Biomacromolecules 2000, 1, 157-161
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Articles Microbial Degradation of Poly(aspartic acid) by Two Isolated Strains of Pedobacter sp. and Sphingomonas sp. Kenji Tabata, Hideki Abe, and Yoshiharu Doi* Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa, Wako-shi, Saitama 351-0198, Japan Received November 22, 1999; Revised Manuscript Received January 18, 2000
Microbial degradation of thermally synthesized poly(aspartic acid) (PAA) was investigated. A PAA-P1 sample (Mn, 7500; Mw, 20 000; number of branched units/100 monomer units, 3.1) was completely degraded in natural river water within 15 days at 25 °C. A new PAA-degrading bacterium (strain KP-2: JCM10638) was isolated together with Sphingomonas sp. KT-1 (JCM10459) from river water, and identified as a member of Pedobacter. A Pedobacter isolate was capable of degrading high-molecular-weight PAA polymers of 5000 to 150,000, and a small amount of low-molecular-weight products of 250 to 5000 was accumulated as residues during the growth of the isolate on PAA. In contrast, the other isolate Sphingomonas sp. KT-1 degraded only low-molecular-weight PAA below 5000. A mixed culturing of Pedobacter sp. KP-2 with Sphingomonas sp. KT-1 resulted in a complete degradation of PAA-P1 sample, but a small amount of low molecular weight components was accumulated during the degradation of highly branched PAA-P2 and PAA-P3 samples. Introduction Poly(aspartic acid) (PAA), belonging to the family of synthetic polypeptides, is a biodegradable water-soluble polymer. PAA polymers have the additional characteristic of each being a polycarboxylate polymer; such polymers have attracted attention as environmentally degradable watersoluble materials to be used as dispersants, as detergent builders, and in biomedical applications.1 PAA is produced by thermal polymerization of L-aspartic acid. Polysuccinimide (PSI) is synthesized as the initial product, and PAA is produced by hydrolysis of PSI,2,3 as shown in Scheme 1. The thermal polymerization of L-aspartic acid with or without catalyst leads to the formation of a mixture of L- and D-succinimide units, and the resulting PAA after hydrolysis of PSI is composed of 70% of β-amide and 30% of R-amide units.1-10 Pivcova´ et al.8 investigated the sequential structure of amide units in thermally synthesized PAA by 13C NMR analysis and concluded that the R- and β-amide units are randomly distributed in the PAA sequence. Wolk et al.10 characterized the thermally synthesized PSI prepolymer by using 1H, 13C, and 15N NMR techniques and reported that the resulting PAA contained branching units. Matsubara et al.11,12 characterized the end groups of thermally synthesized PAA and PSI prepolymer by NMR spectroscopy and demonstrated that the PAA had several irregular end groups resulting from deamination and dicarboxylation of * Corresponding author. Mailing address: Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa, Wako-shi, Saitama 351-0198, Japan. Telephone: +81-48(467)9402. Fax: +81-48(462)4667. E-mail: ydoi@ postman.riken.go.jp.
Scheme 1
aspartic acid. The irregular structures and molecular weights of PAA depend on their synthetic method. The thermal polymerization of L-aspartic acid in the absence of catalyst gives low-molecular-weight PAA below 10 000, while highmolecular-weight PAA of 10 000-90 000 can be produced in the presence of phosphoric acid as a catalyst.13 Wolk et al.10 reported that there was no difference in D/L ratio among PAA polymers synthesized with and without catalyst, while the amount of branched units decreased with an increase in the amount of phosphoric acid catalyst. The biodegradability of thermally synthesized PAA may be affected by the structures of branching and irregular end groups in PAA. Alford et al.14 prepared PAA polymers by the thermal polymerization of 14C-labeled L-aspartic acid in the absence of catalyst and evaluated the biodegradability of 14C-labeled PAA in activated sludge. A portion of PAA polymers remained undegraded in the sludge after degradation. They have suggested that some unusual structures in PAA such as β-amide units and D-aspartic acid units may limit the biodegradation of thermally synthesized PAA. Swift et al.15 and Freeman et al.16 prepared PAA polymers with and without branched units and studied the effect of branching on the biodegradability in activated sludge. A
