Enzymatic Hydrolysis of Bacterial Poly(3-hydroxybutyrate-co-3

Biomacromolecules 2002, 3, 828-834

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Enzymatic Hydrolysis of Bacterial Poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by Poly(3-hydroxyalkanoate) Depolymerase from Acidovorax Sp. TP4 Yi Wang,† Yasuhide Inagawa,† Terumi Saito,‡ Ken-ichi Kasuya,§ Yoshiharu Doi,†,| and Yoshio Inoue*,† Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan, Faculty of Science, Kanagawa University, 2946 Tsuchiya, Hiratsuka, Kanagawa 259-1293, Japan, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan, and The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama 351-0198, Japan Received February 8, 2002; Revised Manuscript Received April 16, 2002

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and AcidoVorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by highperformance liquid chromatography and one- (1D) and two-dimension (2D) 1H NMR spectroscopy. It was found that the PHA depolymerase of AcidoVorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from AcidoVorax Sp. TP4 degraded the P(3HBco-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from AcidoVorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of AcidoVorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1. Introduction Poly(3-hydroxybutyrate) (P(3HB)) with isotactic structure is synthesized and accumulated by a variety of bacteria as a reserve energy source.1,2 A remarkable characteristic of P(3HB) is its biodegradability in various environments. Many extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases have been purified from various microorganisms and characterized.3 More than 10 bacterial PHA depolymerase genes (PhaZ) have been cloned and analyzed since 1989.3-6 All PHA depolymerase proteins have composite domain struc* Corresponding author. Fax: +81-45-924-5827. E-mail address: [email protected]. † Tokyo Institute of Technology. ‡ Kanagawa University. § Gunma University. | The Institute of Physical and Chemical Research (RIKEN).

tures and consist of a signal peptide segment, a large catalytic domain at the N terminus, a C-terminal substrate-binding domain, and a linking domain between catalytic and binding domains. Three strictly conserved amino acids, serine, aspartate, and histidine, constitute the active center of the catalytic domain.3 The extracellular PHA depolymerases are classified into four types (types I, II, III, and IV) by differences in the linker domain structure or in the position of lipase box in the catalytic domain.3 Both types I and II depolymerases have a fibronectin type III module fingerprint as the linker domain, whereas the linker domain of type III depolymerase consists of a threonine-rich region. The type IV PHA depolymerases, which hydrolyze medium-side-chain-length PHAs (6-15 side-chain carbon number) and long-side-chain-length PHA (more than 15 side-chain carbon number), consist of two

10.1021/bm020019p CCC: $22.00 © 2002 American Chemical Society Published on Web 05/15/2002

