Purification and Characterization of Poly (aspartic acid) Hydrolase

Kenji Tabata,† Mariko Kajiyama,‡ Tomohiro Hiraishi,† Hideki Abe,† Ichiro Yamato,‡ and. Yoshiharu Doi*,†. Polymer Chemistry Laboratory, RIK...
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Biomacromolecules 2001, 2, 1155-1160

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Purification and Characterization of Poly(aspartic acid) Hydrolase from Sphingomonas sp. KT-1 Kenji Tabata,† Mariko Kajiyama,‡ Tomohiro Hiraishi,† Hideki Abe,† Ichiro Yamato,‡ and Yoshiharu Doi*,† Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa, Wako-shi, Saitama 351-0198, Japan; and Department of Biological Science and Technology, Science University of Tokyo, 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan Received April 14, 2001; Revised Manuscript Received July 23, 2001

Poly(aspartic acid) (PAA) hydrolase was purified from Sphingomonas sp. KT-1 (JCM10459). The purified hydrolase degraded thermally synthesized PAA to oligomers. The molecular mass of PAA hydrolase was 30 kDa and the isoelectric point was 8.9. The optimum values of pH and temperature for PAA degradation were 10.0 and 40 °C, respectively. The investigation of the effect of inhibitors for the PAA-degrading activities has revealed that the PAA hydrolase is a serine-type hydrolase. The structural analysis of PAAdegraded products using 1H and 13C nuclear magnetic resonances has indicated that the purified enzyme hydrolyzes selectively the β-amide linkage connecting with β-aspartic acid units in PAA. Introduction Poly(aspartic acid) (PAA), belonging to a family of synthetic polypeptide, is a biodegradable water-soluble polymer. PAA can be synthesized by a hydrolysis of polysuccinimide, formed by thermal polymerization of L-aspartic acid with or without catalyst.1-7 The thermal polymerization of L-aspartic acid leads to the formation of a mixture of L- and D-succinimide units, and the resulting PAA after hydrolysis of polysuccinimide is composed of 30% of R-amide and 70% of β-amide units1,8-12 (Structure 1).

The biodegradation of PAA has been investigated in activated sludge and natural freshwater. Alford et al.13 investigated the biodegradation of PAA prepared by the thermal polymerization without catalyst and reported that thermally synthesized PAA without catalyst was partially degraded in activated sludge and contained limited degradable structures. Wolk et al.12 reported that thermally synthesized PAA without catalyst had branched units as byproducts, while thermally synthesized PAA with phosphoric acid catalyst was a linear polymer. Freeman et al.14 and Swift et al.15 investigated the biodegradability of PAA polymers synthesized with and without catalyst and dem* Corresponding author. Telephone: +81-48(467)9402. Fax: +81-48(462)4667. E-mail: [email protected]. † RIKEN Institute. ‡ Science University of Tokyo.

onstrated that a linear PAA sample was completely degraded in activated sludge. In addition, Nakato et al.16 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. Thus, the biodegradability of PAA was affected by the irregular structures of a PAA sequence. On the other hand, there had been a few reports on the isolation of PAA-degrading bacteria and the hydrolysis of PAA using pure cultures and pure enzymes. In our previous studies, we isolated two types of PAA-degrading bacteria (Sphingomonas sp. KT-1 (JCM10459) and Pedobacter sp. KP-2 (JCM10638)) from river freshwater.17,18 Sphingomonas sp. KT-1, first isolated bacterium, could completely degrade PAA of low-molecular-weights below 5000. On the other hand, Pedobacter sp. KP-2 could degrade high-molecularweight PAA of 50 000 to 150 000 to yield oligomers as product. A mixed culturing of Pedobacter sp. KP-2 with Sphingomonas sp. KT-1 resulted in a complete degradation of high-molecular-weight PAA synthesized by thermal polymerization with catalyst. In a previous paper,17 we reported that the cell extract of Sphingomonas sp. KT-1 hydrolyzed high-molecular-weight PAA polymer to yield aspartic acid monomer. This result indicates that Sphingomonas sp. KT-1 has some enzymes capable of hydrolyzing unusual amide linkages such as β-amid linkage. The study of PAA-degrading enzymes will give a greater insight about the mechanism involved in PAA degradation which can further lead to research on selective modification in the polymer structure and precise control of polymer architecture. In this paper, we purify a PAA-degrading enzyme from Sphingomonas sp. KT-1 and characterize its properties. This