10.1021/bm9900038 CCC: $19.00 © 2000 American Chemical Society Published on Web 03/09/2000
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linear PAA polymer prepared with a large amount of phosphoric acid catalyst was completely degraded in activated sludge, while a branched PAA polymer prepared without acid catalyst was partially degraded in activated sludge. They have suggested that the branched structure may limit the biodegradation of PAA. Recently, Nakato et al.17 prepared several PAA polymers, such as poly(R-L-aspartic acid), poly(R-D-aspartic acid), poly(β-L-aspartic acid), and poly(R,β-D,L-aspartic acid)s, and investigated the structural effects on biodegradability of PAA in activated sludge. Both the chirality of monomeric units and amide bond structures in PAA did not affect the biodegradability of PAA, while the biodegradability of PAA decreased with an increase in the amount of irregular end groups in PAA. The biodegradation of PAA chains must be caused by some bacteria in the natural environment. However, there was no report on the isolation of PAA-degrading bacteria from environments. In a previous study,18 we isolated a PAAdegrading bacterium (strain KT-1, JCM10459) from river water and identified as a member of genus of Sphingomonas. This bacterium degraded only low-molecular-weight PAA below 5000. However, high-molecular-weight PAA polymers over 5000 are degraded in activated sludge or in river water, which prompted us to isolate a high-molecular-weight PAAdegrading bacterium. In this paper, we report the isolation of a high-molecular-weight PAA-degrading bacterium from river water and the biodegradation of PAA polymers by isolated bacteria. Materials and Methods Materials. Three samples of poly(aspartic acid) (PAA) were used for the present study. Three PAA-P samples were obtained by hydrolyzing polysuccinimides (PSI) prepared by thermal polymerization of L-aspartic acid with different amounts of phosphoric acid catalyst. PAA-P samples were kindly gifted from Dr. B. Mohr of Polymer Laboratory, BASF, Ludwigshafen, Germany. Polymer analyses. Weight-average molecular weights (Mw) of polymers were determined by the both methods of static light scattering (SLS) and gel permeation chromatography (GPC). SLS was performed using a Wyatt technology DAWN DSP multiangle laser scattering photometer. This instrument, equipped with a He-Ne laser, was operated at a wavelength of 633 nm. A 0.3 M NaCl solution filtered through a 0.02 µm membrane filter was used as a solvent, and samples of PAA solution were filtered through a 0.2 µm membrane filter. Refractive index increment with respect to the change of PAA sample at 633 nm was determined by a refractometer (Ohtsuka, Osaka, Japan) (PAA-P1; 0.154, PAA-P2; 0.167, and PAA-P3; 0.169). Molecular weight analyses from light scattering data, plots of (Kc/Rθ) versus sin2(θ/2) (Zimm plot), were performed with Wyatt Technology DAWN software, where K is an optical constant, c is the polymer concentration, Rθ is the excess Rayleigh ratio of the solvent, and θ is the scattering angle. GPC analysis was performed using a Millennium 486 system (Waters) with a column system of Shodex OHpak SB-804 (Showadenko K. K., Tokyo, Japan) and Superdex
Tabata et al.
peptide (Amersham Pharmacia Biotech). Mw and numberaverage molecular weights (Mn) of PAA polymers were determined using poly(acrylic acid) standard samples for calibration. A 0.4 M solution of sodium nitrate was used for the mobile phase with a flow rate of 0.5 mL/min at 40 °C, and a refractive index detector was used. The solution 1H nuclear magnetic resonance (NMR) spectra of PSI samples in DMSO-d6 were recorded on a JEOL GSX 500 spectrometer. The 500-MHz 1H NMR spectra were recorded at 20 °C on a DMSO-d6 solution of PAA sample (100 mg ml-1) with 5 ms pulse width (45° pulse angle), 2.5 s pulse repetition, 500 Hz spectral width, 16 000 data points, and 16 accumulations. Tetramethylsilane (TMS) was used to construct a calibration curve. The 1H NMR assignments of PSI were made according to the report of Matsubara et