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identical subunits, binding domain at the N terminus and catalytic domain. The extracellular PHA depolymerase of AcidoVorax Sp. Strain TP4 (PhaZaci) has been purified, and the DNA sequence has been determined.5 The sequence of this depolymerase is very similar to that of PHA depolymerase of Comamonas acidoVorans YM1609 (PhaZcom), both of which belong to the type II PHA depolymerase. On the other hand, enzymatic hydrolysis of P(3HB) and its copolymers have been studied on solution-cast films7 and melt-crystallized films,8,9 which demonstrated that the enzymatic hydrolysis occurred first at the amorphous region and subsequently at the crystal region and that for P(3HB) films with a similar degree of crystallinity, the rate of enzymatic hydrolysis is influenced by spherulite and crystal sizes. The biodegradation behavior of copolyesters poly(3hydroxybutyrate-co-3-hydroxypropionate) [P(3HB-co-3HP)]10 and poly(3-hydroxybutyrate-co-6-hydroxyhexanoate)11 by the PHA depolymerase purified from Ralstonia pikettii T1 (PhaZral, previously named Alcaligenes faecalis T1) have been studied. The results suggested that the rates of enzymatic degradation were regulated not only by the crystallinity of polymer but also by the chemical structure of monomeric units and the substrate specificity of PHA depoymerases. The water-soluble degradation products of P(3HB) and its copolymers degraded by several PHA depolymerases have been studied.4,11-14 The results showed that the extracellular PHA depolymerases from R. pikettii, C. acidoVorans, and Pseudomonas lemoignei (PhaZpsel) (defined as types I, II, and III, respectively), degrade the polyester films with endoexo behavior. The difference in the position of the lipase box in the catalytic domain of enzyme was considered to have little effect on the degradation manner of crystal region of P(3HB). The comonomer-unit composition dependence of enzymatic degradability by PhaZral has been investigated for a series of P(3HB-co-3HP) samples with a whole range of the 3HP unit content.10 Since as-produced bacterial P(3HB-co3HP) samples produced by Alcaligenes latus consisted of copolymer fractions with different 3HP contents,15-17 wellcompositionally fractionated copolymer samples have been used in the experiments of enzymatic degradation.10 The crystalline structure and the degree of crystallinity of P(3HBco-3HP) copolymers were confirmed to change with the 3HP content. It has been found that little of fractionated P(3HBco-3HP) with low crystallinity was degraded by PhaZral depolymerase.10 The copolymers forming the P(3HB) homopolymer type crystalline structure could be degraded in a higher rate than those forming the poly(3-hydroxypropionate) (P(3HP)) homopolymer type crystalline structure. The enzymatic hydrolysis behavior of PHA films by PhaZaci depolymerase has not yet been studied in detail. Considering that the PhaZaci depolymerase from AcidoVorax Sp. TP4 belongs to the type II, and degrades P(3HP) easier than P(3HB),5 we wondered whether it would show different degradation behavior with the PhaZral PHA depolymerase. The study described in the present report utilized bacterial P(3HB), comonomer-compositionally fractionated P(3HB-

Table 1. List of the Polyester Samples samplea

3HP mol %b

Mw × 105 c

Mw/Mnc

P(3HB) P(3HP) F-1 F-2 F-3 F-4 F-5 F-6 F-7 F-8 F-9 F-10 F-11 F-12 F-13 F-14 F-15 F-16 F-17 F-18

0 100 86.0 83.9 76.9 74.2 72.7 68.0 65.9 64.7 59.4 56.2 48.8 42.4 33.3 22.5 19.4 18.7 14.9 11.5

5.2 0.2 2.3 2.3 2.9 2.5 2.4 2.2 2.2 2.0 2.1 2.2 2.9 3.8 3.5 4.5 5.0 5.8 5.9 5.4

1.5 2.0 1.9 1.8 1.8 2.1 1.9 2.0 2.4 2.6 3.0 2.6 2.5 2.4 2.6 1.6 2.1 1.9 1.8 1.6

a P(3HB), bacterially synthesized poly(3-hydroxybutyrate); P(3HP), chemosynthesized poly(3-hydroxypropionate); F-n, the comonomer-compositionally fractionated P(3HB-co-3HP) samples. b Determined by 1H NMR spectra. c Mw and Mn, weight- and number-averaged molecular weight, respectively, determined by GPC.

co-3HP)s with varied degree of crystallinity and chemosynthesized P(3HP) to provide evidence for similarity and difference between the enzymatic hydrolysis behavior of the extracellular depolymerases PhaZral and PhaZaci, defined as type I and type II, respectively. The hydrolytic behavior of the PHA films by PhaZaci depolymerase and the degradation products will be investigated, and the results will be compared with those obtained from degradation by PhaZral depolymerase. Experimental Part Materials. P(3HB) and P(3HB-co-3HP) samples were biosynthesized by one-stage fermentation with Alcaligenes latus (ATCC 29713) at 30 °C in a fermentor.17 The bacteria were incubated for 2 days and harvested by centrifugation, followed by lyophilization. Polyesters were extracted from the lyophilized cells using a Soxhlet apparatus with hot chloroform and then purified by precipitating in ethanol and n-hexane. As described in the Introduction, as-produced bacterial P(3HB-co-3HP) samples have more or less distribution of comonomer unit composition, the biosynthesized original P(3HB-co-3HP) samples were compositionally fractionated with chloroform (solvent)/n-heptane four bacterial original P(3HB-co-3HP) samples with 3HP contents of 21.0, 47.5, 56.0, and 62.0 mol %, respectively. P(3HP), chemically synthesized through a ring-opening polymerization of propiolactone, was provided by Tokuyama Co. (Tsukuba, Japan) and purified analogous to P(3HB). The details of the PHA samples used in this study are listed in Table 1. The melt-crystallized polyester films obtained by compression molding were used as the samples for the following analyses. The films cast from chloroform solution were