10.1021/bm0155468 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/29/2001

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is the first report of the purification and characterization of PAA-degrading enzyme. Materials and Methods Materials. Poly(aspartic acid) (PAA) sample was kindly given by Dr. Mohr of the Polymer Laboratory, BASF, Ludwigshafen, Germany. The PAA was obtained by hydrolyzing polysuccinimide prepared by thermal polymerization of L-aspartic acid with phosphoric acid as a catalyst and medium for dissolving crystalline aspartic acid at 160-200 °C. The molecular weight of polymer was determined by gel permeation chromatography (GPC) (Millennium 486 system, Waters) with column system of Shodex OHpak SB804 (Showadenko K. K., Japan) and Superdex peptide (Amersham Pharmacia Biotech). Poly(acrylic acid) standard samples (Polymer science) were used for calibration. Sodium nitrate (0.4 M) was used for mobile phase and flow rate was 0.5 mL/min, and a refractive index detector was used. Other chemicals were purchased from Kanto Chemicals (Tokyo, Japan) or Wako Chemicals (Osaka, Japan). Organism and Growth Condition. A strain of Sphingomonas sp. KT-1 (JCM10459), isolated from river water was reported in our previous study.17 The strain KT-1 was grown in mineral medium at 25 °C. The composition of mineral medium (pH 7.0) 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, 1.5 g of L-aspartic acid, and 0.5 g of yeast extract. The strain KT-1 was aerobically cultivated for 24 h in a 10-L jar fermentor containing 6 L of mineral medium. The agitation speed and aeration rate were 200 rpm and 3 L/min, respectively. Analytical Procedures of Enzyme. The PAA-degrading activity of enzyme was simply determined as follows. First, 0.15 mg of PAA was added in 80 µL of 10 mM sodium borate buffer (pH 8.0) at 30 °C in 96-well microplate. The reaction was started by the addition of 10 µL of enzyme solution. After incubation at 30 °C for 30 min, the reaction was terminated by cooling the plate on ice. Then, 10 µL of 1 M sodium chloride and 100 µL of ethanol were added in the solution to precipitate unreacted PAA polymers, and the solution was mixed for 1 min. The concentration of unreacted polymer was determined by measuring the turbidity at 595 nm using a microplate reader (Bio-Rad), although the value of turbidity was depended on molecular weight and concentration. One unit of enzyme was defined as the amount of protein required to decrease the value of turbidity at 595 nm by 1 per hour. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on slab gels containing 15% (w/v) of acrylamide in the buffer system of Laemmli.19 Precision SDS-PAGE standards (Bio-Rad) were used as molecular mass standards. Protein concentrations were determined by the method of Lowry et al.,20 using the Bio-Rad DC Protein Assay Kit with bovine serum albumin as the standard. Purification of Poly(aspartic acid) Hydrolase. Cells of Sphingomonas sp. KT-1 were harvested by centrifugation at 5000g for 15 min at 4 °C and washed with 10 mM sodium

