Biomacromolecules 2004, 5, 1135-1140
1135
Notes
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
It is known that A. latus produces both P(3HB)13,14 and P(3HB-co-4HB)9,10 effectively. It is necessary to know the detailed comonomer-unit composition and its distribution of bacterial copolyesters to evaluate precise relationships between the comonomer-unit composition and several properties such as thermal and mechanical properties and biodegradability. In this paper, we will report on comonomerunit compositions and their distributions for a series of P(3HB-co-4HB)s biosynthesized by A. latus. The results will be compared with those biosynthesized by R. eutropha.
Received February 13, 2004
Experimental Section
Modal Difference in Comonomer-Unit Compositional Distributions of Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)s Biosynthesized by Two Strains, Ralstonia eutropha and Alcaligenes latus Kazuki Ishida and Yoshio Inoue*
Introduction A wide variety of bacteria are known to synthesize an optically active polyester, poly(3-hydroxybutyrate) [P(3HB)], and accumulate it as carbon and energy sources.1-3 P(3HB) has attracted much industrial attention as a biodegradable and biocompatible material for agricultural, marine, and medical applications.2 Unfortunately, P(3HB) is rather brittle compared with common chemosynthesized plastics. Besides, the thermal stability of P(3HB) is low and it rapidly decomposes at a temperature slightly above its melting point according to a β-elimination mechanism.4 Thus, its application has been extremely restricted. To improve the physical properties of P(3HB), its copolyesters have been widely studied.3 Poly(3-hydroxybutyrateco-4-hydroxybutyrate)s [P(3HB-co-4HB)s] with a wide range of 4HB-unit contents have been synthesized using several strains such as Ralstonia eutropha,5-8 Alcaligenes latus,9,10 and Comamonas acidoVorans.11,12 Several properties of P(3HB-co-4HB)s have been investigated in the previous papers. P(3HB-co-4HB)s have a wide range of physical properties and morphologies from highly crystalline plastic to elastic rubber, and their melting points greatly vary with varying the 4HB-unit content.8,11,12 Moreover, their biodegradation rate is much higher than that of P(3HB) homopolymer especially when the 4HB-unit content is 10-20 mol %, due to the decrease in crystallinity.6,11 Recently, we have reported that as-produced P(3HB-co4HB)s biosynthesized by R. eutropha H16 using 4-hydroxybutyric acid (4HBA), and butyric acid as a mixed carbon source in the presence of ammonium sulfate has bimodal comonomer-unit compositional distributions; namely, these P(3HB-co-4HB)s are mixtures of two types of copolyesters with high 3HB- and high 4HB-unit contents, independent of the average comonomer-unit compositions of as-produced samples.8 From the thermal analysis, these as-produced P(3HB-co-4HB)s were shown to be immiscible blends.8 * To whom correspondence should be addressed. Tel.: +81-45-9245794. Fax: +81-45-924-5827. E-mail:
[email protected].