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inserted between aluminum plates with an aluminum spacer (0.1 mm thickness) and were compression-molded at a temperature 20-30 °C higher than the melting temperature of the samples for 5 min under a pressure of 5 Mpa, using a Toyoseiki Mini Test Press-10 (Toyoseiki Co., Japan). The samples were then cooled to room temperature and kept for at least 3 months to reach equilibrium crystallinity prior to the analyses. Enzymatic Degradation. The extracelluar PHA depolymerase coded as PhaZaci was purified from the cultivation medium of Acid. sp. TP4 according to the method of Kobayashi et al.5 Considering the optimum P(3HB) degradation conditions for the PhaZaci depolymerase5 was different from those for PhaZral depolymerase,18 the degradation experiment by the PhaZaci depolymerase was carried out in 50 mM Tris buffer (pH 8.0) at 30 °C, while that by PhaZral has been carried out in 0.1 M potassium phosphate buffer (pH 7.5) at 37 °C.10 The activity of PHA depolymerase was measured by the decrease in turbidity of a P(3HB) suspension in aqueous buffer solutions.5 The aqueous solution of PhaZaci depolymerase prepared had a P(3HB) degradation activity of 50 U/mg. One unit of enzyme activity is defined as the decrease of the absorbance (at 660 nm) by 0.001 units/min under the assay conditions.13 The polyester film of initial weight about 10 mg and initial dimension 10 × 10 × 0.1 mm was placed in the small bottle containing the given amount of enzyme and 1.0 mL of buffer solution. After the hydrolyzing reaction was allowed to continue for a given period of time, the film was removed, washed with distilled water, and dried to constant weight in a vacuum before weight analysis. For each polymer sample, three films were used and the average value of their weight losses was reported as the result. Control tests, carried out for all samples in buffer solution free from the enzyme, showed no appreciable weight losses under the same condition. After enzymatic reaction, the buffer solution was filtered with a microfilter (core sized, 0.45 µm). The water-soluble degradation products were then analyzed by HPLC (high performance liquid chromatography). The degradation products were also dissolved in deuterated water and were lyophilized twice and then dissolved again in deuterated water (99.96% D2O; Merck Co., USA) before 1H NMR analysis. Analytical Procedures. Measurements of 1H NMR spectra were performed on a JEOL GSX-270 spectrometer operating at 270 MHz and 25 °C. 1H NMR spectra were recorded with 5.0 µs pulse width, 5 s pulse repetition time, 4000 Hz spectral width, 64k data points, and 32 FID accumulations. 3HP mole fractions of the samples were estimated as previous in CDCl3 solution.17 The chemical shifts were referenced to the signal of tetramethylsilane. The NMR analyses of the water-soluble products of enzyme degradation were carried out in D2O solution. DSS (sodium 2,2-dimethyl-2-silapentane-5-sulfate) was added as an internal reference of proton chemical shift. The 1H-1H COSY spectra were collected at room temperature using the π/2 - t1 - π/2 - t2 pulse, with 2000 Hz spectral width and 256 data points for t1 axis and 512 data points for t2 axis.

Wang et al.

