臺灣博碩士論文加值系統

本研究利用日本東京工業大學拓植實驗室(Tsuge Laboratory of Tokyo Institute of Technology in Japan)所提供的pET-15b::PhaCRe質體進行培養，接著利用雙水相系統分離目標酵素聚羥基烷酯聚合酶(PHA polymerase)，本研究共分為兩大部分：1.利用含有PhaCRe蛋白質的澄清菌液(Clarified feedstock)及混濁菌液(Unclarified feedstock)進行雙水相系統，以純化目標酵素並分析活性之最適化條件。雙水相系統測試條件包含選擇不同環境pH值、雙結點曲線(Binodal curve)、節線長度(Tie-line length, TLL)、分離速度(Centrifugation speed)、鹽類(Salt)、操作溫度(Operational temperature)、體積比(Volume ratio)、工作體積(Working volume)及系統中菌液負載濃度(Loading biomass)對PhaCRe蛋白質的影響，進行分離效果的探討。2.利用經最適化條件純化後的PhaCRe蛋白質及利用雙水相系統建立一個酵素反應平台來合成聚羥基烷酯(PHAs)，探討PhaCRe蛋白質催化基質(R)-3HB-CoA之酵素動力學模式(Enzyme kinetics model)。
目前最適化純化結果為將澄清菌液(Clarified feedstock)之pET-15b::PhaCRe經由30% (w/w) PEG 6000 / 8% (w/w) PO4 pH 8.7所組成的雙水相系統於4 oC下操作進行純化程序，其比活性可達到1.76 U mg-1、純化倍率可達到16.23倍及回收率可達到95.32%；將混濁菌液(Unclarified feedstock)之pET-15b::PhaCRe經由30% (w/w) PEG 6000 / 8% (w/w) PO4 pH 8.7所組成的雙水相系統於4 oC下操作進行純化程序，其比活性可達到1.82 U mg-1、純化倍率可達到16.81倍及回收率可達到96.42%。研究結果顯示ATPS是一高效率的蛋白純化及分離技術，可直接從混濁菌液中純化聚羥基烷酯聚合酶。而探討PhaCRe蛋白質之酵素動力學方面，由結果得知經雙水相系統純化後的PhaCRe蛋白質與基質之間的親和力變化雖然不大，但催化速率卻有提升，且酵素催化機制關係呈現”非競爭型”；再藉由實驗結果證實反應催化機制為三級反應，即兩個酵素分子與一個基質分子進行反應。以雙水相系統為酵素反應平台合成的聚羥基烷酯(PHAs)重量平均分子量為8.8 × 103、數目平均分子量為8.5 × 103、分子量分佈指數為1.04，由此結果顯示合成的聚合物分子量集中，易於後續發展應用。

In this study, the recombinant Escherichia coli pET-15b::PhaCRe obtained from Tsuge Laboratory in Tokyo Institute of Technology (Japan) was employed as expression host for PHA polymerase production. Firstly, the expressed enzyme was directly recovered by using aqueous two-phase system (ATPS). The clarified and unclarified feedstock containing PhaCRe protein was introduced to the ATPS for separation. The impacts of pH value, binodal curve, tie-line length (TLL), centrifugation speed, ionic strength, operational temperature, volume ratio of ATPS, Working volume and loading concentration of biomass upon recovery performance were investigated and discussed. Secondly, an integrated aqueous two-phase system contained purified PhaCRe protein was applied to enzymatic reaction of P(3HB) synthesis in vitro. Moreover, the enzyme kinetics models of PhaCRe protein catalyzed with substrate, (R)-3HB-CoA, was determined.
