Microbial synthesis and properties of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in Comamonas acidovorans

Microbial synthesis and properties of poly( 3- hyd roxybutyrate-co-4hydroxybutyrate) in Cornarnonas acidovorans Yuji Saito Research Institute of Innovative Technology for the Earth (RITE), Hirosawa, Wako-shi, Saitama 351-01, Japan and Yoshiharu Doi* Polymer Chemistry Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako-shi, Saitama 351-01, Japan

Received 21 December 1993; revised 12 January 1994 Comamonas acidovorans DS-17 was isolated from activated sludge and found to produce copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB) at 30°C under growth-limited conditions. When 1,4-butanediol or 4-hydroxybutyric acid was used as the sole carbon source, a P(4HB) homopolymer was produced. Random copolymers of 3HB and 4HB units were produced on the addition of glucose or 3-hydroxybutyric acid to the culture solution of 4-hydroxybutyric acid. The physical properties of P(3HB-co-4HB) copolyesters with high 4HB fractions (64-100 mol%) were investigated. The copolyester films with high 4H B fractions exhibited the characteristics of a thermoplastic elastomer, and the tensile strength increased from 17 to 104 MPa as the 4HB fraction was increased from 64 to 100 reel%. The biodegradabilities of P(3HB-co-4HB) films were studied in aqueous solutions of extracellular polyhydroxybutyrate (PH B) depolymerase from Alcaligenes faecalis or of lipase from Rhizopus delemer. The erosion rate of P(3HB-co-4HB) films by PHB depolymerase decreased as the 4HB fraction in copolyester was increased from 64 to 100 mol%. In contrast, the erosion rate of films by lipase increased with the 4HB fraction. Keywords: microbial copolyesters; Comamonasacidovorans; mechanical properties

An optically active polymer of [R]-3-hydroxybutyric acid, P(3HB), is synthesized by a wide variety of micro-organisms as an intracellular carbon and energy storage material ~ s. Recently, many bacteria have been found to produce copolymers of [R]-3-hydroxyalkanoic acids with carbon chains ranging from 4 to 14 carbon atoms from alkanoic acids and alcohols4-12. In addition, 4-hydroxybutyric~3-~s, 3-hydroxypropionic~6, and 4hydroxyvaleric acids ~7 have been found as new constituents of bacterial polyhydroxyalkanoates (PHA). These microbial polyesters are thermoplastic with biodegradable properties, and the physical properties can be regulated by varying the compositions of the copolymers 3,4,18,19. The microbial polyesters have recently attracted much industrial attention as environmentally degradable thermoplastics for a wide range of agricultural, marine and medical applications 2°. * To whomcorrespondenceshould be addressed 0141-8130/94/020099-06 © 1994Butterworth-HeinemannLimited

Copolymers of [R]-3-hydroxybutyrate and 4-hydroxybutyrate, P(3HB-co-4HB), were produced by Alcaligenes eutrophus 13,21,22, A. latus 23 or Pseudomonas acidovorans 24 when 4-hydroxybutyric acid, 1,4-butanediol or ~butyrolactone were used as the carbon source. The copolymer compositions varied from 0 to 60 mol% 4HB, and the copolymers were shown to have a statistically random distribution of 3HB and 4HB units 14. Recently, we have found that P(3HB-co-4HB) copolyesters with a wide range of compositions from 0 to 100reel% 4HB are synthesized by A. eutrophus from 4-hydroxybutyric acid in the presence of additives such as citrate and ammonium sulfate22. However, the yield of P(3HBco-4HB) copolymers with compositions of 70-100 mol% 4HB was very low, and the maximum contents of polyesters in dried cells were below 10 wt%. We have recently isolated Comamonas acidovorans DS-17 from activated sludge. This strain produced P(4HB) homopolymer at a high productivity from

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Poly(3-hydroxybut}/rate-co-4-hydroxybutyrate): Y. Saito and Y. Doi 4-hydroxybutyric acid or 1,4-butanediol. In this study, we report the results of the production of P(3HB-co-4HB) c0polyesters by C. acidovorans DS-17 from several carbon sources, and the physical properties and enzymatic degradation of P(3HB-co-4HB) copolyesters with compositions ranging from 64-100 mol% 4HB.