al.11 Biodegradation Test. The biodegradabilities of PAA samples in river water (Arakawa river, Saitama, Japan) were measured by biochemical oxygen demand (BOD) assay and by GPC analysis. The procedures of BOD-biodegradation test were previously reported.19 Isolation and Identification of PAA-Degrading Bacterium. A freshwater sample 500 mL was taken from the river of Arakawa (Saitama, Japan) and filtered through a 0.45 µm pore size Millipore filter. The filter was incubated aerobically at 25 °C in 100 mL mineral medium containing 0.15% (w/ v) PAA-P1 sample as a substrate. The composition of mineral medium was as follows (per liter); 4.60 g of KH2PO4, 11.60 g of NaHPO4‚12H2O, 1.00 g of NH4Cl, 0.50 g of MgSO4‚ 7H2O, 0.05 g of CaCl2‚2H2O, 0.1 g of FeCl3‚6H2O, and 0.5 g of yeast extract. After 4 days of growth, an aliquot was taken and incubated for 4 days in a new medium of PAAP1. A fourth transfer was performed, and incubations were conducted under the same conditions. Samples from the liquid culture were plated on nutrient agar medium. The composition of nutrient agar medium was as follows (per liter): 5 g of peptone, 8 g of beef extract, and 20 g of agar. After several days, each colony was incubated in the mineral medium containing 0.15% (w/v) PAA-P1 sample at 25 °C, and the degradation of PAA was analyzed by GPC. One PAA-P degrading bacterium was isolated and labeled KP-2. Strain KP-2 has been deposited in the Japan Collection of Microorganisms under accession no. JCM10638. Identification of the isolate was initially performed with API 20 NE test (bio Me´rieux, Nu¨rtrigen, Germany). The sequence of 16S rDNA was determined by PCR using the MicroSeq 16S rRNA gene kit (PE Applied Biosystems).20 Related sequences were obtained from the GeneBank database (National Center for Biotechnology Information, National Library of Medicine) by using the BLAST search program. The sequences were aligned, and phylogenetic tree was constructed with the Genetics program (Software Development Co. Ltd., Japan) by using the neighbor-joining method and the Jukes-Cantor distance correction method.21 A total of 1000 bases available for all related sequences were used. Nucleotide Sequence Accession Numbers. The 16S rDNA sequence of the PAA degrading-strain KP-2 has been deposited in the GeneBank database under accession no.
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Microbial Degradation of Poly(aspartic acid) Table 1. Molecular Weights and Branching of Poly(aspartic acid) Samples Determined by GPC and SLS sample
MwGPCa
Mw/Mn (GPC)
MwSLSb
MwSLS/MwGPC
branched unitc (%)
PAA-P1 PAA-P2 PAA-P3
20000 16000 14000
2.6 2.7 2.8
9700 10500 8900
0.49 0.66 0.63
3.1 4.0 4.9
a Poly(acrylic acid) standards were used for calibration in GPC analysis. Light scattering (SLS) of PAA samples was measured in 0.3M NaCl. c The number of branched units per 100 monomer units was determined by 1H NMR analysis. b
AB033630. The accession numbers of other sequences used for determining levels of 16S rDNA similarity are as follows: FlaVobacterium aquatile; M62797, FlaVobacterium indoltheticum; M58774, Pedobacer heparinas; M11657, Sphingobacterium thalpophilum; M58779, Shingobacterium multiVorum; D14025, Shingobacterium spiritiVorum; M58778, and FlaVobacterium yabuuchiae; D14021. Measurement of PAA-Degrading Activity. Strain KP-2 was grown in a mineral medium containing 0.05%(w/v) yeast extract and 0.15%(w/v) carbon source at 25 °C for 3 days. The cells of strain KP-2 were harvested by centrifugation at 5000g for 15 min at 4 °C and washed with 10 mM sodium phosphate buffer (pH 7.0) and suspended in the same buffer. The suspension was sonically disrupted on ice for 10 min with a 20 kHz ultrasonic oscillator (Tomy Seiko Co. Ltd. Japan). The disrupted cells were centrifuged at 5000g for 15 min at 4 °C, and the resultant supernatant was used as the cell extract. The measuring of PAA-degrading activity was performed as follows. The cell extract was suspended in 10 mM sodium phosphate buffer (pH 7.0) containing 0.15% (w/v) PAA-P1 sample and incubated at 25 °C for 6 h. The molecular weight change of PAA-P1 sample in the solution was analyzed by GPC.
Figure 1. 1H NMR spectra of three polysuccinimide (PSI) samples in DMSO-d6.
