © 2001 American Chemical Society Published on Web 08/08/2001

1046

Biomacromolecules, Vol. 2, No. 3, 2001

that the surface hydrolysis of PHB is a two-step enzymatic reaction. The first step is the adsorption of enzyme to the surface of PHB film via the binding domain, and the second is the surface hydrolysis of the polymer chain to watersoluble products by the catalytic domain of the depolymerase. On the basis of the structure and mechanism of the enzymatic hydrolysis, Abe et al. thus proposed that the PHB depolymerase hardly bound to the mobile PHB chains in an amorphous state at temperatures much higher than the glass transition temperature of PHB and thought that pure a-PHB was not biodegraded because PHB depolymerase could not be adsorbed on the surface of amorphous a-PHB, whereas in a-PHB/PHB blends the stable binding site for the enzyme’s substrate-binding domain was provided by the crystalline PHB component and then the hydrolysis of a-PHB component was induced.13 Scandola et al. further advanced an assumption that the presence of a partially crystalline PHA was a general requirement for the enzymatic hydrolysis of a-PHB component either in a binary blend or in a diblock copolymer.16 Thus it seems interesting to elucidate the following four questions: (i) Can the certain polymers, which are not crystalline polymers but with quite high glass transition temperatures, provide the stable binding sites for the adsorption of PHB depolymerase? (ii) Can these amorphous polymers induce the biodegradation of a-PHB through blending? (iii) How does the degradation temperature affect the enzymatic degradation behavior when the degradation temperature is just higher than the glass transition temperature? (iv) What is the essence of the induced degradation? In this work, poly(methyl methacrylate) (PMMA) and atactic poly(R,S-lactide) (a-PLA) were used as blending partners for a-PHB, as they are amorphous polymers with high glass transition temperature. The binary blends were characterized and subjected to the biodegradation in the presence of PHB depolymerases from A. faecalis T1 and P. stutzeri. On the basis of the results, the biodegradation temperature window was proposed for the blend and the essence of the induced biodegradation was discussed. Some preliminary results of enzymatic degradation of a-PHB induced by PMMA have been reported elsewhere.28,29 Experimental Section Materials. Atactic poly(R,S-3-hydroxybutyrate) (a-PHB, isotactic diad fraction determined by 1H NMR was [i] ) 0.50) was synthesized through anionic polymerization of racemic β-butyrolactone in bulk with potassium oleate/18crown-6 ether complex as an initiator.30 The reagents β-butyrolactone and 18-crown-6 ether (from Tokyo Kasei Industry Co., Japan) were purified as reported in the literature.31,32 The resultant polymer was precipitated twice into methanol from the chloroform solution and dried under vacuum for a week before using. The weight-averaged molecular weight (Mw) and the molecular weight distribution (Mw/Mn) of a-PHB were 4.55 × 104 and 1.17, respectively. Poly(methyl methacrylate) (PMMA) (Mw ) 6.76 × 104, Mw/Mn ) 1.68; Mitsubishi Rayon Co., Japan) was precipitated twice from chloroform solution into ethanol before use. The triad tacticity of this sample was 10% isotactic, 43%

He et al.