The results demonstrated that the optimal conditions for PhaCRe recovery from clarified feedstock are by applying ATPS consisted of 30% (w/w) PEG 6000 and 8% (w/w) PO4 at pH value of 8.7 and 4 oC. The specific activity and purification factor can achieve 1.76 U mg-1 and 16.23, respectively. The efficiency of recovery achieved 95.32%. The identical conditions were also found for PhaCRe protein separation from unclarified feedstock in ATPS. The specific activity compared to the value derived from clarified feedstock increased to 1.82 U mg-1 and the purification factor achieves 16.81. The 96.42% of recovery efficiency was fulfilled. The results illustrated that ATPS can be a promising technique introduced to recover PHA polymerase in one-step operation. In addition, the present study is the first report in purification of PHA polymerase using ATPS technique. In part of kinetics study of PhaCRe polymerase, the results showed that the Km between the purified PhaCRe protein and the (R)-3HB-CoA substrate was similar. However, the Vmax can be improved, and the “non-competitive” enzymatic mechanism was depicted. Determination of polymerization showed a reaction order of 3 where two enzyme molecules react with one substrate molecule. The synthesis P(3HB) polymer in vitro has Mw, Mn and PDI are 8.8 × 103, 8.5 × 103 and 1.04, respectively.

摘要 I
Abstract II
目錄 III
圖目錄 VIII
表目錄 XI
第一章 緒論 1
1.1 前言 1
1.2 蛋白質純化技術 3
1.2.1 膠體過濾技術 4
1.2.2 離子交換層析技術 5
1.2.3 疏水性層析技術 6
1.2.4 親和性層析技術 7
1.2.5 雙水相系統 8
1.2.5.1 雙水相系統的特性 9
1.2.5.2 雙水相系統相圖(Phase diagram) 10
1.2.5.3 雙水相系統的分類 12
1.2.5.4 影響分配係數(K)的操作因子 13
1.2.5.5 雙水相系統的分析 15
1.3 生物可分解性塑膠分類 15
1.4 聚羥基烷酯(PHAs)介紹 17
1.4.1 聚羥基烷酯(PHAs)基本結構 17
1.4.2 聚羥基烷酯(PHAs)性質 18
1.4.3 聚羥基烷酯(PHAs)合成的代謝路徑 19
1.4.4 聚羥基烷酯(PHAs)相關代謝基因 22
1.4.5 聚羥基烷酯(PHAs)合成方式比較 24
1.5 研究動機與策略 24
1.5.1 研究動機 24
1.5.2 研究策略 26
第二章 以雙水相系統分離純化聚羥基烷酯聚合酶(PHA polymerase) 28
2.1 摘要 28
2.2 文獻回顧 29
2.2.1 不同菌株之聚羥基烷酯聚合酶相關研究 29
2.2.2 純化聚羥基烷酯聚合酶相關研究 30
2.3 實驗藥品 32
2.4 實驗儀器設備 33
2.5 實驗方法與分析 33
2.5.1 總蛋白質濃度(Total protein concentration)測定 33
2.5.2 聚羥基烷酯聚合酶活性測定 34
2.5.3 聚丙烯醯胺膠體電泳分析 35
2.5.4 雙水相系統的製備 38
2.5.5 雙水相系統之雙結點曲線製備 38
2.5.6 蛋白質製備(Protein preparation) 39
2.6 研究策略 40
2.7 結果與討論 41
2.7.1 探討pH值對ATPS分離效果的影響 41
2.7.2 不同雙結點曲線與節線長度對ATPS分離效果的影響 43
2.7.3 不同分離速度對ATPS分離效果的影響 49
2.7.4 探討鹽類對ATPS分離效果的影響 51
2.7.5 探討不同操作溫度及體積比對ATPS分離效果的影響 53
2.7.6 探討不同工作體積及節線長度對ATPS分離效果的影響 57
2.7.7 探討不同菌液負載濃度對ATPS分離效果的影響 59
2.7.8 利用SDS-PAGE分析經ATPS純化後的結果分析 60
2.8 結論 63
第三章 以雙水相系統進行聚羥基烷酯(PHAs)體外合成探討 64
3.1 摘要 64
3.2 文獻回顧 64
3.2.1 體外合成(In vitro)路徑 65
3.2.2 體外合成相關研究 67
3.2.3 酵素動力學(Enzyme Kinetics) 69
3.2.3.1 酵素介紹 69
3.2.3.2 酵素的專一性 70
3.2.3.3 酵素分類 70
3.2.3.4 酵素活性(Enzyme activity) 71