Biosyntheses of Copolyesters. P(3HB-co-4HB) copolyesters were produced through two-stage and one-stage fermentations of R. eutropha H16 (ATCC 17699) and A. latus (ATCC 29713), respectively. In the case of R. eutropha H16, bacterial cells were grown under aerobic conditions at 30 °C for 24 h on a shaker in 12 500-mL Sakaguchi flasks (shaking flask with flat bottom, Iwaki Co., Tokyo) with a total of 1.5 L (125 mL per flask) of nutrient-rich medium containing 10 g/L of yeast extract (Oxoid, Ltd., Hampshire), 10 g/L of polypepton (Nihon Seiyaku Co., Ltd., Tokyo), 5 g/L of meat extract (Kyokuto Seiyaku Kogyo Co., Ltd., Tokyo), and 5 g/L of (NH4)2SO4. Then the bacteria were transferred into a fermenter (EYELA Jar Fermenter MBF-250M, Tokyo Rikakikai Co., Ltd.) with 1.5 L of mineral medium. The media contents were described in the previous paper.8 Ten grams per liter of 4HBA, 5 g/L of butyric acid, and 2 g/L of (NH4)2SO4 were added to the mineral medium. The cells were cultivated in the media under an aerobic condition at 30 °C for 48 or 120 h. The pH was regulated at 7.0 with 1 M NaOH and 1 M H3PO4. In the case of A. latus, the inocula for the fermentation were grown on a shaker in a 500-mL Sakaguchi flask with 100 mL of media using sucrose (10 g/L) as a carbon source under an aerobic condition at 30 °C for 24 h. Then the bacteria were transferred into the fermenter with 1.5 L of mineral medium. The media contents were described in the previous paper.15 Seven grams per liter of sucrose and 3 g/L of γ-butyrolactone were added to the mineral medium. The cells were cultivated in the media under an aerobic condition at 30 °C for 48 or 74 h. The pH was regulated at 7.0 with 1 M NaOH and 1 M H3PO4. The bacteria were harvested by centrifugation (5600g, 15 min), washed with distilled water, and lyophilized. Copolyesters were extracted from the lyophilized cells with hot chloroform using a Soxhlet apparatus and were purified by two precipitations in methanol and n-hexane. Fractionation Procedures. The as-produced copolyesters were fractionated by repeated solvent/nonsolvent fractionation procedures. A predetermined constant amount of
10.1021/bm049908y CCC: $27.50 © 2004 American Chemical Society Published on Web 04/03/2004
1136
Biomacromolecules, Vol. 5, No. 3, 2004
Notes
Table 1. Biosyntheses of P(3HB-co-4HB)s by R. eutropha H16 (Samples 1, 2) and A. latus (Samples 3, 4) at 30 °C and pH 7 sample
bacterial strain
carbon source
incubation time, h
cell dry weight, g L-1
polyester content,c wt %
4HB-unit content,d mol %
10-5Mwe
Mw/Mne
1 2 3 4
R. eutropha R. eutropha A. latus A. latus
a a b b
48 120 48 74
7.59 8.63 1.89 2.32
12.9 10.5 29.0 47.6
20.9 39.6 31.2 21.7
10.6 9.1 4.6 4.8
2.5 3.2 2.2 2.4
a 4HBA (10 g/L) and butyric acid (5 g/L) in the presence of ammonium sulfate (2 g/L). b Sucrose (7 g/L) and γ-butyrolactone (3 g/L). c Content of polyester in the dry cell. d Determined by 1H NMR. e Determined by GPC.
Table 2. Fractionation of Bacterial P(3HB-co-4HB)s (Samples 1, 3 and 4) with Chloroform/n-Heptane. samplea
fraction no.
concentration of n-heptane, vol %
weight ratio, wt %
4HB-unit content,b mol %
1
original 1 2 3 4
44 46 48 50
100 12.0 33.4 38.1 2.6
20.9 94.1 18.6 8.7 11.0
5 6
52 54
13.1 0.8
7.0 13.3
original 1 2 3 4 5e
50 54 56 64
100 4.4 38.4 32.4 17.4 7.4
original 1 2 3 4 5 6e
53 55 57 59 64
100 6.0 22.6 39.7 11.8 6.7 13.2
3
4
a
10-5Mwc
Mw/Mnc
10.6 4.3 10.0 10.5 12.2 0.7 9.4 5.6 0.3
2.5 1.9 2.7 1.5 1.4 1.3 1.5 1.6 1.2
31.2 46.9 37.0 24.1 23.8
4.6 7.8 6.5 3.8 1.1
2.2 2.0 1.3 1.7 1.9
21.7 35.4 21.1 15.7 14.3 15.9
4.8 7.7 6.7 5.4 1.8 0.7
2.4 1.7 1.7 1.4 1.4 1.3
Tm,d °C
Tg,d °C
146.4 53.0 50.4 149.6 140.8 137.9
-44.8 -46.3 3.6 2.8 1.3
133.7 128.5
0.6 -1.3
50.8 165.9 48.2 47.3 47.3 114.4
-11.5 -42.0 -15.1 -9.2 -9.8
56.1
55.0
56.5 52.8 48.8 52.1 49.5 49.4
127.8 152.8 134.3 120.2 124.2 119.4
-5.2 -0.2 -7.2 -4.2 -4.4 -4.9
See Table 1. b Determined by 1H NMR. c Determined by GPC. d Measured by DSC. e Unrecoverd fraction.