Experimental Screening of Comamonas acidovorans DS-17from activated sludge Activated sludge from the municipal wastewater treatment plant of Narashino-shi, Japan, was diluted and spread on nutrient agar plates (polypeptone, 10 g l - l ; meat extract, 5 g 1-1; NaCI, 5 g 1-1: pH 7.0). The plates were incubated at 30°C for 3days. Several colonies appeared on the plates and were transferred to replica agar plates containing sodium 4-hydroxybutyric acid as a sole carbon source (sodium 4-hydroxybutyrate, 5 g 1-1; (NH4)2SO4, 0.033 g 1-1; KH2PO4 ' 2.65 g 1-1; Na2HPO4"12H20, 7.16g1-1; MgSO4"7H20, 0.33 gl-1; microelement solution, 10 ml 1-1; agar, 18 g 1-1: pH 7.0). The replica agar plates were incubated at 30°C for 4days. All colonies on the plates were stained by Nile blue-A solution ( 5 m g p e r 100ml ethanol). Red fluorescent colonies at an excitation wavelength of 254nm were selected 25. One of the selected microorganisms accumulated P(3HB-co-4HB) copolyester within its cells from 4-hydroxybutyric acid. The isolated stain, DS-17, a Gram negative rod, was identified as C. acidovorans and used in this study. Media and culture conditions The microbial synthesis of P(3HB-co-4HB) was carried out by a two-stage cultivation. The DS-17 strain was first grown under aerobic conditions at 30°C for one day on a reciprocal shaker in a 500 ml flask containing 100 ml of a nutrient-rich medium (pH 7.0) containing 1 g polypeptone, 0.5 g meat extract and 0.5 g NaCI. The cells were harvested by centrifugation at 7000g for 10min. Accumulation of polyester in the cells was not observed under these culture conditions. The centrifuged cells were transferred into 100 ml of nitrogen-free medium (pH 7.0) containing 0.265 g K H 2 P O 4, 0.716 g Na2HPO4.12H20, 0.033 g MgSO4.7H20 and 1 ml of microelement solution. The microelement solution contained 119 mg CoC12, 9.7 g FeC13, 7.8 g CaCI2, 118 mg NiCI2"6H20, 62.2 mg CrCI2 and 156.4mg CuSO4.5H20 (per litre of 0.1M HCI). Different carbon substrates were added to the nitrogenfree medium as carbon sources, and the cells were incubated for 48 h at 30°C, harvested by centrifugation, washed with water, and lyophilized. Analytical procedures Polyesters were extracted from the lyophilized cells with hot chloroform in a Soxhlet apparatus for 5 h. The extract was concentrated to 3 ml using a rotary evaporator, and polyesters were precipitated by the addition of 100 ml hexane. The compositions and sequence distributions of P(3HB-co-4HB) copolyesters were determined by analysis of 1H- and 13C-NMR spectra 14. The solution 1H- and 13C-NMR spectra of P(3HB-co-4HB) in chloroform were recorded on a Jeol GX-400 spectrometer. The 400 MHz I H - N M R spectra were recorded at 27°C