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phosphate buffer (pH 7.0). The cells were suspended in the same buffer. The suspension was sonically disrupted on ice for 30 min with a ultrasonic oscillator (20 kHz, 70W, Tomy Seiko Co. Ltd. Japan). The disrupted cells were centrifuged at 150000g for 1 h at 4 °C, and the resultant supernatant was used as soluble fraction. All purification steps were carried out at 0-4 °C. The soluble fraction was applied to a cation exchange chromatography column (2.6 cm o.d. by 10 cm) containing SP Sepharose HP (Amersham Pharmacia Biotech), equilibrated with 10 mM sodium phosphate buffer (pH 7.0). The column was washed with two bed volumes of the buffer, and adsorbed proteins were eluted at a flow rate of 5 mL/min with a linear gradient (500 mL) from 0 to 250 mM NaCl in 10 mM sodium phosphate buffer. The fractions with PAAdegrading activity were pooled and dialyzed using regenerated cellulose membrane (10 000 Mw cut off) against 10 mM sodium phosphate buffer (pH 7.0). The dialyzed fractions were applied to a hydroxyapatite chromatography column (1.6 cm o.d. by 10 cm) containing ceramic hydroxyapatite (BioRad), equilibrated with 10 mM sodium phosphate buffer (pH 7.0). The column was washed with two bed volumes of the equilibrated buffer, and the enzyme was eluted at a flow rate of 2 mL/min with a linear gradient (100 mL) from 0 to 250 mM potassium phosphate buffer (pH 7.0). The fractions with PAA-degrading activity were pooled and dialyzed using regenerated cellulose membrane (10 000 Mw cut off) against 10 mM sodium phosphate buffer (pH 7.0). The dialyzed fractions were applied to a cation exchange column (0.5 cm o.d. by 5 cm) containing Mono S (Amersham Pharmacia Biotech), equilibrated with 10 mM sodium phosphate buffer (pH 7.5). The column was washed with five bed volumes of the buffer, and the enzyme was eluted at a flow rate of 0.5 mL/min with a linear gradient (40 mL) from 0 to 150 mM NaCl in 10 mM sodium phosphate buffer (pH 7.5). The enzyme was eluted at 50-80 mM NaCl. The enzyme was dialyzed using regenerated cellulose membrane (10 000 Mw cut off) against 10 mM sodium phosphate buffer (pH 7.0). Peptide Sequencing. For amino-terminal amino acid sequencing, the proteins separated by SDS-PAGE were transferred electrophoretically onto Immobilon-P transfer membrane and visualized by staining with Coomassie brilliant blue R-250. The band of PAA hydrolase was excised out and subjected to amino-terminal amino acid sequence analysis on an Applied Biosystems 473A protein sequencer. Determination of Structure of PAA Degraded Products. First, 20 mg of PAA in 1 mL of 10 mM sodium borate buffer (pH 8.0) was incubated in the presence of 3 units of purified PAA hydrolase at 30 °C for 24 h. The solution was mixed with 25 mL of acetonitrile in order to precipitate the degraded products. After centrifugation (15 min at 5000g), precipitate was dried in vacuo. The precipitate was dissolved in 200 µL of D2O (pH 8.0), and the spectra of 1H and 13C nuclear magnetic resonances (NMR) were measured. The solution 1H NMR spectra of reaction products in D O were recorded 2 on a JEOL GSX 500 spectrometer. The 500 MHz 1H NMR spectra were recorded on a D2O solution of reaction product (100 mg/mL) with 1 µs pulse width (45° pulse angle), 2.2 s pulse repetition, 500 Hz spectral width, 16 000 data points,

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Figure 1. SDS-PAGE of the purified PAA hydrolase: lane 1, molecular mass standards, with the mass indicated on the left; lane 2, crude extract of Sphingomonas sp. KT-1; lane 3, active fraction after chromatography on SP-Sepharose; lane 4, active fraction after chromatography on hydroxyapatite; lane 5, purified PAA hydrolase after chromatography on Mono S. The arrowhead indicates the position of the PAA hydrolase. Table 1. Purification of PAA Hydrolase from Sphingomonas sp. KT-1 step soluble fraction SP Sepharose HP hydroxyapatite Mono S

total activity total specific activity yield (units) protein (mg) (units/mg) (%) 5300 720 820 640