Results and Discussion Characterization of Poly(aspartic acid) Structures. The molecular weights of PAA samples were determined by both GPC and SLS analyses. Table 1 shows the Mw and polydispersities (Mw/Mn) of PAA samples. The Mw values determined by GPC (MwGPC) were larger than those by SLS (MwSLS) for all samples, and the ratios of MwSLS to MwGPC of PAA samples were in the range 0.49-0.66. It is well-known that the molecular weight of a polymer defined by GPC depends on its hydrodynamic volume23 and that hydrodynamic volume of a branched polymer is smaller than that of a linear polymer having the same molecular weight.24 In contrast, the Mw vales determined by SLS are independent of polymer structure. Therefore, the ratio of MwSLS to MwGPC for a branched polymer should be larger than that for a linear polymer. Three PAA samples have similar values of MwSLS, while the ratio (0.49) of MwSLS/MwGPC for PAA-P1 is smaller than those (0.63-0.66) for the other PAA-P2 and PAA-P3 samples. This result suggests that PAA-P1 has a smaller amount of branched units. To investigate the branching structure of PAA samples, 1H NMR analyses of polysuccinimide (PSI) prepolymers were performed. Figure 1 shows the 1H NMR spectra of PSI samples in DMSO-d6. The
Figure 2. BOD-biodegradation curve of PAA-P1 in fresh river water at 25 °C. Inset shows time-dependent changes in molecular weights of PAA-P1 in fresh river water. A 0.175 mg/mL sample of PAA-P1 was incubated in the fresh river water at 25 °C, and the molecular weight was analyzed by GPC. Each line indicates incubation time as follows: s, 0 day; - - - , 2 days; ‚‚‚, 4 days; - ‚ -, 20 days.
methine proton resonances of PSI at 4.3-4.8 ppm have been assigned to those in succinimide units connecting with the branched units or ring-opening sites.11 Assuming that all of the methine protons arises from succinimide units connecting with branched units, the amount of branched units in PSIP1, -P2, and -P3 samples were estimated to be 3.1, 4.0, and 4.9 units per 100 monomeric units, respectively (Table 1). Isolation and Characterization of a PAA-Degrading Bacterium. The biodegradability of the PAA-P1 sample in river water was measured by BOD assay and GPC analysis. Figure 2 shows a typical BOD-biodegradation curve of PAAP1 in river water at 25 °C. The BOD-biodegradability of PAA-P1 sample increased with time to reach about 78% within 15 days, and PAA-P1 sample was completely degraded within 12 days as measured by GPC. In a previously study,18 we isolated a PAA-degrading bacterium
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Tabata et al. Table 2. Yields and PAA Degradation Activities of Strain KP-2 Grown on Various Carbon Sources
Figure 3. Phyrogenic tree based on the partial 16S rDNA gene sequence of strain KP-2 and the seven species belonging to the genus of Flavobactrium. The bar insert represents 5% sequence divergence as determined by measuring lengths of horizontal lines connecting any species.
(strain KT-1) belonging to a genus of Sphingomonas from river water. This bacterium was isolated using a lowmolecular-weight PAA-T sample (MwSLS ) 3200) as a carbon source, and could degrade only low-molecular-weight PAA below 5000. However, in river water, high-molecular-weight PAA-P polymers over 5000 were completely degraded. In this study, isolation of a high-molecular-weight PAAdegrading bacterium was attempted from river water using PAA-P1 sample as a carbon source. After the fourth transfer of the enrichment procedure in mineral media containing 0.15%(w/v) PAA-P1 and 0.05%(w/v) yeast extract, several bacterial colonies appeared on the nutrient agar plate. The PAA-P1-degrading activity of each colony was examined, and one bacterium capable of degrading PAA-P1 was isolated and labeled strain KP-2. Strain KP-2 grew aerobically, and oxidase activity was present. Nitrate was not reduced to nitrite, and indole was not produced. Strain KP-2 grew on glucose, N-acetyl-D-glucosamine, maltose, and D-mannose, while L-arabinose, D-mannitol, gluconate, n-capric acid, adipic acid, malic acid, citrate, and phenyl acetate did not support growth. β-Galactosidase and β-glucosidase were present, while urease, arginine dehydrolase and gelatinase were absent. The colony was circular and white. A partial 16S rDNA sequence of strain KP-2 (ca. 1000-bp) was determined and compared with the sequences of seven species belonging to the genus of FlaVobacterium. The GeneBank database was used to search for 16S rDNA sequences homologous to that of strain KP-2. Figure 3 illustrates the phylogenetic relationship of bacteria in the FlaVobacterium group. The closest relative to strain KP-2 was Pedobacter heparinas (92.3% similarity). On the basis of above taxonomic studies, strain KP-2 was identified as a member of Pedobacter, which belongs in the CytophagaFlaVobacterium-Bacteriods-Sphingobacterium group.22 The effect of carbon source on the growth of strain KP-2 was investigated by measurement of turbidity at 660 nm of culture media (Table 2). The expression of the PAAdegrading activity of strain KP-2 was examined with the cell extract prepared by an ultrasonic disintegration of KP-2 cells.