Molecular weights of polyester samples were measured by a TOSO HLC-8020 gel permeation chromatography (GPC) system with a TriSEC Data Acquisition System (TOSO CO. Ltd., Japan) at a flowing rate of 1.0 mL/min and at 40 °C. Chloroform was used as an eluent solvent. Polystyrene standards with low polydispersity were used to construct the calibration curve. Differential scanning calorimetry (DSC) measurements were conducted on a SEIKO EXSTAR6000 system equipped with a DSC 220U, using 1-3 mg of samples. Melting temperature, Tm, and the heat of fusion, ∆H, were determined by heating the crystallized samples from -50 to 200 °C at a heating rate of 20 °C/min (first scan). The Tm values were determined from the maxima of the endothermic peaks, and the values of ∆H were calculated from the integral of endothermic curve observed by the first scan. The samples were melted in the DSC apparatus at 200 °C for 2 min, quenched to -80 °C by using liquid nitrogen, and then reheated to 200 °C at a heating rate of 20 °C/min (second scan). The glass transition temperature (Tg) values were taken at the midpoint of the transition observed by the second heating scan. Wide-angle X-ray diffraction (WAXD) patterns were recorded on a Rigaku RU-200 (40 kV/200 mA) and a Rigaku IP R-AXIS-DS3 system. The nickel-filtered Cu KR X-ray beam with a pinhole graphite monochromator (λ ) 0.15418 nm) was used as the source. The WAXD pattern was measured in a 2θ range of 8-40° at a scanning speed of 1 deg/min. The degree of crystallinity, Xc, was estimated according to the method developed by Vonk.19 HPLC analyses were performed on a SHIMAZU Organic Acid Analysis System equipped with two Shim-pack SPR-H (250 mm × 7.8 mm) columns at 40 °C. The mobile phase was 4 mM p-toluenesulfonic acid aqueous solution with a flow rate of 0.6 mL/min. Results Physical Properties of Compositionally Fractionated P(3HB-co-3HP)s. Thermal properties of the fractionated P(3HB-co-3HP) samples are summarized in Figure 1. The values of Tm and ∆H plotted against the 3HP content showed a ravine at the 3HP content between 48 and 78 mol %. The values of the lowest Tm and ∆H are about 45 °C and near 0 J/g. The Tg values decreased continuously with the increase of the 3HP content. Figure 2 shows the WAXD patterns of four fractionated P(3HB-co-3HP) samples with 22.5, 48.8, 68.0, and 83.9 mol % 3HP, P(3HB), and P(3HP). The peak at 2θ ) 17° corresponds to the (110) diffraction of the P(3HB) homopolymer-type lattice, and that at 2θ ) 25.4° corresponds to the (002) of P(3HP).9,16,20-22 Comparing all the fractionated samples, it was found that the samples with 3HP content lower than 42 mol % formed only the P(3HB)-type crystalline structure, while in the samples with 3HP higher than 56 mol %, only the P(3HP)-type crystalline structure was observed. The weak diffractions corresponding to both the P(3HB)-type and P(3HP)-type crystals were observed in the WAXD pattern of P(3HB-co-48.8 mol % 3HP) fractionated sample, which may be caused by the ineffective fractionation.



Figure 3. The degree of crystallinity (Xc) obtained from WAXD patterns of P(3HB), P(3HP), and fractionated P(3HB-co-3HP) samples plotted against the 3HP content.

Figure 1. The thermal properties of the fractionated P(3HB-co-3HP) samples with 3HP content varied from 0 (P(3HB)) to 100 mol % (P(3HP)) determined by DSC: heat of fusion ∆H (O) and melting temperature Tm (b) obtained from the first scan; glass transition temperature Tg (4) obtained from the second scan.

Figure 4. The 3HP content dependences of weight loss of P(3HB), P(3HP), and fractionated P(3HB-co-3HP) samples hydrolyzized by 2 µg of PhaZaci depolymerase in 50 mM Tris buffer (pH 8.0) at 30 °C for 24 h.

Figure 2. The WAXD patterns of P(3HB), P(3HP), and four fractionated P(3HB-co-3HP) films with 3HP content of 22.5, 48.8, 68.0, and 83.9 mol %.

The degrees of crystallinity (Xc, (5%) of P(3HB), P(3HP), and fractionated P(3HB-co-3HP)s were calculated from the WAXD patterns, and the results were plotted in Figure 3. Being different from poly(3-hydroxybutyrate-co- 3-hydroxyvalerate) (P(3HB-co-3HV)) copolymer,23 the Xc values decreased from about 68% to lower than 10% when the 3HP contents of the copolymers increased from 0 (P(3HB)) to 48.8 mol %. The Xc values are nearly constant over the 3HP content range from 48.8 to 74.2 mol % and then increased with the increase of 3HP content from 74.2 to 100 mol % (P(3HP)). These results are consistent with the 3HP content dependence of Tm and ∆H values obtained by DSC, which indicate that incorporating a secondary comonomer unit into the chain of either P(3HB) or P(3HP) will cause a suppressing effect on the crystallization of the major comonomer unit.