3100 100 6.4 4.6

1.8 72 130 140

100 14 15 12

and 16 accumulations. Dimethylsilane pentasulfonate was used to construct a calibration curve. The solution 13C NMR spectra of reaction products in D2O were recorded on a JEOL GSX 500 spectrometer. The 125 MHz 13C NMR spectra were recorded on a D2O solution of reaction product (100 mg/ mL) with 1 µs pulse width (45° pulse angle), 1.2 s pulse repetition, 500 Hz spectral width, 33 000 data points, and 36 000 accumulations. The 1H and 13C NMR assignments of reaction product and PAA were made according to the reports of Wolk et al.12 and Matsubara et al.21 Results and Discussion Purification of Poly(aspartic acid) Hydrolase from Sphingomonas sp. KT-1. We purified a PAA hydrolase to homogeneity from strain KT-1 as described in the Materials and Methods. As listed in Table 1, PAA hydrolase was purified 78-fold, recovering 12% of the activity present in the initial soluble fraction after purification with Mono S column. SDS-PAGE analysis of combined fractions containing PAA hydrolase activity, eluted from the Mono S column, revealed a single polypeptide band at 30 kDa (Figure 1). The amino-terminal amino acid sequence of PAA hydrolase was determined as APAAASKGKAAALPDLKPGAGSFLFTG by automated Edman degradation. The result of a BLASTP search has indicated that the amino-terminal amino acid sequence of purified PAA hydrolase has no similarity to other proteins. The isoelectric point of the enzyme was 8.9. The pH dependency on PAA hydrolysis by the purified PAA hydrolase was determined in 10 mM sodium phosphate buffer (pH 5-8) or 10 mM sodium borate buffer (pH 8-10.5) (Figure 2a). The PAA-degrading activi-

Figure 2. Effects of pH (a) and temperature (b) on PAA-degrading activity. (a) Reactions of PAA degradation were performed at different pH values at 30 °C in 10 mM sodium phosphate buffer (b) or 10 mM sodium borate buffer (O). (b) Reactions of PAA degradation were performed at different temperatures in 10 mM sodium borate buffer (pH 8.0). PAA-degrading activity at 30 °C in 10 mM sodium borate buffer (pH 8.0) was defined as 100%. Table 2. Effect of Protease Inhibitors on the Activity of PAA Hydrolasea inhibitor

relative activity (%)

none DFP (3 mM) PMSF (10 mM) EDTA (1 mM) pepstatin (8 µM) aprotinin (1 µM) E64 (0.1 mM)

100 ( 8 23 ( 5 30 ( 3 100 ( 5 99 ( 9 98 ( 8 100 ( 2

a PAA hydrolase (0.4 µM) was incubated with each inhibitor for 15 min at 30 °C in 10 mM sodium borate buffer (pH 8.0). After incubation, PAA degrading activities were measured. DFP: diisopropyl fluorophosphates; PMSF: phenylmethane sulfonyl fluoride; E64: N-[N-(L-3-trans-carboxirane-2-carbonyl)-L-leucyl]agmatine.

ties were found in the buffer between pH 6.0 and 10.5. The optimum pH for enzymatic activity was 10.0, and the activity at pH 8.0 was 53% of maximum activity. As shown in Figure 2b, the optimum temperature was 40 °C. The PAA hydrolase activity remained unchanged after 3 h incubation at 40 °C. The effects of protease inhibitors on PAA-degrading activity of purified PAA hydrolase were investigated. The result is shown in Table 2. The PAA-degrading activity was significantly inhibited by the addition of diisopropyl fluorophosphates (DFP) or phenylmethane sulfonyl fluoride (PMSF), while EDTA and the other protease inhibitors hardly inhibited the enzyme activity. It is well-known that DFP and PMSF strongly inhibit a serine-type hydrolase.22,23 This result indicates that the PAA hydrolase from Sphingomonas sp. KT-1 is a serine-type hydrolase. Enzymatic Degradation of Poly(aspartic acid). The degradation of PAA by purified PAA hydrolase was examined by GPC. The time-dependent changes in GPC profile of PAA by treatment with purified PAA hydrolase are shown in Figure 3. The molecular weight of PAA sample before degradation was distributed from 1000 to 150 000, and the number-average and weight-average molecular weights were respectively 7500 and 20 000. The elution curves of degraded products were shifted toward lower molecular weights. After incubation for 24 h, the peak value of product corresponded to the oligomers with five to six aspartyl residues. As shown in Figure 3, the production of aspartic acid monomer was

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Figure 3. Time-dependent changes in molecular weights of PAA treated by PAA hydrolase. PAA was incubated with PAA hydrolase in borate buffer (pH 8.0) at 30 °C.