n-heptane (ethanol for sample 2; see Table 4) was carefully added to the copolyester/chloroform solution (initial polymer concentration, 10 mg/mL) with stirring at room temperature, and the precipitate was isolated by centrifugation (10 000g, 15 min). When no precipitates appeared, the same amount of nonsolvent was further added. After the precipitate was collected, the same amount of nonsolvent was again added to the remaining solution and the precipitate was isolated. This procedure was repeated until adding any amount of nonsolvent did not cause a precipitation. Six, three, four, and five fractions were obtained by these fractionations from samples 1-4 (original 4HB-unit content 20.9, 39.6, 31.2, and 21.7 mol %), respectively. Some fractions obtained with the chloroform/n-heptane and with chloroform/ethanol fractionation procedures turned out not to be well-fractionated by comonomer-unit composition, because they still showed multimelting peaks in the differential scanning calorimetry (DSC) thermograms. These fractions were dissolved again (ca. 1 mg/mL) and were refractionated with chloroform/ethanol (when the first fractionation was done with chloroform/n-heptane) or with chloroform/n-heptane (when the first one was done with chloroform/ethanol). In the case of refractionation with chloroform/ethanol, ethanol was carefully dropped into the chloroform solution with stirring at 0 °C until the solution
became slightly turbid (final ethanol concentration, 64 vol %). Then it was cooled to -20 °C with stirring and stored overnight at this temperature. The precipitate was isolated by centrifugation (10 000g, 15 min), and the solvent (chloroform/ethanol) was removed by rotoevaporation. By this method, two P(3HB-co-4HB) fractions, soluble and insoluble in chloroform/ethanol mixed solvent, were obtained. In the case of refractionation with chloroform/n-heptane, n-heptane was carefully dropped into the chloroform solution with stirring at room temperature. Three fractions (named r1, r2, and r3) were sampled at the n-heptane concentration of 44, 46, and 50 vol %, respectively, by the same method as that of the first-stage fractionation. Analytical Procedures. The 4HB-unit contents were estimated from 1H NMR spectra. The number-average (Mn) and the weight-average (Mw) molecular weights and the polydispersity index (Mw/Mn) were measured by gel permeation chromatography (GPC). The melting (Tm) and the glass transition (Tg) temperatures were measured by DSC. The details of the analytical procedures have been described in the previous paper.8 Results and Discussion Microbial Syntheses of P(3HB-co-4HB)s. Table 1 lists the results of biosyntheses of copolyesters by R. eutropha
Notes
Biomacromolecules, Vol. 5, No. 3, 2004 1137
Figure 1. DSC traces during the first heating scan of (a-c) the bacterial as-produced samples and their fractions obtained with chloroform/ n-heptane and (d) fraction 2 of sample 4 and its refractionated samples obtained with chloroform/ethanol. (a) Obtained for bacterial P(3HBco-4HB) with the 4HB-unit content of 20.9 mol % produced by R. eutropha H16; (b and c) obtained for those with the 4HB-unit contents of 21.7 and 31.2 mol % produced by A. latus, respectively.