The Enzymatic Hydrolysis of Fractionated P(3HB-co3HP) Copolymers. In our previous paper, the degradation by PhaZral depolymerase isolated from R. pikettii T1 has been investigated for a series of compositionally fractionated P(3HB-co-3HP) samples.10 It has been found that PhaZral depolymerase degraded the copolymers forming the P(3HB)homopolymer type crystalline structure relatively faster than those forming P(3HP)-homopolymer type crystal. The maximum degradation rate of P(3HB) crystalline type copolymer has been observed for the P(3HB-co-3HP) fraction with the 3HP content of about 20-30 mol %. This was considered to be the delicate balance between the increasing of degradation rate with the decrease in the crystallinity and the decreasing of enzyme adsorption on the polymer surface with the increase in the amorphous part.10,24,25 In the case of degradation by PhaZaci depolymerase, the results are very different, as shown in Figure 4, the changing of the weight loss (or the degradation rates) of the fractionated P(3HB-co-3HP) copolymers exhibited the opposite tendency as that of the crystallinity with the increase in the 3HP content (Figure 3). The degradation rates of the fractionated P(3HB-co-3HP) samples increased continuously with the 3HP content from 0 (P(3HB)) to 56.2 mol %. The samples with 3HP content from 56.2 to 83.9 mol % show almost similar degradation rates. When the 3HP content was higher than 84 mol %, the degradation rate decreased with the increase of the copolymer crystallinity. The water-soluble degradation products was characterized by the 1D 1H NMR and 2D 1H-1H COSY spectroscopy. Figure 5 shows the 1D and 2D 1H NMR spectra of the

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Figure 6. 1D 1H NMR and 2D 1H-1H COSY NMR spectra of watersoluble degradation products from P(3HP) after 24 h of hydrolysis by 2 µg of PhaZaci PHA depolymerase at 30 °C. Figure 5. 1D 1H NMR and 2D 1H-1H COSY NMR spectra of watersoluble degradation products from P(3HB) after 24 h hydrolysis by 2 µg of PhaZaci PHA depolymerase at 30 °C.

degradation products of P(3HB). In the 2D 1H-1H COSY spectra, the cross-peaks of methine (2) with methylene (3) and methyl (1) of the 3HB monomer, and those of methine (8) with methylene (9) and methyl (7) of the 3HB dimer were observed. It is obvious that the products from the P(3HB) film were a mixture of the 3HB monomer (major product) and the 3HB dimer (minor product). Any resonances corresponding to the trimer were not observed in this figure and in the other degradation products. Figure 6 shows the 1D and 2D 1H NMR spectra of the products after 24 h of degradation of P(3HP). The crosspeaks of R- and β-methylene of the 3HP monomer (2 and 1) and the dimer (4 and 3, 6 and 5) were observed. Any resonance of the 3HP trimer was not detected in the NMR spectra shown in this figure, although it could be observed in the products produced from P(3HP) degraded by PhaZaci depolymerase for 15 min. This result indicated that the hydrolysis of the 3HP trimer by PhaZaci depolymerase was preferable to the 3HP dimer. Figure 7 shows the 1H NMR spectrum of the degradation products of bacterial unfractionated P(3HB-co-21.0 mol % 3HP) by PhaZaci PHA depolymerase for 30 days. Only 3HB and 3HP monomers were observed. This result indicated that the PhaZaci depolymerase could recognize two monomeric units as hydrolysis substrates. It also indicated that this

Figure 7. 1H NMR spectra of the products from bacterial P(3HBco-21.0 mol % 3HP) after 30 days hydrolysis by 2 µg of PhaZaci PHA depolymerase solution at 30 °C.