carbon sourcea
OD660b
PAA degradation activityc
glucose succinate L-alanine L-isoleucine L-valine L-aspartic acid D-aspartic acid L-asparagine L-glutamic acid PAA-P1 controld
0.37 0.31 0.49 0.16 0.12 0.58 0.30 0.49 0.48 0.34 0.30
+ -
a Strain KP-2 was grown in mineral medium containing 0.05% (w/v) yeast extract and 0.15% (w/v) carbon source at 25 °C for 3 days. b The turbidity of culture medium was measured at 660 nm. c PAA-degrading activity by cell extract was determined by GPC analysis. d The growth of strain KP-2 in a mineral medium containing only 0.05% yeast extract was regarded as a control.
PAA-degrading activities of strain KP-2 cell extract were evaluated from the time-dependent changes in the molecular weight of PAA-P1 by GPC analysis. As shown in Table 2, the growth of bacterium and PAA-degrading activities were affected by the kind of carbon substrates. The strain KP-2 grew on glucose, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, and PAA-P1. PAA-degrading activity was only detected in the presence of PAA-P1, while L-aspartic acid monomer did not induce any PAA-degrading activity, which suggests that a PAA-degrading activity of strain KP-2 is induced by PAA polymer. Microbial Degradation of PAA Samples. Strain KP-2 was incubated in the mineral medium containing 0.15% PAA sample and 0.05% yeast extract, and the time-dependent changes in the molecular weight of PAA samples in the medium were analyzed by GPC (Figure 4). The amount of PAA-P1 sample in the medium decreased with incubation time, and an original GPC peak of PAA-P1 sample disappeared after 20 days (Figure 4a). This result indicates that strain KP-2 is capable of degrading high-molecular-weight PAA polymers over 5000. During the course of incubation, low-molecular-weight products of 250-5000 were accumulated and their amounts increased with time. When strain KP-2 was incubated with other samples PAA-P2 and PAA-P3, the majority of the PAA polymers were degraded for 20 days, and low-molecular-weight products were also accumulated in the cultivation media. At present, the molecular structure of low-molecular-weight products is being investigated. As reported in a previous paper,18 Sphingomonas sp. KT-1 degraded low-molecular-weight PAA polymers below 5,000, which suggests us that Sphingmonas sp. KT-1 may be able to degrade the low-molecular-weight products accumulated in the media after PAA degradation by strain KP-2. The microbial degradations of PAA-P samples were carried out by a mixed cultivation of strain KP-2 and Sphingomonas sp. KT-1. Figure 5 shows the GPC profiles of PAA-P samples in during the course of mixed culture with strain KP-2 and Sphingomonas sp. KT-1. The amount of PAA-P1 polymer in the medium decreased with incubation time, and no product was accumulated (Figure 5a). After 12 days, all
Microbial Degradation of Poly(aspartic acid)
Figure 4. Time-dependent changes in molecular weights of PAAP1 (a), PAA-P2 (b), and PAA-P3 (c) in the culture media incubated with strain KP-2 at 25 °C. Each line indicates incubation time as follows: s, 0 day; - -, 2 days; - - -, 4 days; ‚‚‚, 20 days.