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Int. J. Biol. Macromol. Volume 16 Number 2 1994

for a CDCI 3 solution of P(3HB-co-4HB) (3 mg m1-1) with 5ms pulse width, 32000 data points and 16 accumulations. The 100MHz 13C-NMR spectra were recorded at 27°C for a CDC13 solution of P(3HB-co-4HB) (20 mg m l - 1) with 5 ms pulse width (45 ° pulse angle), 0.7 s pulse repetition, 23 000 Hz spectral width, 32000 data points, and 8000-20 000 accumulations. Tetramethylsilane (Me4Si, ~=0) was used as an internal chemical shift standard. All molecular weight data were obtained at 40°C by using a Shimazu 6A gel permeation chromatography (GPC) system with a Shodex 80M column. Chloroform was used as eluant at a flow rate of 0.8 ml min- 1, and a sample concentration of 1.0mgm1-1 was used. Polystyrene standards with a low polydispersity were used to create a calibration curve. The differential scanning calorimetry (DSC) data for the polyesters were recorded on a Shimazu DSC-50 under a nitrogen flow of 30 ml min- 1. The melting temperature (Tin) and enthalpy of fusion (Anm) were determined from the DSC endotherms. For measurement of the glasstransition temperature (T~), the melt samples were rapidly cooled to - 150°C. They were heated from - 150 to 200°C at heating rate of 20°C min- 1. The Tg was taken as the midpoint of the heating capacity change. Wide-angle X-ray diffraction measurements of P(3HBco-4HB) samples were carried out using a Rigaku RAD-1VB system. CuK~ radiation (2=0.1542nm) was used as the source. The X-ray diffraction patterns of polyesters were recorded at 27°C in the range 20 = 6-40 ° at a scan speed of 1 degree per min. X-ray crystallinities were measured for polyester films that had been cast from chloroform solution and allowed to stand for 3 weeks at room temperature. The percentage of crystallinity was calculated from diffracted intensity data according to Vonk's method 31. Stress-strain curves of cast films of P(3HB-co-4HB) samples were obtained at 23°C with a strain rate of 20 mm min- ~ using an Imada SV-50 tensile machine. The mechanical tensile data were calculated from such curves for an average of three specimens.

Enzymatic degradation Lipase from Rhizopus delemer 26 and extracellular PHB depolymerase purified from Alcaligenesfaecalis T127 were used in this study. Enzymatic degradations of P(3HBco-4HB) films were carried out at 37°C in a 0.1 M phosphate buffer (pH 7.4). The P(3HB-co-4HB) films (initial weight, 6 mg; initial film dimensions, 10 x 10 mm; initial thickness, 0.05 mm) were placed in small bottles containing 1.0 ml of the buffer. The reactions were started by the addition of an aqueous solution of PHB depolymerase (1.5 #g) or lipase (285/~g). The reaction solutions were incubated at 37°C with shaking. The films were periodically removed, washed with water, and dried to constant weight in vacuo before analysis.

Results and discussion Microbial synthesis of P(3HB-co-4HB) Table 1 lists the results of copolyester production by C. acidovorans from several carbon substrates at 30°C. C. acidovorans produced P(3HB-co-4HB) copolyesters when 4-hydroxybutyric acid, 1,4-butanediol or 1,10decanediol were used as the sole carbon source. P(4HB)

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)." Y. Saito and Y. Doi Table 1

Production of polyesters from different carbon sources by C. acidovorans DS-17 (48 h, 30°C)

Carbon source Glucose Acetate Propionate But yrate Pentanoate 1,4-Butanediol 1,4-Butanediol 1,5-Pentanediol 1,6-Hexanediol 1,8-Octanediol 1,10-Decanediol 4-Hydroxybutyrate 4-Hydroxybutyrate y-Butyrolactone fl-Acetylpropionate

Content (g 1- i)

Dry cell weight (g I- l)

Polyester content" (wt%)

Polyester compositionb (mol%) 3HB

3HV

4HB

10 10 10 10 10 10 15 10 10 10 10 10 15 10 10

2.4 1.9 1.9 2.1 2.4 2.1 2.3 1.7 1.5 1.3 1.9 2.2 2.1 1.6 2.2

7 1 1 14 18 17 28 trace trace trace 3 21 18 trace 17

100 100 63 100 39 3 61 2 36

37 61 60

97 100

4HV

39 100 98 -

a Polyester content in dry cells b Determined by 1H-NMR spectra: 3HB, 3-hydroxybutyrate; 3HV, 3-hydroxyvalerate; 4HB, 4-hydroxybutyrate; 4HV, 4-hydroxyvalerate

