Global Journal of Environmental Research, 1 (2): 69-73, 2007 ISSN 1992-0075 IDOSI Publications, 2007

Physical and Chemical Properties of Polyhydroxyalkanotes Biodegradable Polymers Produced in Transgenic Yeasts
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Desouky A.M. Abd-El-Haleem, 2M.A. AlMa'adeed and 3N. Al-Thani

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Department of Biological Sciences, Department of Math and Physics Sciences, College of Arts and Sciences, Qatar University, 2713 Doha, State of Qatar 3 Environmental Studies Research Center, Department of Physical Sciences, Qatar University, 2713 Doha, State of Qatar
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Abstract: Polyhydroxybutyrate Homopolymers (PHB) were produced by transgenic yeasts Saccharomyces cerevisiae INVSc1/PHA1 and Schizosaccharomyces pombe Q01/PHB equipped in their cytoplasm and chromosome with the phaABCRe operon of the PHA biosynthetic pathway of Ralstonia eutropha, respectively. The polyesters were purified and physically characterized by Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy (IR). The physical properties of the PHB homopolymers were dependent on the type of transgenic yeast. Key words: Polyhydroxybutyrate % transgenic yeasts % differential scanning calorimetry % infrared spectroscopy % physical properties INTRODUCTION Polyhydroxybutyrate, (PHB), synthesized by various microorganisms, serves as an intracellular carbon and energy reserve materials [1, 2]. It is known as a biodegradable polymer having some potential applications in different fields as biomedical and environments. However, the major commercial drawback of the bacterial PHBs is their high production cost, making them substantially more expensive than synthetic plastics [3]. Therefore, engineering of novel pathways in eukaryotic cell systems seems to be a beneficial alternative to the production of PHBs in bacteria. For this purpose, yeast cells were used as models to gain information on PHBs synthesis in eukaryotes [4, 5]. Yeasts as hosts for synthesis of PHBs have certain advantages over bacteria. First, yeasts have been studied intensively from physiology, molecular biology and biotechnology points of views. Second, yeasts are physiologically flexible and they are larger than bacteria [4, 5]. Moreover, yeast like, Saccharomyces cerevisiae, Klyveromyces marxianus, Candida utilis and others, has been approved as a GRAS microorganism by Food and Drug Administration [6]. Generally, Polyhydroxyalkanotes (PHA) homopolymers are classified into two groups based on the number of carbon atoms in the monomer units incorporated into the polymer chain [7, 8]. Short-chainLength (SCL) PHA consists of 3-hydroxyalkanoate (3HA) monomers with 3-5 carbon atoms in length. Medium-chain-length (MCL) PHA consists of 3HA monomers with 6-14 carbon atoms in length. However, the monomer structures and contents of PHA have considerable effects on their physical properties [8]. As a typical bacterial SCL PHA, Polyhydroxybutyrate (PHB) is a stiff crystalline material and has a high melting temperature. Bacterial PHB is too brittle to be processed, hence, limiting its industrial applications[8]. Recently Abuelhamd et al. [9] reported a PHB homopolymer production in two transgenic yeasts. One of them (Saccharomyces cerevisiae INVSc1/PHA1) harboring the PHB synthase genes of R. eutropha in its cytoplasm, while in the second (Schizosaccharomyces pombe Q01/PHB), PHB biothensyzie genes were integrated into the chromosome. In this study, the PHB homopolymer produced by both transgenic yeasts were purified and investigated for their physical properties.

Corresponding Author: Dr. Desouky A.M. Abd-El-Haleem, Department of Biological Sciences, College of Arts and Sciences, Qatar University, Doha, Post Code 2713, Qatar