of the PAA-P1 polymers were degraded. Thus, a mixed culturing of strain KP-2 with Sphingomonas sp. KT-1 resulted in a complete degradation of the PAA-P1 sample containing a small amount of branched units. In the mixed culture, majority of PAA-P2 and PAA-P3 polymers were degraded, but a small portion of PAA polymers (Mr; 500010 000) and a small amount of low-molecular-weight products were still present after cultivation of 12 days. In conclusion, a mixed culturing of Pedobacter sp. KP-2 with Sphingomonas sp. KT-1 resulted in a complete degradation of PAA-P1 sample for 12 days. Acknowledgment. We gratefully acknowledge Dr. Bernhard Mohr of BASF, Ludwigshafen, Germany for supplying PAA and PSI samples. This work was supported by CREST (Core Research for Evolutional Science and Technology) of the Japan Science and Technology Corporation (JST). References and Notes (1) Roweton, S.; Huang, S. J.; Swift, G. J. EnViron. Polym. Degrad. 1997, 5, 175. (2) Harada, K. J. Org. Chem. 1959, 24, 1662. (3) Vegotsky, A.; Harada, K.; Fox, S. W. J. Am. Chem. Soc. 1959, 80, 3361. (4) Kokufuta, E.; Suzuki, S.; Harada, K. Bull. Chem. Soc. Jpn. 1978, 51, 1555. (5) Kovacs, J.; Kovacs, H. N.; Ko¨nyves, I.; Csa´sza´r, J.; Vajda, T.; Mix H. J. Org. Chem. 1961, 26, 1084. (6) Matsuyama, M.; Kokufuta, E.; Kusumi, T.; Harada, K. Macromolecules 1980, 13, 196.
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Figure 5. Time-dependent changes in molecular weights of PAAP1 (a), PAA-P2 (b), and PAA-P3 (c) in the culture media incubated with a mixture of strain KP-2 and Sphingomonas sp. KP-1 at 25 °C. Each line indicates incubation time as follows: s, 0 day; - -, 2 days; - - -, 4 days; ‚‚‚, 12 days. (7) Pivcova`, H.; Saudek, V.; Dronbnik, H. Polymer 1982, 23, 1237. (8) Pivcova`, H.; Saudek, V.; Dronbnik, J.; Vlasak, J. Biopolymer 1980, 20, 1605. (9) Rao V. S.; Lapointe, P.; McGregor, D. N. Makromol. Chem. 1993, 194, 1095. (10) Wolk, S. K.; Swift, G.; Paik, Y. H.; Yocom, K. M.; Smith, R. L.; Simon, E. S. Macromolecules 1994, 27, 7613. (11) Matsubara, K.; Nakato, T.; Tomida, M. Macromolecules 1997, 30, 2305. (12) Matsubara, K.; Nakato, T.; Tomida, M. Macromolecules 1998, 31, 1466. (13) Neri, P.; Antoni, G.; Benvenuti, F.; Cocola, F.; Gazzei, G. J. Med. Chem. 1973, 16, 893. (14) Alford, D. D.; Wheeler, A. P.; Pettigrew, C. A. J. EnViron. Polym. Degrad. 1994, 2, 225. (15) Swift, G.; Freeman, M. B.; Paik, Y. H.; Simon, E.; Wolk, S. K.; Yocom, K. M. Macromol. Symp. 1997, 123, 195. (16) Freeman, M. B.; Paik, Y. H.; Swift, G.; Wilczynski, R.; Wolk, S. K.; Yocom, K. M. Polym.r Repr. 1994, 35, 423. (17) Nakato, T.; Toshitake, M.; Matsubara, K.; Tomida, M.; Kakuchi, T. Macromolecules 1998, 31, 2107. (18) Tabata, K.; Kasuya, K.; Abe, H.; Masuda, K.; Doi, Y. Appl. EnViron. Microbiol. 1999, 65, 4268. (19) Ohura, T.; Aoyagi, Y.; Takagi, K.; Yoshida, Y.; Kasuya, K.; Doi, Y. Polym. Degrad. Stab. 1999, 63, 23. (20) Sakaki, R. K.; Gelfand, D. H.; Stoffe, S.; Scharf, S. J.; Higuch, R.; Horn, G. T.; Mullis, K. B.; Erlich, A. Science 1988, 239, 487. (21) Saitou, N.; Nei, M. Mol. Biol. EVol. 1987, 4, 406. (22) Steyn, P. L.; Segers, P.; Vancanneyt, M.; Sandra, P.; Kersters, K.; Joubert. J. J. Inter. J. System. Bacteriol. 1998, 48, 165. (23) Grubisic, Z.; Rempp, P.; Benoit, H. J. Polym. Sci., B, Polym. Lett. 1968, 5, 753. (24) Kurata, M.; Fukatsu, M. J. Chem. Phys. 1964, 41, 2934.
BM9900038