hardly detected during the course of degradation reaction with purified enzyme. The distribution of low-molecularweight products remained almost unchanged by further incubation of 24 h with the addition of fresh PAA hydrolase. These results indicate that the purified PAA hydrolase hydrolyzes a portion of amid linkages in PAA to yield oligomers of aspartic acid. The thermally synthesized PAA contains both R-unit and β-unit in a main chain. Additionally, R,β-aspartyl residues and R,β-dicarboxylic acid residues are present at the chain end of PAA. Figure 4 shows the structures of R,β-monomeric units in a main chain and of end groups of PAA polymer and degradable products. The structures of degraded products and PAA polymer were characterized by using NMR spectroscopy.12,21 1H NMR spectra of PAA polymer (a) and degraded products (b) are shown in Figure 5. The methylene proton resonances of PAA polymer were observed between 2.3 and 3.0 ppm. The two peaks at 4.47 and 4.66 ppm have been assigned to the methine protons of R- and β-units in a main chain, respectively. The mole ratio of R-unit to β-unit calculated from the peak areas was 30 to 70 before degradation. The peaks arising from the methine protons of amino end groups and dicarboxylic acid end groups were hardly detected before enzymatic degradation. In the 1H NMR spectrum (b) of degraded products, a new peak was detected at 4.01 ppm arising from the methine proton (14) of β-aspartyl residues. The peak of the methine proton (10) from R-aspartyl residues was not detected at 4.22 ppm.21 The peaks arising from the methine proton (18, 22) of dicarboxylic acid end groups were overlapped at 4.35-4.55 ppm with the peak (6) from the β-unit in the main chain. The amount of dicarboxylic acid end groups in the degraded products was almost consistent with that of amino end group since both the amino end and the dicarboxylic acid end groups are produced by the hydrolysis of an amino linkage in PAA by enzyme. On the basis of the 1H NMR spectrum

Figure 4. Structures of monomeric units in main chain and end groups for PAA polymer and degraded products.

Figure 5. 1H NMR spectra of PAA and degraded products in D2O. (a) PAA; (b) degraded products after 24 h of enzymatic hydrolysis.

(b) of degraded products, the mole ratios of aspartic acid units for R-unit in main chain: β-unit in main chain:

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Figure 6. Sequential structures of diad units in PAA polymer.

dicarboxylic acid end groups: β- aspartyl residues were determined as 32:44:12:12 from the intensities of methine proton resonances of degraded products, respectively. The fraction of β-monomeric units in main chain of degraded products was decreased to 44 mol % from 70 mol % (the fraction in original PAA polymer) by the enzymatic hydrolysis for 24 h, while the fractions (30-32 mol %) of R-monomeric units in main chain remained almost unchanged during the course of enzymatic hydrolysis. Since both R- and β-units are connecting randomly in a polymer chains, the four types of sequential diads are present in PAA polymer prepared by thermal polycondensation (Figure 6). The 13C resonances of carbonyl carbon in amide linkages are resolved into three peaks due to diad sequences as shown in Figure 7. The largest intense peak at 172.0 ppm is assigned to carbonyl resonance of β-β diad sequence, while the smallest peak at 173.3 ppm is arising from the carbonyl carbon of R-R diad sequence.12 The middle peak is the sum of amide carbonyl carbons of R-R and β-R diads. Before degradation, the ratios of these peak areas were 1:4:5 in the spectrum of Figure 7a. In contrast, the ratios of three peak areas were 1:4:3 in the spectrum obtained after 24 h of enzymatic hydrolysis of PAA (Figure 7b). Thus, the peak area of amide carbonyl carbon of β-β diad sequence was only reduced during the enzymatic hydrolysis of PAA with purified enzyme. In contrast, the peak area of carbonyl carbons (13, 16) of β-aspartic residues increased during the enzymatic hydrolysis. These results indicate that the purified PAA hydrolase is an endo-type hydrolase and hydrolyzes selectively the amide linkage of β-β diad sequence in a PAA chain. In a previous paper,17 we reported that the cell extract of Sphingomonas sp. KT-1 hydrolyzed high-molecular-weight PAA of 5000 to 150 000 to yield aspartic acid monomer as a main product for 9 h. However, in this study, highmolecular-weight PAA was hydrolyzed into oligomers by the purified enzyme, and the production of aspartic acid monomer was not observed (Figure 3). In the purification procedure, the total activity was significantly decreased at the first cation exchange chromatography step (Table 1). The loss of activity may be caused by the removal of other enzymes relating to the PAA degradation. The purified PAA hydrolase should be one of the enzymes involving in the degradation of PAA in Sphingomonas sp. KT-1 cells. Thermally synthesized PAA is composed of both the R- and