Table 2

Production of copolyesters from mixtures of different substrates by C. acidovorans DS-17 (48 h, 30°C)

Sample

Carbon sources" (0.05 M/0.05 M)

Dry cell weight (g 1-1)

1 2 3 4 5

Glucose/4HB Butyrate/4HB Crotonate/4HB [S]-3HB/4HB [R]-3HB/4HB

3.3 3.4 3.5 3.6 4.1

Polyester content b (wt%) 18 15 22 33 36

Composition c (mol%)

Diad sequence distribution d

3HB

4HB

F33

F34

11 45 46 63 67

89 55 54 37 33

0.01 0.40 0.41 0.48 0.53

0.10 0.05 0.05 0.15 0.14

F43 0.12 0.10 0.12 0.27 0.19

F44 0.77 0.45 0.42 0.10 0.14

D' 0.6 36.0 28.7 1.2 2.8

a 4HB, sodium 4-hydroxybutyrate; [S]-3HB, sodium [S]-3-hydroxybutyrate; [R]-3HB, sodium [-R]-3-hydroxybutyrate b Polyester content in dry cells ¢ Determined by I H - N M R spectra aThe relative peak areas of carbonyl resonances in 13C-NMR spectra e D = F33F44/F34F43

40H3

H .O. % /* '

~, 0

II C

8 OH 2

)xV, O

C"2

3HB

6

,'O , ,H

C7H2

4HB

2

I~;.]y O

4*4

7 6

CDCI3 t 4*3

3*4

172 171 170 169 5 4 2

_lll 160

140

120

100 80 in ppm

60

40

20

Figure 1 100MHz 13C-NMR spectrum of P(3HB-co-89%4HB) (sample 1) in CDCI3 at 27°C

homopolyester was produced from 15g1-1 of 1,4butanediol or 10gl -~ of 4-hydroxybutyric acid, and the polyester contents in dried cells were as high as 21-28 wt %. This is the first example of efficient production of P(4HB) homopolymer by a micro-organism. P(3HB) homopolyester was produced by C. acidovorans from glucose, acetate or butyrate. Copolymers of 3HB and 3-hydroxyvalerate (3HV) were produced from propionate or pentanoate. When fl-acetyl-propionic acid was used as the sole carbon source, a terpolyester of 3HB, 3HV and 4-hydroxyvalerate (4HV) was produced. In order to control the copolymer compositions of P(3HB-co-4HB), substrate mixtures of 4-hydroxybutyric acid and different compounds were used as carbon sources for the production of copolyesters by C. acidovorans. Table 2 lists the results of polyester production from mixtures of different substrates. Figure 1 shows the 100 MHz 13C-NMR spectrum of sample 1 (P(3HB-co-89%4HB)) produced from a mixture of 4-hydroxybutyric acid and glucose, together with the 13C chemical shift assignments 14. The carbonyl resonances (6= 169-173) were resolved into four groups of peaks, arising from the different diad sequences of 3HB and 4HB units (3,3(3HB-3HB), 3,4(3HB-4HB), 4,3(4HB-3HB) and 4,4(4HB-4HB)) 14. The diad sequence distributions of the five samples in Table 2 were determined from the relative peak areas of

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Poly(3-hydroxybutyrate-co-4-hydroxybutyrate): Y. Saito and Y, Doi order to determine whether each sample is a r a n d o m copolymer or not, a parameter D is defined as21:

carbonyl c a r b o n resonances. The sequence distribution data of 3HB and 4HB units were c o m p a r e d with the Bernoullian statistics applicable to a statistically r a n d o m copolymerization. In the Bernoullian model, the mole fraction Fij of diad sequence ij can be expressed using the mole fractions F; and Fj o f / a n d j units as F I j = F i F J. In