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MATERIALS AND METHODS Transgenic yeasts and culture conditions: Transgenic yeasts Saccharomyces cerevisiae INVSc1/PHA1 and Schizosaccharomyces pombe Q01/PHB [9] harboring the PHB synthesis genes in their cytoplasm and chromosome, respectively were routinely maintained in leucine-deficient media (0.67% yeast nitrogen base without amino acids [Difco, Detroit, Mich.], 0.5% ammonium sulfate, 2% glucose and 0.4 g of leucine dropout supplement (Clontech, Palo Alto, Calif). For PHB production, a stationary-phase culture grown in leucine-deficient media was harvested by centrifugation and cells were washed once in water and resuspended in Mineral Salts Medium (MSM) [10] supplemented with 0.1% glucose, 0.5% of the detergent Tween 60 (Sigma, St. Louis, Mo.) and 0.1% fatty acid (oleic acid). Cells were grown at 30C for an additional 1 to 6 days before harvest of the cells for PHB analysis. The pH of the growth media was 6.0. PHB extraction: PHB extraction procedure was performed according to Findlay and White [11]. Lyophilized yeast cell sediments were placed in a Soxhlet extractor lined with glass wool and wrapped with a resistance strip heater. Enough chloroform to cover the sample was added and the sample was sonicated for 10 min. The sample was extracted overnight in a total of 125 ml of chloroform. The extraction thimble of the Soxhlet extractor was heated so that the chloroform present boiled, maintaining solubility of the polymers. The chloroform was recovered and removed in a rotary evaporator in vacuum. Subsequently, the polymer was redissolved in hot chloroform and PHA was recovered from the chloroform by non-solvent precipitation and filtration. Methanol was used as the nonsolvent (4-6 volumes). Chemical and physical characterizations of the PHB extracted polymers: IR spectra were recorded with the IR-Bruker spectrometer. The NMR spectra were recorded on a JOEL-ECA 500 spectrometer. The 500 MHz 1H-NMR spectra were recorded from a CDCl3 solution of the PHA (30 mg mlG1) at 20C, 1.30809856 s acquisition times and 12.5250501 KHz spectral width. Differential scanning calorimetry (DSC) was performed with a Perkin-Elmer DSC7 thermal analyzer calibrated by high purity Indium in the temperature range from 20C to 200C at a heating rate of 10C/min. The melting Temperature (Tm) was taken as the peak temperature of the melting endothermic peak; the crystallization temperature Tc was taken as the peak

temperature of the exothermic peak. Enthalpy of fusion for the melting ()Hm) and enthalpy of fusion for the cold crystallization ()Hc) was calculated. The crystallinity percentage was calculated using the following equation Degree of Crystallinity (%) = H H * Where )H* is a completely crystalline material 146 Jg G1 [12]. RESULTS AND DISCUSSION The IR spectroscopic analysis gave further insights into the chemical structure without a previous hydrolysis of the polymer and reflects the monomeric units. However, NMR is an important and very sensitive method for determining the domain size and miscibility, which is not easy to identify using conventional microscopic [13]. As shown in Fig. 1, the IR spectra of both polymers revealed the presence of marked peaks at wave numbers 3440, 2920-2980, 1720 and 1240-1370 cmG1 representing the presence of O-H bending, two bands of C-H stretching, strong absorption band of aliphatic carbonyl C=O and C-H band of aliphatic compound, respectively. The presence of C-H stretch and C-H of aliphatic compound is detected at peaks of 2920-2980 cmG1 and 1240-1370 cmG1, respectively. However, the higher area emerging of 1720 cmG1 in PHB of the transgenic strain INVSc1/PHA1 is attributed to carbonyl groups in the material itself or resulting from oxidative degradation of the chains [14]. The strong transmission band in PHB of strain INVSc1/PHA1 compared to strain Q01/PHB due to OH of water at around 3500-3300 cmG1 indicated the presence of bound H2O. The higher absorption at 2920 for INVSc1/PHA1 indicates the presence of several methyl groups in the material more than strain Q01/PHB. As shown in Fig. 2, the 1H NMR spectra obtained from the PHBs extracted from both transgenic yeasts S. pombe Q01/PHB and S. cerevisiae INVSc1/PHA1 had the characteristic peaks at 1.2, 2.4-2.6 and 5.2 ppm found in the PHB standard [15]. The molecular composition of the polyesters as indicated by chemical shifts, generates a structure of (CH2-CH) backbone and assigned the presence of (CH3) group. The characteristic signals for other hydroxyalkanoaic acids, however, were totally lacking and confirmed the homopolymeric nature of the extracted PHBs.

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Global J. Environ. Res., 1 (2): 69-73, 2007

Fig. 1: IR spectra analysis of PHB polymers produced in both strains Q01/PHB and INVSc1/PHA1

Fig. 2: 1 H NMR analysis of the PHB produced in both yeast strains Q01/PHB and INVSc1/PHA1
55 Q01/PHB INVSc1/PHA1

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Heat flow mW

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Temperature C

Fig. 3: The DSC thermograms of the first heatingcooling cycle of PHB produced in both yeast strains Q01/PHB and INVSc1/PHA1
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Table 1: Shows both crystallinity percentage and melting temperature First heating cooling cycle PHB of strain Q01/PHB PHB of strain INVSc1/PHA1 155.17 171.33 57.97 27.13 36.03 152.84 163.42 168.33 59.66 31.90 50.05 Tm (1)C Tm (2)C Tm (3)C Tc C Crystallinity Crystallinity % (heating) % (cooling)