H16 and A. latus at 30 °C. In this study, the two strains were used to biosynthesize P(3HB-co-4HB)s with similar comonomer-unit compositions. As the incubation time increased, the average 4HB-unit content of P(3HB-co-4HB) produced by R. eutropha increased but that produced by A. latus decreased. Molecular weights of bacterial P(3HB-co4HB)s produced by R. eutropha were relatively large compared with those by A. latus, though both were obtained after almost the same fermentation time. Polyester contents in the cells of A. latus were much greater than those in the cells of R. eutropha. It has been reported that A. latus produces P(3HB-co-4HB) more efficiently than R. eutropha.16 As previously suggested,16 A. latus may be unable to convert efficiently 4-hydroxybutyryl-CoA, which is produced by metabolizing γ-butyrolactone, to acetyl-CoA via β oxidation. As a result, much of 4-hydroxybutyryl-CoA may be introduced into the copolyester chain as the 4HB units. On the other hand, R. eutropha is able to metabolize efficiently 4-hydroxybutyryl-CoA to acetyl-CoA, and, thus, a large amount of 4-hydroxybutyryl-CoA may be converted into the 3HB units and also be used for energy and metabolic intermediate generation in the tricarboxylic acid cycle. The 4HB-unit contents of bacterial P(3HB-co-4HB)s from the two strains were very similar, as shown in Table 1. This should be explained from the difference of the comonomerunit compositional distribution. Fractionation of Bacterial P(3HB-co-4HB)s. Tables 2-4 list the results of solvent/nonsolvent fractionations for bacterial P(3HB-co-4HB)s. Crystalline copolymers are known to be fractionated by molecular weight, comonomer-unit composition, and crystallizability with the solvent/nonsolvent
system.17 Three as-produced copolyesters, that is, P(3HBco-20.9 mol % 4HB) produced by R. eutropha H16 and P(3HB-co-4HB)s with the 4HB-unit contents of 21.7 and 31.2 mol % by A. latus, were fractionated with chloroform/ n-heptane, while P(3HB-co-39.6 mol % 4HB) by R. eutropha H16 was fractionated with chloroform/ethanol. The tendencies of fractionations with respect to molecular weight and comonomer-unit composition were investigated. Table 2 shows the relationships between the n-heptane concentration in the mixed solvent and the molecular characteristics of the fractions, that is, 4HB-unit content, molecular weight, Tm, and Tg. Four to six fractions were obtained from the as-produced copolyesters by these fractionation procedures. In general, the higher the chloroform volumetric content in the mixed solvent, the higher were the 4HB-unit content and molecular weight of the resulted fraction. This tendency corresponds well with the fractionation results obtained for P(3HB-co-4HB)s with chloroform/ n-hexane.8 As listed in Table 2, each of the fractions 4 and 6 of sample 1 consist of two components with different molecular weights. One of them has a molecular weight more than 10 times larger than that of the other. We have reported such coexistence of two components with significantly different molecular weights in some precipitates obtained by the fractionation with chloroform/n-hexane for bacterial samples from R. eutropha H16, that is, the one with larger molecular weight and higher 3HB-unit content and the other with the smaller molecular weight and higher 4HB-unit content.8 However, the relative amount of the fraction with 4HB-rich and a small molecular weight was very small. On the other
1138
Biomacromolecules, Vol. 5, No. 3, 2004
Notes
Table 3. Refractionation with Chloroform/Ethanol of Fraction 2 of Sample 4 Obtained by Fractionation with Chloroform/n-Heptane sample
concentration of ethanol, vol %
fraction by chloroform/ethanol
weight ratio, wt %
4HB-unit content,a mol %
10-5Mwb
Mw/Mnb
Tm,c °C
Tg,c °C
fraction 2 of sample 4
64
soluble insoluble
62.4 37.6
28.2 7.9
5.1 5.7
1.8 1.9
52.7 134.7
-12.4 -0.2
a
Determined by 1H NMR. b Determined by GPC. c Measured by DSC.