heterotactic, and 47% syndiotactic as determined by 1H NMR.33 Atactic P(R,S-lactide) (Mw ) 1.06 × 105, Mw/Mn ) 2.45; isotactic diad fraction [i] ) 0.50) was provided by Shimadzu Co., Japan. This sample was also purified twice as PMMA. Bacterial PHB (Mw ) 4.73 × 105, Mw/Mn ) 1.99) purchased from Aldrich Chemical Co. was used after purification by precipitation in ethanol from 1,2-dichloroethane solution. Preparation of Blend. The films of a-PHB/PMMA and a-PHB/a-PLA blends were prepared by casting the 5 wt % CH2Cl2 solution to a Teflon Petri dish, allowing the solvent to evaporate at room-temperature overnight. After drying under vacuum at 60 °C for 2 days to remove the residual solvent, the casting films were subsequently compressionmolded between Teflon sheets for 3 min at 200 °C under a pressure of 5 MPa, using a laboratory press (Mini Test Press10, Toyoseiki Co., Japan), following with a fast cooling to room temperature between two iron plates. Before physical characterization and enzymatic degradation were attempted, the molded films were aged at room temperature for 4 weeks. The following codes are used in this paper: BM40, BA50 and so on, where BM and BA refer to a-PHB/PMMA and a-PHB/a-PLA blends, respectively. The numbers refer to the weight percent content of PMMA or a-PLA in the blends. Experimental Techniques. 1H NMR measurement was employed at 270 MHz on a JEOL GSX270 NMR spectrometer (JEOL, Japan). The molecular weights were characterized by a Tosoh HLC-8020 GPC system (Tosoh, Japan), using chloroform as the eluent and polystyrene samples with a narrow molecular distribution as the standards. Differential scanning calorimetry (DSC) analysis was performed on a SEIKO DSC 220 system (Seiko Instruments, Japan). The polymer samples packed in aluminum pans were heated from -100 to 195 °C at a heating rate of 20 °C/min. The value of the end point in the transition was taken as the glass transition temperature. Adsorption Measurements of PHB Depolymerase. The adsorption of PHB depolymerase onto the surface of the sample films was carried out at 37 °C in phosphate buffer. The film with an initial dimension of 10 × 10 × 0.1 mm was placed in a test tube containing 1 mL of potassium phosphate buffer (0.1 M, pH ) 7.4) and 2.9 µg of PHB depolymerase of A. faecalis T1. After the mixture solution was incubated at 37 °C for 15 min, the supernatant was separated and the amount of PHB depolymerase in the supernatant was determined from the hydrolysis rate of p-nitrophenylbutyrate.27 The amount of enzyme adsorbed on a test film was calculated from those of the added and unadsorbed enzyme. Enzymatic Degradation. The extracellular PHB depolymerase was purified from A. faecalis T1 and P. stutzeri as reported in the literatures.27,34,35 The enzymatic degradation was carried out in 0.1 M phosphate buffer (pH ) 7.5). The film of initial weight about 10 mg and initial dimension 10 × 10 × 0.1 mm was placed in the small bottle containing 2.0 µg of enzyme and 1.0 mL of buffer solution. After the reaction was allowed to continue for a period of time, the films were removed, washed with distilled water, and dried to constant weight in a vacuum before weight analysis. For

Biomacromolecules, Vol. 2, No. 3, 2001 1047

Enzymatic Degradation of Polymer Blends

Figure 1. DSC traces of pure a-PHB, PMMA, and their blends. The numbers in the sample codes refer to the weight percent content of PMMA in the blends.

Figure 2. DSC traces of pure a-PHB, a-PLA, and their blends. The numbers in the sample codes refer to the weight percent content of a-PLA in the blends.

Table 1. Glass Transition Temperatures (Tgs) of a-PHB, PMMA, a-PLA, and Their Blends sample

Tg/°C

sample

Tg/°C

a-PHB BM20 BM40 BM50 BM60

5 6 7.58 5.60 40

BM75 PMMA BA50 BA60 a-PLA

63 115 29 32 58

each polymer sample, three films were used and the average value of their weight loss was reported as the result. Control tests, carried out for all samples in buffer solution free from the enzyme, showed no appreciable weight losses over the time scale of the experiments. Results Physical Properties of the Blends. The DSC curves are summarized in Figure 1 for a-PHB, PMMA, and their blends. The thermograms of a-PHB and PMMA showed distinct and narrow glass transition. The glass transition temperatures (Tgs) of plain a-PHB and PMMA were about 5 and 115 °C (Table 1), respectively. For the blends with PMMA content ranging from 40 to 50 wt %, it showed the presence of two phases in these blends: one was the pure a-PHB phase with Tg of around 5 °C; the other was the blend phase with a broad glass transition (Tg ≈ 60 °C, Figure 1). For the blends with PMMA content of 60 and 75 wt %, only one broad glass transition was observed. The thermograms of the blends indicated that a-PHB and PMMA were partially miscible at the PMMA content of 40 and 50 wt % while they were miscible when the PMMA content is higher than 60 wt %. For BM20 blend, although only one Tg was observed, the Tg was almost the same as that of plain a-PHB. Hence, it was difficult here to estimate the miscibility of BM20. Lotti et al. have reported that bacterial PHB/PMMA blends containing PHB up to 20 wt % were single-phase amorphous materials, while at high PHB content all of the PMMA was involved in the 20/80 PHB/ PMMA miscible phase and the excess PHB segregated to form a partially crystalline phase.36 Siciliano et al. have also pointed out that PHB/PMMA blend showed an upper critical solution temperature behavior.37 Thus, it seemed that a-PHB/