D = (F33F44)/(F34F43) The value of D is equal to 1.0 for a statistically r a n d o m copolymer. The D value for a block copolymer should be larger than 1.0. The D values of samples 1-5 are given in Table 2. The D values of samples 1 and 4 were 0.6 and 1.2, respectively, suggesting that the samples have a r a n d o m distribution of 3HB and 4HB units. In contrast, the D values of samples 2 and 3 were 36.0 and 28.7, indicating that samples 2 and 3 are either block polymers or mixtures of 3HB- and 4HB-rich r a n d o m copolymers. In a previous paper 21, we reported that A. eutrophus p r o d u c e d a mixture of r a n d o m P ( 3 H B - c o - 4 H B ) c o p o l y m e r s with two different 4HB fractions from a mixture of ),-butyrolactone and butyric acid as the c a r b o n source. Table 3 lists the results of polyester production from substrate mixtures of [ R ] - 3 - h y d r o x y b u t y r i c and 4-hydroxybutyric acids. The composition and productivity of P(3HB-co-4HB) copolyesters were dependent on the composition of c a r b o n sources in the culture solution. We carried out solvent extraction of the produced copolyesters (samples 7-10) with boiling acetone for 5 h in a Soxhlet apparatus 21, and obtained acetone-soluble and acetone-insoluble fractions. Table 4 gives the result of the fractionated P(3HB-co-4HB) samples. The 4HB contents of the acetone-soluble fractions decreased from 90 to 64 m o l % as the fraction of [ R ] - 3 - h y d r o x y b u t y r i c acid in the c a r b o n source was increased. In contrast, the 4HB contents of the acetone-insoluble fractions were in the range 18-32 m o l % , independent of the composition of the c a r b o n source. The diad sequence distributions of boiling acetonesoluble fractions of samples 7-10 are listed in Table 5. The D values of these samples range from 1.2 to 2.2, suggesting that samples 7-10 have a r a n d o m distribution of 3HB and 4HB units.

Table 3 Production of polyesters from [R]-3-hydroxybutyric and 4-hydroxybutyric acids by C. acidovorans DS-17 (48 h, 30°C)

Carbon sources" (g I- 1) Sample

[R]-3HB

6 7 8 9 10

4HB

0 0.5 1.0 1.5 2.0

Compositionb (mol%)

Dry cell Polyester weight content (gl-l) (wt%)

10.0 9.5 9.0 8.5 8.0

2.6 3.3 3.2 3.5 3.4

3HB

17 23 24 26 27

0 17 20 27 44

4HB 100 83 80 73 56

aI-R]-3HB, sodium [R]-3-hydroxybutyrate; 4HB, sodium 4-hydroxybutyrate bDetermined from IH-NMR spectra Table 4 Fractionation of polyester samples 7-I0 with boiling acetone

Composition (mol%) Fraction by acetonea

Sample 7

Weightratio (wt%)

Soluble Insoluble Soluble Insoluble Soluble Insoluble Soluble Insoluble

8 9 10

90 10 88 12 91 9 92 8

3HB

4HB

10 82 18 68 22 81 36 71

90 18 82 32 78 19 64 29

Polyester samples were fractionated with boiling acetone for 5 h Table 5 Sequence distributions of acetone-soluble P(3HB-co-4HB) samples

Compositionb (mol%) Sample" 7-S 8-S 9-S IO-S

3HB

4HB

10 18 22 36

90 82 78 64

Physical properties of P(3HB-co-4HB) copotyesters In previous papers la'zl, we reported the physical properties of P(3HB-co-4HB) copolyesters with c o m p o sitions ranging from 0 to 28 m o l % 4HB, In this paper, we report the physical properties of five P(3HB-co-4HB) samples containing 64, 78, 82, 90 and 100 m o l % 4HB. The polyester films were prepared by conventional solvent casting techniques from chloroform solutions of polyesters, as described in the experimental section. Table 6 summarizes molecular weights, thermal properties and X-ray crystallinities of the P(3HB-co-4HB) samples. The X-ray crystallinities of P(3HB-co-4HB)