Table 4. (a) Fractionation of Bacterial Sample 2, P(3HB-co-39.6 mol % 4HB), with Chloroform/Ethanol and (b) Refractionation of Fraction 1 of Sample 2 with Chloroform/n-Heptane Part a samplea 2
fraction no.
concentration of ethanol, vol %
weight ratio, wt %
4HB-unit content,b mol %
10-5Mwc
Mw/Mnc
original 1 2 3 4e
60 64 70
100 50.6 45.8 1.7 1.9
39.6 60.9 14.5 83.9
9.1 7.4 12.8 0.5
3.2 2.5 1.4 1.3
Tm,d °C
Tg,d °C
144.7 147.6 143.2 58.3
-45.1 -44.4 2.9 -42.9
Tm,d °C
Tg,d °C
53.8 53.6
Part b fraction no. fraction 1 of sample 2 r1 r2 r3 r4e a
concentration of n-heptane, vol %
weight ratio, wt %
4HB-unit content,b mol %
10-5Mwc
Mw/Mnc
44 46 50
100 42.4 17.9 35.4 4.3
60.9 96.0 72.8 9.0
7.4 3.7 5.9 13.0
2.5 1.6 2.5 1.2
53.6
147.6 59.4 52.3 149.8 146.5
-44.4 -44.5 -43.2 2.0
See Table 1. b Determined by 1H NMR. c Determined by GPC. d Measured by DSC. e Unrecovered fraction.
hand, as listed in Table 2, a relatively large amount of a 4HB-rich and large-molecular-weight fraction also exists (fraction 1 of sample 1; 12.0 wt %, 94.1 mol % 4HB, and Mw of 4.3 × 105). In this and in the previous study,8 it was found that the molecular weight distribution of 4HB-rich P(3HB-co-4HB)s from R. eutropha H16 was very broad compared with that of the 3HB-rich ones. The Tm values of the fractions are also listed in Table 2. Even after the fractionation, some fractions as well as bacterial unfractionated samples still show two melting points. In Figure 1 are shown the DSC thermograms of the fractionated samples together with those of the original unfractionated ones recorded during the first heating scan (a-c). As can be seen, some multi-endothermic peaks were observed in the low and high temperature regions (approximately 40-60 and 120-170 °C, respectively) for several fractions as well as some bacterial original samples. In the high-temperature region, two melting peaks were observed for some fractions and original samples. The peak at higher temperature was due to the recrystallized phase, in which the lattice of copolyester had enough time to be rearranged at a given DSC heating rate, because the intensity of this peak relative to that of the peak at a lower temperature decreased with increasing the heating rate.8 As described in Experimental Section, the refractionation with chloroform/ ethanol was carried out for a representative fraction having two melting points (except for the peak due to the recrystallization), that is, fraction 2 of sample 4 (21.1 mol % 4HB). Table 3 and Figure 1d show the refractionation results. Figure 1d displays the DSC thermograms of the fraction and its refractionated samples recorded during the first heating scan. As clearly seen in Table 3 and Figure 1d, the fraction was compositionally well-fractionated with chloroform/ethanol.
Figure 2. GPC curves of (a) the original as-produced sample 2 (39.6 mol % 4HB) and its fractions obtained by the first fractionation with chloroform/ethanol and (b) fraction 1 of sample 2 and its refractionated samples obtained with chloroform/n-heptane.