3.2.3.5 酵素催化反應 71
3.3 實驗藥品 72
3.4 實驗儀器設備 73
3.5 實驗方法與分析 73
3.5.1 總蛋白質濃度測定 73
3.5.2 聚羥基烷酯聚合酶活性測定 73
3.5.3 雙水相系統的製備 73
3.5.4 反應動力學 74
3.5.5 酵素的抑制作用 79
3.5.6 聚羥基丁酯(PHB)之定量與定性分析 82
3.5.7 聚羥基烷酯(PHAs)之分子量分析 84
3.6 研究策略 87
3.7 結果與討論 89
3.7.1 探討不同CoA濃度對ATPS分離效果的影響 89
3.7.2 探討不同金屬離子對PhaCRe蛋白質活性的影響 90
3.7.3 探討不同pH及反應溫度對PhaCRe活性的影響 93
3.7.4 Michaelis-Menten酵素動力學 95
3.7.5 探討聚合聚羥基烷酯(PHAs)之酵素催化機制 98
3.7.6 聚羥基烷酯(PHAs)之定性分析 99
3.7.7 聚羥基丁酯(PHB)之分子量分析 100
3.7.8 聚羥基丁酯(PHB)之定量及定性分析 101
3.8 結論 103
第四章 總結與未來展望 105
4.1 總結 105
4.2 未來展望 107
第五章 參考文獻 108

Agus, J., Kahar, P., Hyakutake, M., Tomizawa, S., Abe, H., Tsuge, T., Satoh, Y., and Tajima, K. (2010). Unusual change in molecular weight of polyhydroxyalkanoate (PHA) during cultivation of PHA-accumulating Escherichia coli. Polymer Degradation and Stability 95, 2250-2254.
Akbari, N., Daneshjoo, S., Akbari, J., and Khajeh, K. (2011). Isolation, characterization, and catalytic properties of a novel lipase which is activated in ionic liquids and organic solvents. Applied Biochemistry and Biotechnology 165, 785-794.
Albertsson, P. A. (1986). Partition of cell particles and macromolecules. 3rd edition. Wiley &; Sons, New York.
Anderson, A. J., and Dawes, E. A. (1990). Occurrence, metabolism, metabolic role, and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews 54, 450-472.
Andrews, B. A., and Asenjo, J. A. (2010). Theoretical and experimental evaluation of hydrophobicity of proteins to predict their partitioning behavior in aqueous two phase systems: A review. Separation Science and Technology 45, 2165-2170.
Andrews, B. A., Schmidt, A. S., and Asenjo, J. A. (2005). Correlation for the partition behavior of proteins in aqueous two-phase systems: effect of surface hydrophobicity and charge. Journal of Biotechnology and Bioengineering 90, 380-90.
Antov, M. G., Pericin, D. M., and Pejin, S. N. (2004). Pectinases partitioning in aqueous two-phase systems an integration of the systems poly(ethylene glycol)crude dextran and poly(ethylene glycol)ammonium sulphate. Journal of the Serbian Chemical Society 69, 299-307.
Asenjo, J. A., and Andrews, B. A. (2011). Aqueous two-phase systems for protein separation: a perspective. Journal of Chromatography A 1218, 8826-35.
Baskir, J. N., Hatton, T. A., and Suter, U. W. (1989). Thermodynamics of the partitioning of biomaterials in two-phase aqueous polymer systems: Comparison of lattice model to experimental data. Journal of Physical Chemistry 93, 2111-2122.
Bassani, G., Farruggia, B., Nerli, B., Romanini, D., and Pico, G. (2007). Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems. J Chromatogr B Analyt Technol Biomed Life Sci 859, 222-8.