Diad sequence distribution c F33

F34

F4a

F44

DO

0.01 0.03 0.06 0.14

0.08 0.13 0.15 0.18

0.08 0.14 0.16 0.18

0.83 0.70 0.63 0.50

1.3 1.2 1.6 2.2

a7-S, 8-S,9-S and 10-S are acetone-soluble fractions of polyester samples 7-10 b Determined by 1H-NMR spectra c The relative peak areas of carbonyl resonances in ~aC-NMR spectra dD = Fa3F44/F34F43

Table 6 Molecular weights, thermal properties and X-ray crystallinities of random copolyesters

Composition (mol%) Sample 6 7-S 8-S 9-S 10-S

3HB

4HB

0 10 18 22 36

100 90 82 78 64

Thermal propertiesa Molecular weight 10- 3 h~,

Mw/Mo

Tm (°C)

AHm (cal g- 1)

2.3 2.8 3.1 3.8 3.8

53 50 52 49 50

8.6 7.2 5.4 4.8 4.0

339 207 191 163 155

a Determined by DSC. The heats of fusion AHm were not corrected using the measured X-ray crystallinities bDetermined by X-ray diffraction

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Int. J. Biol. Macromol. Volume 16 Number 2 1994

(°C) -48 -42 -39 - 37 - 35

Crystallinityb (%) 34+ 5 28 + 5 18+5 17_ 5 15 _ 5

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate): Y. Saito and Y. Doi samples with compositions of 64-100 mol% 4HB decreased from 34 to 15% with a decrease in the 4HB fraction. Figure 2 shows the X-ray diffraction patterns of P(3HB-co-4HB) films varying from 64 to 100 m o l % 4HB. All samples showed the P(4HB) crystal lattice, and the P(3HB) crystal lattice was not detected. The X-ray crystallinity of P(3HB) homopolymer was 60___5 % ~s, and

100% 4HB

the value for the P(4HB) homopolymer was 3 4 _ 5%. As reported in a previous paper is, the crystallinities of P(3HB-co-4HB) samples with compositions of 0-49 tool% 4HB decreased from 60 to 14% as the 4HB fraction increased from 0 to 49mo1%. The crystallinities of P(3HB-co-4HB) copolyesters with compositions ranging from 50 to 70 m o l % 4HB were as low as 1 5 + 5 % . Table 7 shows the results of stress-strain tests of P(3HB-co-4HB) films at 23°C. The stresses are calculated on the cross-section. As reported in a previous paper 21, the tensile strength of P(3HB-eo-4HB) films with compositions of 0-16 m o l % 4HB decreased from 43 to 26 M P a with an increase in the 4HB fraction, while the

6 '~

[] 100%4HB • 90%4HB

0 82%4HB

5

•

90% 4HB

.~"

78%4Ha

,-""

2 82% 4HB

O!

0

10

20

30

Time (h) Figure3 Enzymaticdegradation (erosion) profiles of P(3HB-co-4HB) films in an aqueous solution of PHB depolymerase from A. faecalis (pH 7.4, 37°C)

78% 4HB 6 t"~

S

4

Z

30

,..,/ .,,,,,I-.~//I..J / i , , ~

2

0

I

20 20

/ /

3

64% 4HB

If0

R

[] l~%4HB • 90%4HB "0 82%4HB • 78%4HB A 64%4HB

40

Figure2 X-ray diffraction patterns of P(3HB-co-4HB)filmscast from CHCI3 solutions. Films had been aged for 3 weeksat room temperature after evaporation of the solvent