depolymerase degraded P(3HB), P(3HB-co-3HP) or P(3HP) films first into the trimers or the dimers then into the monomers. The water-soluble degradation products of the enzymatic hydrolysis of the fractionated P(3HB-co-3HP), P(3HB), and P(3HP) films by PhaZaci depolymerase were analyzed by HPLC and confirmed by 1H NMR. The results are shown in Figure 8. The values of the relative concentrations of the dimers and monomers were calculated from the corresponding peak’s area in the HPLC chromatograms. In the figure, b, p, bb, bp, and pb correspond to 3HB and 3HP monomer,



Figure 8. Relative amounts of the water-soluble hydrolysis products from P(3HB), P(3HP), and fractionated P(3HB-co-3HP)s degraded by 2 µg of PhaZaci PHA depolymerase at 30 °C for 24 h: b (3HB monomer, white circle); p (3HP monomer, black circle); bb (3HB dimer, black triangle); bp or pb (3HB-3HP dimer, white triangle); pp(3HP dimer, black square).

3HB dimer, and the dimer containing one 3HB unit and one 3HP unit, respectively. In all of the cases, both monomer and dimer were observed. The relative amount of monomer is larger than that of dimer in the products from P(3HB) and the fractionated P(3HB-co-3HP)s. The degradation products from the P(3HP) film contained more of the 3HP dimer than the monomer. The 3HP dimer was only observed in the products of the hydrolysis of P(3HP), which may be caused by the high crystallinity of the P(3HP) film. With the increase in the 3HP content of the fractionated P(3HBco-3HP)s from 30 to 90 mol %, the relative concentration of the 3HB monomer in the hydrolysis products decreased while that of the 3HP monomer increased. The relative concentrations of the dimers with both 3HB and 3HP units were about 10%. For the fractionated copolyester samples with 3HP content lower than 30 mol %, the amounts of 3HB monomer and dimer in the hydrolysis products decreased while those of 3HP monomer and 3HB-3HP dimer increased. Compared with the degradation products from copolyesters with 3HP content higher than 30 mol %, the proportion of dimer to monomer was larger in the products from copolyesters with 3HP content lower than 30 mol %. Discussion In this paper, the thermal and crystalline properties were investigated for bacterial P(3HB), chemosynthesized P(3HP), and compositionally fractionated bacterial P(3HB-co-3HP) with 3HP content ranging from 11.5 to 86.0 mol %. The 3HP-content dependence of crystallinities of these samples obtained from DSC were similar to those observed by WAXD measurements. The crystallinities of the fractionated P(3HB-co-3HP) samples with 3HP content ranging from 48 to 78 mol % were very low. It has been reported that the P(3HB-co-3HP) samples with 3HP content at about 48-75 mol % were in the almost amorphous state.16 After being crystallized for more than 3 months, the films of fractionated P(3HB-co-3HP) with 3HP content ranging from 48 to 78