The 4HB-unit content of “soluble” fraction (28.2 mol %) was higher than that of “insoluble” fraction (7.9 mol %). This tendency was contrary to the fractionation using chloroform/n-heptane. Table 4a shows the relationships between the ethanol concentration in the mixed solvent and the molecular characteristics of the fractions obtained from P(3HB-co-39.6
Notes
Biomacromolecules, Vol. 5, No. 3, 2004 1139
composition and molecular weight) of these fractions. Figure 2 shows the GPC curves of the fractionated and the refractionated samples obtained for sample 2. Apparently, fraction 1 of sample 2 (60.9 mol % 4HB) had two peaks of molecular weight. By refractionation of this “fraction 1” with chloroform/n-heptane, it was found that one of two components having a higher molecular weight was 3HB-rich, while the other having a lower molecular weight was 4HB-rich (Table 4b, Figure 2b), corresponding well to the fractionation result of sample 1. As we can see in the GPC curve, “fraction r2” (refractionated sample, 72.8 mol % 4HB) from fraction 1 of sample 2 was not compositionally well-fractionated because it showed still two fractions with different molecular weights. This was confirmed from Figure 3, which shows “fraction r2” as well as fraction 1 of sample 2 having two melting peaks. So, this two-step fractionation procedure with first chloroform/ethanol fractionation and then chloroform/ n-heptane refractionation seems to be less efficient than that with first chloroform/n-heptane fractionation and then chloroform/ethanol refractionation. Figure 3. DSC traces during the first heating scan of (a) the original as-produced sample 2 (39.6 mol % 4HB) and its fractions obtained by the first fractionation with chloroform/ethanol and (b) fraction 1 of sample 2 and its refractionated samples obtained with chloroform/ n-heptane.
mol % 4HB) (sample 2) with chloroform/ethanol. Three fractions were obtained by this fractionation procedure. The amounts of fractions 1 and 2 were very large (50.6 and 45.8 wt %, respectively) compared with that of fraction 3 (1.7 wt %). There was no obvious relationship between the ethanol concentration and molecular characteristics (comonomer-unit
Comonomer-Unit Compositional Distributions of Bacterial P(3HB-co-4HB)s. As a result of fractionation with alternative solvent/nonsolvent systems, six and five fractions were obtained from bacterial P(3HB-co-4HB)s with the 4HBunit contents of 20.9 and 39.6 mol % produced by R. eutropha H16, respectively, and six and four fractions were obtained from bacterial P(3HB-co-4HB)s with those of 21.7 and 31.2 mol % produced by A. latus, respectively. Figure 4 displays comonomer-unit compositional distributions of P(3HB-co-4HB)s from R. eutropha H16 (a, b) and from A. latus (c, d), in which the relationships between the 4HB-
Figure 4. Comonomer-unit compositional distributions of (1) P(3HB-co-4HB)s biosynthesized by R. eutropha H16 using 4HBA and butyric acid as a mixed carbon source in the presence of ammonium sulfate and (2) those biosynthesized by A. latus using sucrose and γ-butyrolactone. Original 4HB-unit contents are (a) 20.9, (b) 39.6, (c) 21.7, and (d) 31.2 mol %.
1140
Biomacromolecules, Vol. 5, No. 3, 2004
unit content and the weight ratio (wt %) for all fractions are shown. The former copolyesters had bimodal distributions independent of the average comonomer-unit composition of unfractionated original samples; that is, as-produced samples from R. eutropha were mixtures of 3HB- and 4HB-rich copolyesters. On the other hand, the latter copolyesters had monomodal distributions. Because 3HB- and 4HB-rich P(3HB-co-4HB)s are immiscible in their blend,8 one needs to compositionally fractionate the bacterial P(3HB-co-4HB)s produced by R. eutropha for the evaluation of the precise relationships between the comonomer-unit composition and several properties, such as thermal and mechanical properties and biodegradability, which are known to depend on the solid-state phase structure. The difference in the comonomer-unit compositional distributions of the copolyesters biosynthesized by the two strains may be explained by their different metabolic systems. As mentioned previously, A. latus may be unable to metabolize 4-hydroxybutyryl-CoA to acetyl-CoA, so the 3HB and the 4HB units of P(3HB-co-4HB) may be supplied respectively from sucrose and γ-butyrolactone, while in the case of R. eutropha the 3HB units may be supplied from both 4HBA and butyric acid.16 Even in the case where 4HBA was used as a single carbon source for R. eutropha, the comonomer-unit compositional distribution was extremely broad and complicated (4HB-unit contents of three main fractions were 1, 28, and 82 mol %).7 Therefore, as long as the carbon source that is metabolized via β oxidation to acetyl-CoA is used for the incubation of R. eutropha, the resulting copolyester P(3HB-co-4HB) has broad comonomerunit compositional distribution, maybe bimodal distribution, although this kind of carbon source is essential for the production of P(3HB-co-4HB) by R. eutropha. As a result, it is unavoidable for R. eutropha to biosynthesize P(3HBco-4HB)s with a broad and complex comonomer-unit compositional distribution, while A. latus can produce P(3HB-co-4HB)s with a narrower and monomodal comonomer-unit compositional distribution. Conclusion In this paper, comonomer-unit compositional distributions of P(3HB-co-4HB)s biosynthesized by A. latus and by R.