Biazus, J. P. M., Severo, J. B., Santana, J. C. C., Souza, R. R., and Tambourgi, E. B. (2006). Study of amylases recovery from maize malt by ion-exchange expanded bed chromatography. Process Biochemistry 41, 1786-1791.
Bowen, W. R., and Pan, L. C. (1997). Ion exchange of bovine serum albumin at a natural organic anion exchanger: Thermodynamics and energetics. Journal of Colloid and Interface Science 189, 328-336.
Boyer, R. F. (2006). Concepts in Biochemistry. 3rd edition. Wiley &; Sons, Hoboken, New Jersey.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72, 248-254.
Burns, K. L., Oldham, C. D., Thompson, J. R., Lubarsky, M., and May, S. W. (2007). Analysis of the in vitro biocatalytic production of poly-(β)-hydroxybutyric acid. Enzyme and Microbial Technology 41, 591-599.
Chanprateep, S. (2010). Current trends in biodegradable polyhydroxyalkanoates. Journal of Bioscience and Bioengineering 110, 621-32.
Chavez-S, A., Benavides, J., Vermaas, W., and Rito-Palomares, M. (2010). Application of Aqueous Two-Phase Systems for the Potential Extractive Fermentation of Cyanobacterial products. Chemical Engineering and Technology 33, 177-182.
Cheng, M. L., and Sun, Y. M. (2009). Effect of Thermal History on the Free Volume Properties of Semi-Crystalline Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Membranes by Positron Annihilation Lifetime Spectroscopy. Journal of Polymer Science Part B: Polymer Physics 47, 855-865.
Chinwetkitvanich, S., Randall, C. W., and Panswad, T. (2004). Effects of phosphorus limitation and temperature on PHA production in activated sludge. Water Science and Technology 50, 135-143.
Clapés, P., and Infante, M. R. (2002). Amino acid-based surfactants: Enzymatic synthesis, properties and potential applications. Biocatalysis and Biotransformation 20, 215-233.
De Souza, R. L., Barbosa, J. M. P., Zanin, G. M., Lobão, M. W. N., Soares, C. M. F., and Lima, Á. S. (2010). Partitioning of porcine pancreatic lipase in a two-phase systems of polyethylene glycol/potassium phosphate aqueous. Applied Biochemistry and Biotechnology 161, 288-300.
Fisher, D., and Sutherland, I. A. (1989). Separations using aqueous phase system: applications in cell biology and biotechnology. Plenum press, New York.
Forciniti, D., Hall, C. K., and Kula, M. R. (1991). Influence of polymer molecular weight and temperature on phase composition in aqueous two-phase systems. Fluid Phase Equilibria 61, 243-262.
Gerngross, T. U., Snell, K. D., Peoples, O. P., Sinskey, A. J., Csuhai, E., Masamune, S., and Stubbe, J. (1994). Overexpression and purification of the soluble Polyhydroxyalkanoate synthase from Alcaligenes eutrophus: Evidence for a required posttranslational modification for catalytic activity. Biochemistry 33, 9311-9320.
H-Kittikun, A., Prasertsan, P., Zimmermann, W., Seesuriyachan, P., and Chaiyaso, T. (2012). Sugar ester synthesis by thermostable lipase from Streptomyces thermocarboxydus ME168. Applied Biochemistry and Biotechnology 166, 1968-1982.
Harris, J. M. (1992). Poly(ethylene glycol) chemistry: Biotechnical and biomedical applications. Plenum press, New York.
Hatti-Kaul, R. (2000). Aqueous Two-Phase Systems: Methods and Protocols. Humana Press, Totowa, New Jersey.
Hatti-Kaul, R. (2001). Aqueous two-phase systems: A general overview. Applied Biochemistry and Biotechnology - Part B Molecular Biotechnology 19, 269-277.
Hong, S. G., Lin, Y. C., and Lin, C. H. (2008). Improvement of the Thermal Properties of Polyhydroxybutyrate by Grafted Chemicals. Journal of Applied Polymer Science 110, 2718-2726.