Figure 3. Amount of enzyme bound on the test film at 37 °C in the presence of PHB depolymerase from A. faecalis T1: 1 mL of potassium phosphate buffer (0.1 M, pH ) 7.4); 2.9 µg of enzyme; adsorption time of 15 min.

PMMA blends showed a similar miscibility behavior to that of PHB/PMMA blends. For a-PHB/a-PLA blends with a-PLA content from 20 to 80 wt %, a single composition-dependent glass transition temperature was observed (Figure 2, only the DSC curves for BA50 and BA60 blends were shown), indicating they were miscible blends as a-PHB/PLA blends.15,38 The glass transition temperature was 29 °C for blend BA50 and 32 °C for blend BA60 (Table 1). No melting peak appeared in Figures 1 and 2, which confirmed that all polymers and blends investigated here were free from the crystalline phase. Analysis of the Enzyme Binding. The amount of bound protein was reported in Figure 3. As expected, the film of plain a-PHB cannot adsorb the depolymerase due to the experiment temperature (37 °C) is much higher than its Tg (5 °C). However, the binding of the enzyme to the films of plain a-PLA, PMMA, blend BA50, and blend BM40 was confirmed, suggesting a high Tg is favorable for enzyme binding. An interesting fact was that the amount of the bound enzyme on the PMMA film was the highest among the studied samples although PMMA was a nonbiodegradable amorphous polymer. Beside that, another point should be noted was that the amount of bound protein on the BA50

1048

Biomacromolecules, Vol. 2, No. 3, 2001

Figure 4. Biodegradation results of a-PHB/PMMA blends at 37 °C and pH 7.5 in the presence of PHB depolymerase purified from A. faecalis T1. The biodegradations of plain a-PHB and PMMA are also shown for comparison.

Figure 5. Biodegradation results of a-PHB/PMMA blends at 37 °C and pH 7.5 in the presence of PHB depolymerase purified from P. stutzeri.

blend film was quite high at 37 °C, which was 8 °C higher than its Tg. The DSC thermograms of BM40 and BM50 indicated the presence of two phases in the blend, the pure a-PHB phase with Tg of about 5 °C and the blend phase with Tg of around 60 °C. This meant that some of the a-PHB chains were in the rubbery phase and the others in the glassy phase of the film at 37 °C. Thus, in these blends, both the a-PHB chains in the glassy phase and the rigid chains of PMMA were possible to provide the stable binding site for the enzyme adsorption. It is difficult here to estimate that the binding site was provided by PMMA chains only or by both PMMA and a-PHB chains. Induced Enzymatic Hydrolysis. The biodegradation results of a-PHB/PMMA and a-PHB/a-PLA blends using PHB depolymerase from A. faecalis T1 and P. stutzeri are plotted in Figures 4-6 as normalized weight loss vs exposure time. The biodegradation extent of plain a-PHB, PMMA, and a-PLA is also shown in the figures for the sake of comparison. As expected, neither of the pure components showed any appreciable weight loss on the experiment time

He et al.