Notes
eutropha H16 were studied. By carrying out a series of solvent/nonsolvent fractionations, as-produced P(3HB-co4HB) samples were compositionally well-fractionated into several fractions with different comonomer-unit compositions. There was a remarkable difference in the mode of comonomer-unit compositional distribution between P(3HBco-4HB)s produced by R. eutropha H16 and those by A. latus; the former had a bimodal distribution and the latter had a monomodal one. It was probably caused by their different metabolic systems. The evaluations of not only the comonomer-unit composition but also its distribution were found to be very important for the studies on the comonomerunit composition-dependent structure and properties of bacterial P(3HB-co-4HB)s. References and Notes (1) Doi, Y. Microorganisms and poly(3-hydroxyalkanoates). Microbial polyester; VCH Publishers: New York, 1990; pp 33-62. (2) Inoue, Y.; Yoshie, N. Prog. Polym. Sci. 1992, 17, 571-610. (3) Steinbu¨chel, A.; Valentin, H. E. FEMS Microbiol. Lett. 1995, 128, 219-228. (4) Kopinke, F.-D.; Remmler, M.; Mackenzie, K. Polym. Degrad. Stab. 1996, 52, 25-38. (5) Kunioka, M.; Nakamura, Y.; Doi, Y. Polym. Commun. 1988, 29, 174-176. (6) Nakamura, S.; Doi, Y.; Scandola, M. Macromolecules 1992, 25, 4237-4241. (7) Shi, F.; Ashby, R. D.; Gross, R. A. Macromolecules 1997, 30, 25212523. (8) Ishida, K.; Wang, Y.; Inoue, Y. Biomacromolecules 2001, 2, 12851293. (9) Hiramitsu, M.; Koyama, N.; Doi, Y. Biotechnol. Lett. 1993, 15, 461464. (10) Zhu, Z.; Dakwa, P.; Tapadia, P.; Whitehouse, R. S.; Wang, S.-Q. Macromolecules 2003, 36, 4891-4897. (11) Saito, Y.; Doi, Y. Int. J. Biol. Macromol. 1994, 16, 99-104. (12) Mitomo, H.; Hsieh, W.-C.; Nishiwaki, K.; Kasuya, K.; Doi, Y. Polymer 2001, 42, 3455-3461. (13) Wang, F.-L.; Lee, S.-Y. Appl. EnViron. Microbiol. 1997, 63, 37033706. (14) Grothe, E.; Moo-Young, M.; Chisti, Y. Enzymol. Microb. Technol. 1999, 25, 132-141. (15) Cao, A.; Ichikawa, M.; Kasuya, K.; Yoshie, N.; Asakawa, N.; Inoue, Y.; Doi, Y. Polym. J. 1996, 28, 1096-1102. (16) Saito, Y.; Nakamura, S.; Hiramitsu, M.; Doi, Y. Polym. Int. 1996, 39, 169-174. (17) Wang, Y.; Yamada, S.; Asakawa, N.; Yamane, T.; Yoshie, N.; Inoue, Y. Biomacromolecules 2001, 2, 1315-1323.
BM049908Y