Hustedt, H. (1986). Extractive enzyme recovery with simple recycling of phase forming chemicals. Biotechnology Letters 8, 791-796.
Jaeger, K. E., and Eggert, T. (2004). Enantioselective biocatalysis optimized by directed evolution. Current Opinion in Biotechnology 15, 305-13.
Jossek, R., Reichelt, R., and Steinbuchel, A. (1998). In vitro biosynthesis of poly(3-hydroxybutyric acid) by using purified poly(hydroxyalkanoic acid) synthase of Chromatium vinosum. Applied Microbiology and Biotechnology 49, 258-266.
Jossek, R., and Steinbuchel, A. (1998). In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system. FEMS Microbiology Letters 168, 319-324.
Kennedy, J. F., and Cabral, J. M. S. (1993). Recovery processes for biological materials. Wiley &; Sons, New York.
Khanna, S., and Srivastava, A. K. (2005). Recent advances in microbial polyhydroxyalkanoates. Process Biochemistry 40, 607-619.
Koller, M., Salerno, A., Dias, M., Reiterer, A., and Braunegg, G. (2010). Modern biotechnological polymer synthesis: A review. Food Technology and Biotechnology 48, 255-269.
Kunasundari, B., and Sudesh, K. (2011). Isolation and recovery of microbial polyhydroxyalkanoates. Express Polymer Letters 5, 620-634.
Lacki, K. M., and Brekkan, E. (2011). Protein Purification: Principles, High Resolution Methods, and Applications. 3rd edition. Wiley &; Sons, USA.
Lazreg-Aref, H., Mosbah, H., Fekih, A., Mars, M., and Said, K. (2012). Purification and Biochemical Characterization of Lipase from Ficus carica Latex of Tunisian East Coast Zidi Variety. Journal of the American Oil Chemists' Society 1-9.
Lee, S. Y. (1996). Plastic bacteria? Progress and prospects for Polyhydroxyalkanoate production in bacteria. Trends in Biotechnology 14, 431-438.
Liebergesell, M., Sonomoto, K., Madkour, M., Mayer, F., and Steinbuchel, A. (1994). Purification and characterization of the poly(hydroxyalkanoic acid) synthase from Chromatium vinosum and localization of the enzyme at the surface of poly(hydroxyalkanoic acid) granules. European Journal of Biochemistry 226, 71-80.
Lima, Á. S., Alegre, R. M., and Meirelles, J. A. (2002). Partitioning of pectinolytic enzymes in polyethylene glycolpotassium phosphate aqueous two-phase systems. Carbonhydrate Polymers 50, 63-68.
Liu, S. J., and Steinbuchel, A. (2000). Exploitation of butyrate kinase and phosphotransbutyrylase from Clostridium acetobutylicum for the in vitro biosynthesis of poly(hydroxyalkanoic acid). Applied Microbiology and Biotechnology 53, 545-552.
Lo, C. W., Wu, H. S., and Wei, Y. H. (2009). Optimizing acidic methanolysis of poly(3-hydroxyalkanoates) in gas chromatography analysis. Asia-Pacific Journal of Chemical Engineering 4, 487-494.
Lu, Q., Han, J., Zhou, L., Zhou, J., and Xiang, H. (2008). Genetic and Biochemical Characterization of the Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) Synthase in Haloferax mediterranei. Journal of Bacteriology 190, 4173-4180.
Madeira, P. P., Teixeira, J. A., Macedo, E. A., Mikheeva, L. M., and Zaslavsky, B. Y. (2008). Correlations between distribution coefficients of various biomolecules in different polymer/polymer aqueous two-phase systems. Fluid Phase Equilibria 267, 150-157.
Madison, L. L., and Huisman, G. W. (1999). metabolic engineering of poly(3-hydroxyalkanoates) from dna to plastic. Microbiology and Molecular Biology Reviews 63, 21-53.