Figure 6. Biodegradation results of a-PHB/a-PLA blends at 20 °C and pH 7.5 in the presence of PHB depolymerase purified from A. faecalis T1.

scale. Also, blends BM20 and BM75 were hardly degraded during the experiment. However, the biodegradation results of Figures 4-6 demonstrated that the blends of BM40, BM50, and BA50 continuously lost weight upon exposure to PHB depolymerase. BM40 lost 16 wt % of its initial weight after the degradation at 37 °C for 72 h and BA50 lost 10 wt % after the degradation at 20 °C for 144 h in the presence of depolymerase from A. faecalis T1 (Table 2). The average biodegradation rates of BM40 in the presence of the depolymerases from A. faecalis T1 and P. stutzeri were 0.027 and 0.010 mg cm-2 h-1, respectively, which were of the same order of magnitude as those reported for a-PHB/ PCL blends15 and the diblock copolymer of poly(R,S-βbutyrolactone-b- pivalolactone).16 To reveal which component in the blends was degraded during the experiments, the blend composition after degradation as well as the degradation products was analyzed by 1H NMR. The blend composition after the degradation was determined for BM40 and BA50 and was compared with the value calculated on the assumption that only the a-PHB component undergoes enzymatic degradation (Table 2). The excellent agreement between experimental and calculated composition suggested that only a-PHB component was hydrolyzed in both a-PHB/PMMA and a-PHB/a-PLA blends. 1H NMR analysis of the enzymatic degradation products showed the presence of only the monomer and oligomers of a-PHB, free from any evidence of PMMA or a-PLA fragments, which further confirmed that only a-PHB underwent enzymatic hydrolysis in the studied blends. The above results confirmed the enzymatic degradation of BM40, BM50, and BA50 blends and demonstrated that blending with an amorphous polymer can provide the stable binding site for the enzyme and induce the biodegradation of a-PHB component under appropriate conditions regardless whether the amorphous polymer was a biodegradable one such as a-PLA or a nonbiodegradable one such as PMMA, regardless whether the blend was a miscible system such as BA50 or partially miscible system such as BM40 and regardless whether the depolymerase was purified from A. faecalis T1 or P. stutzeri.

Biomacromolecules, Vol. 2, No. 3, 2001 1049

Enzymatic Degradation of Polymer Blends

Table 2. Enzymatic Degradation Results of BM40 and BA50 Blends Exposed to PHB Depolymerase from A. faecalis T1 a-PHB content after degradation (wt %)

a

sample

exposure time (h)

degradation temp (°C)

wt loss after degradation (wt %)

BM40 BA50

72 144

37 20

16 10

from NMR

1H

54 43

calcda 54 44

From the weight loss data, assuming biodegradation of the a-PHB component only.

Figure 7. Enzymatic degradation profiles of BA50 blend in the presence of PHB depolymerase purified from A. faecalis T1 at temperatures of 20, 25, 30, and 37 °C.

As mentioned above, BM20 and BM75 blends were hardly hydrolyzed by the depolymerases. The Tg of BM20 was about 6 °C, which was much lower than the temperature of the degradation experiment, 37 °C (Figure 1 and Table 1). It is quite reasonable that BM20 was not hydrolyzed under an action of the enzyme since BM20 was in the rubbery state at 37 °C. As for the BM75 blend, no biodegradation in this experiment was possibly due to the following two aspects: (i) the number of the a-PHB chains accessible for the enzyme was quite small as a-PHB content is low in the blend; (ii) the a-PHB chain might be too rigid to be hydrolyzed by the enzyme because the Tg of BM75 is high compared with the temperature of the degradation experiment. Scandola et al. observed that the biodegradation of a-PHB15 and PHB39 components in miscible blends was completely prevented when the Tg values of the blends were much higher than the degradation temperature. Effect of the Temperature of Degradation Experiment. As revealed above, a-PHB component underwent enzymatic erosion when the glass transition temperature Tg of the blend was close to the degradation temperature, while it did not undergo enzymatic erosion when the Tg was much lower than the degradation temperature. In this section, the effect of the temperature of the degradation experiment on the degradation behavior of a-PHB component is investigated when the experimental temperature is a little higher than the Tg of the blend. In Figure 7 are summarized the degradation results of a BA50 blend at degradation temperature from 20 to 37 °C in the presence of PHB depolymerase from A. faecalis T1. It was observed that BA50 continuously lost weight during the course of the whole experiment at these temperatures. At

Figure 8. Effect of experimental temperature on the enzymatic degradation of blend BA50 and natural PHB.