CONCLUSION PHB biopolymers are produced by both transgenic yeasts Saccharomyces cerevisiae INVSc1/PHA1 and Schizosaccharomyces pombe Q01/PHB and characterized by IR, 1H NMR and DSC. IR and 1H NMR confirmed the existence of the PHB homopolymer compositions. PHB of strain Q01/PHB showed more crystallites regions confirmed by the existence of two small shoulders and one large peak, while strain INVSc1/PHA1 showed two crystallites regions which are more stabilized than the three crystallites in PHB of strain Q01/PHB. Also the total crystallinity percentage and nucleation density of crystallites are more in strain Q01/PHB. More absorption of water is noticed in the strain INVSc1/PHA1. The high amount of water may cause more degradation in the polymer and showed that strain INVSc1/PHA1 is much sensitive to the air with more absorption of water which acts as a plasticizer. The crystallinity behavior of the PHB produced by the yeast have the same behavior as that produced by bacteria with the difference that the produced by the yeast have in addition to the main crystalline regions other smaller crystallites with lower melting points while the produced by bacteria have larger thickness, but strain Q01/PHB has a higher crystallinity percentage than strain INVSc1/PHA1 due to the existence of more crystallites regions. REFERENCES 1. Jendrossek, D., A. Schirmer and H.G. Schlegel, 1996. Biodegradation of polyhydroxyalkanoic acids. Applied Microbiology and Biotechnology, 46: 451-463. Sudesh, K., H. Abe and Y. Doi, 2000. Factors affecting the freeze-fracture morphology of in vivo polyhydroxyalkanoate granules. Can. J. Microbiol., 46: 304-311. Arcana, M., O. Giani-Beaune, F. Schue, W. Amass and A. Amass, 2000. Structure and morphology of polyhydroxybutyrate synthesized by ring-opening polymerization of racemic (R, S) butyrolactone with distannoxane derivatives. Polymer International, 49: 1348-1355. Carlson, R., D. Fell and F. Srienc, 2002. Metabolic Pathway analysis of recombinant yeast for rational strain development. Biotechnol. Bioeng., 79: 121-134.

Figure 3 shows the DSC thermograms of the first heating-cooling cycle. PHB polymer sample of the transgenic strain Q01/PHB revealed existing of two shoulders in the peak, which means the existence of a main crystallite regions with more perfect structure [16] and other two smaller thinner crystallites regions with some defects and lower melting points. However, PHB of the transgenic strain INVSc1/PHA1 shows a different type of endothermic peak where, there are two peaks one is smaller than the other indicating the existing of two crystallites with different sizes. Melting behavior of bacterial poly (3hydoxybutyrate) (PHB) has been investigated by Gunaratne et al. [16] found the melting point and crystallinity in the same range but with a shoulder at higher temperature rather than lower temperature as in our case. This may be due to the smaller lamellar thickness and less stable crystallites in the yeast production than in bacteria production. As shown in Table 1, PHB of strain INVSc1/PHA1 has a higher thermal stability than strain Q01/PHB, where melting temperatures are higher. Crystallinity percentage is higher in PHB of strain Q01/PHB. Gunaratne et al. [16] found the crystallinity percentage in the same heating ray for 71% which is much higher than that for yeast production, which is shown in Table 1. The higher melting points in PHB of strain INVSc1/PHA1 cause the presence of new secondary force and ensure that the molecules are packed closely to increase the crystallinity, however the presence of more crystallites region (even with lower melting points) in the PHB of strain Q01/PHB increase the crystallinity percentage, this is confirmed by the higher crystallization percentage during cooling and lower crystallization temperature due to increase in the nucleation density in PHB of strain INVSc1/PHA1. To study the effect of heating and cooling on the samples, Table 1 shows that both crystallinity percentage and melting temperature decrease with increasing the cycles, which means more degradation is occurring during the heating and the forces between the chains are less with more flexible bonds. 72

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11. Findlay, R.H. and D.C. White. 1983. Polymeric beta-hydroxyalkanoates from environmental samples and Bacillus megaterium. Applied and Environ. Microbiol., 45: 71-78. 12. Gogolewski, S., M. Jovanovic, S.M. Perren, J.G. Dillon, M.K. Hughes, 1993. J. Biomed. Mater. Res., 27: 1135-1148. 13. Doi, Y., M. Kunioka, Y. Nakamura and K. Soga, 1986. Nuclear magnetic resonance studies on poly (L-hydroxybutyrate) and a copolyester of Lhydroxybutyrate and L-hydroxyvalerate isolated from Alcaligenes eutrophus H16., Macromolecules, 19: 2860-2864. 14. Miguez-Suarez, J.C., E.B. Mano and R.A. Pereira, 2000. Thermal behavior of gamma-irradiated recycled polyethylene blends. Polymer Degradation and Stability, 69: 217-222. 15. Yoshie, N, Y. Goto, M. Sakurai, Y. Inoue, R. Chujo and Y. Doi, 1992. Biosynthesis and n.m.r. studies of deuterated poly(3-hydroxybutyrate) produced by Alcaligenes eutrophus H16. Intl. J. Biol. Macromol., 14: 81-86. 16. Gunaratne, L., R. Shanks and G. Amarasinghe, 2004. Thermal history effects on crystallisation and melting of poly(3-hydroxybutyrate). Thermochimica Acta 423: 127-135.

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