Figure 7. 13C NMR spectra of carbonyl carbon in PAA and degraded products in D2O: (a) PAA; (b) degraded products after 24 h of enzymatic hydrolysis.

β-units of aspartic acid. The four types of diad sequences exist in a polymer chain, and the β-β diad is major sequence. The present study has demonstrated that the purified PAA hydrolase hydrolyzes selectively β-β diad sequence in PAA to yield oligomers and is inactive for the hydrolysis of R-R diad sequence. From these results, it is concluded that the PAA hydrolase purified in this study is one of enzymes involving in the PAA degradation. Acknowledgment. We gratefully acknowledge Dr. Bernhard Mohr of BASF, Germany for supplying PAA sample.This work was supported by Ecomolecular Science Research Project of RIKEN Institute. 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) Tomida, M.; Nakato, T.; Kuramachi, M. Polymer 1996, 37, 4435. (6) Nakato, T.; Kusuno, A.; Kakuchi, T. J. Polym. Sci., Part A 2000, 38, 117. (7) Kovacs, J.; Kovacs, H. N.; Ko¨nyves, I.; Csa´sza´r, J.; Vajda, T.; Mix, H. J. Org. Chem. 1961, 26, 1084. (8) Neri, P.; Antoni, G.; Benvenuti, F.; Cocola, F.; Gazzei, G. J. Med. Chem. 1973, 16, 893. (9) Matsuyama, M.; Kokufuta, E.; Kusumi, T.; Harada, K. Macromolecules 1980, 13, 196. (10) Pivcova`, H.; Saudek, V.; Dronbnik, J.; Vlasak, J. Biopolymer 1980, 20, 1605. (11) Pivcova`, H.; Saudek, V.; Dronbnik, H. Polymer 1982, 23, 1237.

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(12) Wolk, S. K.; Swift, G.; Paik, Y. H.; Yocom, K. M.; Smith, R. L.; Simon, E. S. Macromolecules 1994, 27, 7613. (13) Alford, D. D.; Wheeler, A. P.; Pettigrew, C. A. J. EnViron. Polym. Degrad. 1994, 2, 225. (14) Freeman, M. B.; Paik, Y. H.; Swift, G.; Wilczynski, R.; Wolk, S. K.; Yocom, K. M. Polym. Reprints 1994, 35, 423. (15) Swift, G.; Freeman, M. B.; Paik, Y. H.; Simon, E.; Wolk, S. K.; Yocom, K. M. Macromol. Symp. 1997, 123, 195. (16) Nakato, T.; Yoshitake, M.; Matsubara, K.; Tomida, M.; Kakuchi, T. Macromolecules 1998, 31, 2107. (17) Tabata, K.; Kasuya, K.; Abe, H.; Masuda, K.; Doi, Y. Appl. EnViron. Microbiol. 1999, 65, 4268.

Tabata et al. (18) Tabata, K.; Abe, H.; Doi, Y. Biomacromolecules 2000, 1, 157. (19) Leammli, U. K. Nature 1970, 227, 680. (20) Lowry, O. H.; Roserbrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193, 265. (21) Matsubara, K.; Nakato, T.; Tomida, M. Macromolecules 1998, 31, 1466. (22) Kita, K.; Ishimaru, K.; Teraoka, M.; Yanase, H.; Kato, N. Appl. EnViron. Microbiol. 1995, 61, 1727. (23) Nakamura, K.; Tomita, T.; Abe, H.; Kamio, Y. Appl. EnViron. Microbiol. 2001, 67, 345.

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