37 °C the weight loss was fairly low. However, BA50 showed a stable enzymatic degradation even at this temperature (8 °C higher than the Tg). In Figure 8 are plotted the average weight loss rates of a BA50 blend and PHB as a function of temperature. The degradation rate of PHB increased with the testing temperature from the 20 to 37 °C range as well-known. Conversely, the degradation rate of BA50 decreased rapidly with the degradation temperature. The two steps of the hydrolytic reaction are the adsorption of enzyme on the film via its binding domain and surface hydrolysis of the polyester chain via its catalytic domain. In general, the increase in the temperature weakens the adsorption interaction while it promotes the surface hydrolysis.25 Here, PHB was a crystalline polymer with high crystallinity (about 60 wt %) and high melting point (around 180 °C), while BA50 was an amorphous blend with a glass transition temperature of just 29 °C. In the investigated temperature range (20-37 °C), the chain mobility of PHB in the crystalline phase (which provides the stable adsorption site for the enzyme) should change little with the temperature as the temperature was much lower than the melting point, resulting in little effect on the adsorption interaction of the enzyme. As a result, the enzymatic degradation rate of PHB increased evidently as the hydrolysis activity of the catalytic domain heightened with the temperature. However, BA50 changed from a glass to a rubber in this temperature range and the mobility of the chain rose drastically with the temperature, greatly weakening the adsorption interaction between the bound enzyme and film surface and decreasing the amount of bound enzyme. Hence, the increase of degradation temperature led to an evident drop of the degradation rate.

1050

Biomacromolecules, Vol. 2, No. 3, 2001

It is noteworthy that the average degradation rate of BA50 (6 × 10-4 mg cm-2 h-1) was 2 orders of magnitude lower than that of PHB (0.208 mg cm-2 h-1) and also 1 order lower than that of BM40 (0.027 mg cm-2 h-1) at 37 °C in the presence of PHB depolymerase from A. faecalis T1, although the amount of enzyme adsorbed on BA50 is much higher than that on PHB and BM40 (Figure 3). This result indicated that the higher amount of bound enzyme need not mean a higher degradation rate. It is quite possible that the degradation rate is also related to the strength of adsorption interaction. Discussion Temperature Window of Enzymatic Degradation. As revealed in the previous section, plain a-PHB could not be hydrolyzed by extracellular PHB depolymerase due to its too low glass transition temperature. However, the enzymatic degradation of a-PHB component could be induced when the Tg was heightened to be close to the temperature of degradation experiment through blending with amorphous polymer with high Tg. In the previous section, it was also found that the biodegradation rate of the a-PHB component droped rapidly with temperature when the Tg of the blend was lower than the degradation temperature. Beside these, it has been reported that the biodegradation of a-PHB components was completely prevented in the miscible blends with Tg much higher than the degradation temperature.15 These facts suggest that a-PHB component underwent enzymatic hydrolysis when the Tg was close to the degradation temperature while the biodegradation was restrained when the Tg was far from the temperature of degradation experiment. In other words, the temperature window for the a-PHB component in an amorphous blend to undergo effectively a two-step enzymatic degradation was [Tg - ξ, Tg + σ], where ξ and σ > 0 and their values varied with the blend and enzyme. A good guess of the values should be ξ and σ ≈ 10-20 °C. Also, estimation of the biodegradation window can be obtained from the mechanism of the enzymatic degradation. As mentioned in the Introduction, the surface enzymatic hydrolysis is a two-step heterogeneous reaction. The first step is the adsorption of enzyme via its binding domain and the second is the surface hydrolysis by its catalytic domain. As the degradation temperature was higher than the Tg of the amorphous system, the mobility of the polymer chain rose drastically with the temperature, greatly weakening the adsorption interaction between the binding domain of the enzyme and polymer chain and thus restraining the degradation. In the case of a degradation temperature lower than the Tg, the polymer chain was frozen with the decrease of the temperature, preventing the second step reaction, the surface hydrolysis by the catalytic domain. In other words, the heterogeneous enzymatic degradation of an amorphous system was only possible in a limited temperature range around its Tg. It should be pointed out that the biodegradation window just means that the enzymatic hydrolysis of a-PHB is possible only in this temperature range but does not mean that the