mol % were found to form P(3HB)- or P(3HP)-type crystalline structure, although their crystallinity were very low. This result shows that it is possible for these P(3HBco-3HP) samples to form partially crystalline structure. Among the various extracellular PHA depolymerases isolated from a variety of bacteria, the properties of PhaZral PHA depolymerase have been studied in great detail.10,11,13,18,26 It has been demonstrated that PhaZral hydrolyzed P(3HB) through endo-exo modes and could not hydrolyze the 3HB dimer.12,13,27 The active site of PhaZral recognized at least three monomeric units as substrate for the hydrolysis of ester bonds in the P(3HB) polymer chain.12,27 The rate of enzymatic hydrolysis depended on the sequential structures of polyester chain and the crystallinity of the polyester films.12,13,27,28 The behavior of PHA degradation by PHA depolymerase of C. acidoVorans YM16094,12 is little different from that by PHA depolymerase of P. lemoignei,14 although they belong to the different types of PHA depolymerase. Another PHA depolymerase purified from P. stutzeri (PhaZpses) exhibited a low level of genetic homology to those of other PHA depolymerases (below 10%), while relatively high degrees of similarities were found in the regions surrounding putative active sites and the substrate-binding domain at the C terminus.6 The active site of the catalytic domain of the PhaZpses PHA depolymerase recognized at least two monomeric units as substrates for the hydrolysis of ester bonds in the 3HB sequence.6,29 Thus, it was considered to be different in specificities from those of other PHA depolymerases. In this paper, we studied the behavior of PHA hydrolysis by PhaZaci PHA depolymerase, defined as type II, on a series of fractionated P(3HB-co-3HP)s with different degree of crystallinity (Figure 4). The decrease of crystallinity increased the hydrolysis rate of PhaZaci PHA depolymerase without the influence of 3HP content. In contrast, the maximum degradation rate of P(3HB-co-3HP) copolyesters has been observed at the fraction with 3HP content of about 20-30 mol % when hydrolyzed by PhaZral PHA depolymerase.10 That is, the PhaZaci PHA depolymerase hydrolyzed PHA films with low degree of crystallinity in a much high rate than the PhaZral PHA depolymerase.27 It has been known that PhaZral depolymerase gave 3HB dimer as a major product in low enzyme concentration.4,18 However, P(3HB) was degraded into more 3HB monomer than the dimer by PhaZaci PHA depolymerase. Furthermore, the P(3HB-co3HP) films could be degraded into 3HB and 3HP monomers at last by PhaZaci PHA depolymerase, which indicated that the active site of its catalytic domain recognized at least two monomer units for hydrolysis, which is similar with PhaZpsus depolymerase. No trimer but both the 3HB-3HP and the 3HP-3HB dimers were observed in the degradation products from P(3HB-co-3HP) hydrolyzed by PhaZaci PHA depolymerase, which indicated PhaZaci PHA depolymerase could hydrolyze the ester bonds between 3HB-3HP and 3HP3HB. Conclusions The behavior of hydrolysis by two types of extracellular PHA depolymerase purified from R. pikettii T1 and Aci. Sp.

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TP4, defined as types I and II depolymerase, were compared for bacterial P(3HB), chemosynthesized P(3HP), and compositionally fractionated P(3HB-co-3HP) films. Different from PhaZral PHA depolymerase, the PhaZaci depolymerase hydrolyzed P(3HB-co-3HP) films with lower degree of crystallinity in a higher rate. The enzymatic hydrolysis of P(3HB) by PhaZaci depolymerase produced the 3HB monomer as the major product, while mainly the 3HB dimer was produced by PhaZral. The PhaZaci depolymerase could hydrolyze P(3HB-co-3HP)s into 3HB and 3HP monomers, indicating that this depolymerase could recognize at least two monomeric units as hydrolysis substrate. On the basis of these results, the behavior of PHA degradation by PhaZaci depolymerase could be considered to be different from that by PhaZral PHA depolymerase. References and Notes (1) Doi, Y. Microbial Polyesters; VCH: New York, 1990. (2) Inoue, Y.; Yoshie, N. Prog. Polym. Sci. 1992, 17, 571. (3) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451. (4) Kasuya, K.; Inoue, Y.; Tanaka T.; Akehata, T.; Iwata, T.; Fukui, T.; Doi, Y. Appl. EnViron. Microbiol. 1997, 63, 4844. (5) Kobayashi, T.; Sugiyama, A.; Kawase, Y.; Saito, T.; Mergaert, J.; Swings J. J. EnViron. Polym. Degrad. 1999, 7, 9. (6) Ohura, T.; Kasuya, K.; Doi, Y. Appl. EnViron. Microbiol. 1999, 65, 189. (7) Doi, Y.; Kitamura, S.; Abe, H. Macromolecules 1995, 28, 4822. (8) Tomasi, G.; Scandola, M.; Briese, B. H.; Jendrossek, D. Macromolecules 1996, 29, 507. (9) Koyama, N.; Doi, Y. Macromolecules 1997, 30, 826. (10) Cao, A.; Arai, Y.; Yoshie, N.; Kasuya, K.; Doi, Y.; Inoue, Y. Polymer 1999, 40, 6821.

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BM020019P

Enzymatic Hydrolysis of Bacterial Poly(3-hydroxybutyrate-co-3

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