1

2

3

4

5

6

7

8

Time (h) Figure4 Enzymaticdegradation (erosion) profiles of P(3HB-co-4HB)

films in an aqueous solution of lipase from R. delemer (pH 7.4, 37°C)

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Poly(3-hydroxybutyrate-co-4-hydroxybutyrate): Y. Saito and Y. Doi Table 7 Stress-strain results of P(3HB-co-4HB) films at 23°C

4HB fraction (mol%) Properties Crystallinity (%)b Young's modulus (MPa) Stress at yield (MPa) Elongation at yield (%) Tensile strength (MPa) Elongation to break (%)

0=

3"

10~

16=

64

60

55

45

45

43 5

34 4 28 45

28 5 24 242

19 7 26 444

15 30 . . 17 591

78

. .

17 24 . . 42 1120

82

90 18 45

28 100

58 1320

65 1080

. .

100 34 149 14 17 104 1000

=From Reference 21

bDetermined by X-ray diffraction elongation to break increased from 5 to 444%. The tensile strength of the films with compositions of 64--100 m o l % 4HB increased from 17 to 1 0 4 M P a with increasing 4HB fraction. The true tensile strength of P(4HB) h o m o p o l y m e r film is calculated to be as large as 1 G P a if the cross-section is corrected. Thus, the P(3HB-co-4HB) copolyesters with high 4 H B fractions are very strong thermoplastic elastomers. All data on physical properties were obtained on the solution-cast films of P(3HB-co4HB) copolyesters. It is k n o w n that crystallization p h e n o m e n a in bacterial polyesters depend strongly on the crystallization and annealing temperatures 2°'29'3°. It is necessary to study the differences between the mechanical properties of melt and solution-cast films of P(3HB-co-4HB).

Enzymatic degradation o f P(3HB-co-4HB) copolyesters

The enzymatic degradations of P(3HB-co-4HB) films (initial weight, 6rag) were carried out at 37°C in 0.1 M phosphate buffer (pH 7.4) using extracellular P H B depolymerase purified from A. faecalis or lipase from R. delemer. In this study, six samples of P(3HB-co-4HB) containing 0, 64, 78, 82, 90 and 1 0 0 t o o l % 4 H B were used. Fi#ure 3 shows the weight loss (erosion) profiles of the polyester films as a function of time using the extracellular P H B depolymerase from A. faecalis. The a m o u n t of film erosion increased proportionally with time for all the samples. N o erosion of films was observed for 30 h at 30°C without enzyme. The film of P(4HB) h o m o p o l y m e r was eroded by P H B depolymerase, but its degradation rate was slower than that of P(3HB) h o m o p o l y m e r . The erosion rate of P(3HB-co-4HB) films by P H B depolymerase increased as the 4HB fraction decreased from 100 to 64mo1%. In a previous paper 22, we reported that the rate of enzymatic degradation by P H B depolymerase of P(3HB-co-4HB) films with compositions of 0 - 2 8 m o 1 % 4 H B increased with an increase in the 4HB fraction. It has been found that the rate of enzymatic degradation of P(3HB) films by P H B depolymerase increases with a decrease in crystallinity 28. The acceleration of enzymatic degradation by P H B depolymerase for the P(3HB-co-4HB) films m a y be caused by the decrease in crystallinity. Figure 4 shows the weight loss profiles of the polyester films as a function of time using the lipase from R. delemer. It is of interest to note that the film of P(3HB) h o m o p o l y m e r was not eroded by the lipase, and that the erosion rate of P(4HB) h o m o p o l y m e r was highest. The erosion rate of films by lipase increased with an increase in the 4HB fraction. Thus, P(3HB-co-4HB) films are hydrolysed by both P H B depolymerase and lipase.

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Acknowledgements This work was performed under the management of the Research Institute of Innovative Technology for the Earth (RITE) as part of the Development of

Biodegradable Plastics project supported by the New Energy and Industrial Technology Development Organization (NEDO).

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