He et al.

hydrolysis is bound to take place in this temperature range. The temperature window is proposed here only considering the mobility of polymer chains and not considering the activity of the depolymerase and other factors. Thus, it is quite possible that the a-PHB component does not undergo hydrolysis even at an experimental temperature included in the temperature window due to activity of the depolymerase and other factors. Also, it should be pointed out that extrapolation of the concept of the enzymatic temperature window to other kinds of degradation systems should require some caution as the characteristics of the enzyme and the enzymatic degradation mechanism may be quite different from those of the system studied in this paper. Essence of the Induced Biodegradation. In essence, the induced biodegradation through blending is the result of a shift of the temperature window of enzymatic degradation. For pure a-PHB, its enzymatic degradation temperature window is located on the low-temperature side due to its low Tg. It could not be hydrolyzed by the depolymerase at a given temperature such as 37 or 20 °C, which was not included in the temperature window. Through blending with an amorphous glassy polymer with high glass transition temperature, the Tg increased and the temperature window was up shifted. When the given degradation temperature was in the up shifted window, the enzymatic degradation of a-PHB was induced by the blending partner polymer. Conclusions The enzymatic degradation of a-PHB can be induced by blending with amorphous polymers to heighten the glass temperature, regardless whether the amorphous polymer is a biodegradable one such as a-PLA or a nonbiodegradable one such as PMMA, regardless whether the blend is a miscible one such as a-PHB/a-PLA blend or a partially miscible one such as a-PHB/PMMA blend, and regardless whether the depolymerase is purified from A. faecalis T1 or P. stutzeri. Furthermore, this paper revealed that the enzymatic degradation rate of an amorphous blend decreased drastically with the degradation temperature when it was just higher than the glass transition temperature of the blend. On the basis of these results, a temperature window of the enzymatic degradation was first proposed to be [Tg - ξ, Tg + σ] for the amorphous blend and the induced degradation was suggested to be the result of the shift of the enzymatic degradation window in essence. References and Notes (1) Holmes, P. A. In DeVelopments in crystalline polymers; Bassett, D. C., Eds.; Elsevier: Amsterdam, 1981; Vol. 2. (2) Doi, Y. Microbial Polyesters; VCH: New York, 1990. (3) Inoue, Y.; Yoshie, N. Prog. Polym. Sci. 1992, 17, 571. (4) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450. (5) Lusty, C. J.; Doudoroff, M. Proc. Natl. Acad. Sci. U.S.A. 1966, 56, 960. (6) Yamada, K.; Mukai, K.; Doi, Y. Int. J. Biol. Macromol. 1993, 15, 215. (7) Mukai, K.; Yamada, K.; Doi, Y. Polym. Degrad. Stab. 1994, 43, 319. (8) Tanio, T.; Fukui, T.; Shirakura, Y.; Saito, T.; Tomita, K.; Kaiho, T.; Masamune, S. Eur. J. Biochem. 1982, 124, 71. (9) Mukai, K.; Yamada, K.; Doi, Y. Polym. Degrad. Stab. 1993, 41, 85.

Enzymatic Degradation of Polymer Blends (10) Kasuya, K.; Inoue, Y.; Tanaka, T.; Akehata, T.; Iwata, T.; Fukui, T.; Doi, Y. Appl. EnViron. Microbiol. 1997, 63, 4844. (11) Jedlinski, Z.; Kowalczuk, M.; Kurcok, P.; Brzoskowska, L.; Franek, J. Makromol. Chem. 1987, 188, 1575. (12) Jedlinski, Z.; Kurcok, P.; Kowalczuk, M.; Kasperczyk, J. Makromol. Chem. 1986, 187, 1651. (13) Abe, H.; Matsubara, I.; Doi, Y. Macromolecules 1995, 28, 844. (14) Scandola, M.; Focarete, M. L.; Adamus, G.; Sikorska, W.; Baranowska, I.; Swierczek, S.; Gnatowiski, M.; Kowalczuk, M.; Jedlinski, Z. Macromolecules 1997, 30, 2568. (15) Focarete, M. L.; Ceccorulli, G.; Scandola, M.; Kowalczuk, M.; Macromolecules 1998, 31, 8485. (16) Scandola, M.; Focarete, M. L.; Gazzano, M.; Matuszowicz, A.; Sikorska, W.; Adamus, G.; Baranowska, I.; Swierczek, S.; Gnatowiski, M.; Kurcok, P.; Kowalczuk, M.; Jedlinski, Z. Macromolecules 1997, 30, 7743. (17) Jendrossek, D.; Schiremer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451. (18) Shirki, M.; Iwata, A.; Saito, T. J. EnViron. Polym. Degrad. 1993, 2, 99. (19) Jendrossek, D.; Backhaus, M.; Andermann, M. Can. J. Microbiol. 1995, 41 (Suppl. 1), 160. (20) Shinohe, T.; Nojiri, M.; Saito, T.; Stanislawski, T.; Jendrossek, D. FEMS Microbiol. Lett. 1997, 141, 10. (21) Jaeger, K. E.; Steinbuchel, A.; Jendrossek, D. Appl. EnViron. Microbiol. 1995, 61, 3113. (22) Jaeger, K. E.; Ransac, S.; Dijkstra, B. W.; Colson, D.; van Heuvel, M.; Misset, O. FEMS Microbiol. ReV. 1994, 15, 29. (23) Schrag, J. D.; Li, Y.; Wu, S.; Cygler, M. Nature 1991, 351, 761.

Biomacromolecules, Vol. 2, No. 3, 2001 1051 (24) Hansen, C. K. FEBS Lett. 1992, 305, 91. (25) Kasuya, K.; Inoue, Y.; Yamada, K.; Doi, Y. Polym. Degrad. Stab. 1995, 48, 167. (26) Scandola, M.; Focarete, M. L.; Frisoni, G. Macromolecules 1998, 31, 3846. (27) Kasuya, K.; Inoue, Y.; Doi, Y. Int. J. Biol. Macromol. 1996, 19, 35. (28) He, Y.; Shuai, X.; Cao, A.; Kasuya, K.; Doi, Y.; Inoue, Y. Macromol. Rapid Commun. 2000, 21, 1277 (29) He, Y.; Shuai, X.; Cao, A.; Kasuya, K.; Doi, Y.; Inoue, Y. Polym. Degrad. Stab. 2001, 73, 193. (30) Kurcok, P.; Kowalczuk, M.; Hennek, K.; Jedlinski, Z. Macromolecules 1992, 25, 2017. (31) Jedlinski, Z.; Stolarzewicz, A.; Grobelny, Z.; Szwarc, M. J. Phys. Chem. 1984, 88, 6094. (32) Kurcok, P.; Dubois, P.; Sikorska, W.; Jedlinski, Z.; Jerome, R. Macromolecules 1997, 30, 5591. (33) Inoue, Y.; Nishioka, A.; Chujo, R. Polym. J. 1971, 2, 535. (34) Shirakura, Y.; Fukui, T.; Saito, T.; Okamoto, Y.; Narikawa, T.; Koide, K.; Tomita, K.; Takemasa, T.; Masamune, S. Biochim. Biophys. Acta 1986, 880, 46. (35) Uefuji, M.; Kasuya, K.; Doi, Y. Polym. Degrad. Stab. 1997, 58, 275 (36) Lotti, N.; Pizzoli, M.; Ceccorulli, G.; Scandola, M. Polymer 1993, 34, 3368. (37) Siciliano, A.; Seves, A.; Macro, T.; Cimmino, S.; Martuscelli, E.; Silveestre, C. Macromolecules 1995, 28, 8065. (38) Ohkoshi, I.; Abe, H.;. Doi, Y. Polymer 2000, 41, 5985. (39) Scandola, M. Can. J. Microbiol. 1995, 41, 310.

BM010087W