Miller, J. M. (2004). Chromatography: Concepts and Contrasts. 2nd edition. Wiley &; Sons, Hoboken, New Jersey
Nguyen, A. L., Grothe, S., and Luong, J. H. T. (1988). Applications of pullulan in aqueous two-phase systems for enzyme production, purification and utilization. Applied Microbiology and Biotechnology 27, 341-346.
Ojumu, T. V., Yu, J., and Solomon, B. O. (2004). Production of polyhydroxyalkanoates, a bacterial biodegradable polymer. African Journal of Biotechnology 3, 18-24.
Ooi, C. W., Tey, B. T., Hii, S. L., Kamal, S. M. M., Lan, J. C. W., Ariff, A., and Ling, T. C. (2009). Purification of lipase derived from Burkholderia pseudomallei with alcohol/salt-based aqueous two-phase systems. Process Biochemistry 44, 1083-1087.
Pan, I. H., Yao, H. J., and Li, Y. K. (2001). Effective extraction and purification of β-xylosidase from Trichoderma koningii fermentation culture by aqueous two-phase partitioning. Enzyme and Microbial Technology 28, 196-201.
Pantazaki, A. A., Ioannou, A. K., and Kyriakidis, D. A. (2005). A thermostable β-ketothiolase of polyhydroxyalkanoates (PHAs) in Thermus thermophilus: Purification and biochemical properties. Molecular and Cellular Biochemistry 269, 27-36.
Piculell, L., Nilsson, S., Falck, L., and Tjerneld, F. (1991). Phase separation in aqueous mixtures of similarly charged polyelectrolytes. Polymer Communications Guildford 32, 158-160.
Ploux, O., Masamune, S., and Walsh, C. T. (1988). The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli. European Journal of Biochemistry 174, 177-182.
Qi, Q., Rehm, B. H. A., and Steinbuchel, A. (1997). Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: Comparison of PhaC1 and PhaC2. FEMS Microbiology Letters 157, 155-162.
Qi, Q., Steinbuchel, A., and Rehm, B. H. A. (2000). In vitro synthesis of poly(3-hydroxydecanoate): Purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa. Applied Microbiology and Biotechnology 54, 37-43.
Rehm, B. H. A. (2003). Polyester synthases: Natural catalysts for plastics. Biochemical Journal 376, 15-33.
Rehm, B. H. A., Antonio, R. V., Spiekermann, P., Amara, A. A., and Steinbüchel, A. (2002). Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: In vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model. Biochimica et Biophysica Acta - Protein Structure and Molecular Enzymology 1594, 178-190.
Rito-Palomares, M. (2004). Practical application of aqueous two-phase partition to process development for the recovery of biological products. Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Science 807, 3-11.
Satoh, Y., Murakami, F., Tajima, K., and Munekata, M. (2005). Enzymatic synthesis of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) with CoA recycling using polyhydroxyalkanoate synthase and acyl-CoA synthetase. Journal of Bioscience and Bioengineering 99, 508-11.
Satoh, Y., Tajima, K., Tannai, H., and Munekata, M. (2003). Enzyme-catalyzed poly(3-hydroxybutyrate) synthesis from acetate with CoA recycling and NADPH regeneration in vitro. Journal of Bioscience and Bioengineering 95, 335-341.
Sheu, D. S., Lai, Y. W., Chang, R. C., and Chen, W. M. (2009). Detection of polyhydroxyalkanoate synthase activity on a polyacrylamide gel. Analytical Biochemistry 393, 62-6.
Smith, S. (1994). The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. The FASEB Journal 8, 1248-1259.
Solaiman, D. K., Ashby, R. D., Foglia, T. A., and Marmer, W. N. (2006). Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates). Applied Microbiology and Biotechnology 71, 783-9.
Song, J. J., Zhang, S., Lenz, R. W., and Goodwin, S. (2000). In vitro polymerization and copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from Ralstonia eutropha. Biomacromolecules 1, 433-439.
Steinbuchel, A. (2001). Perspectives for biotechnological production and utilization of biopolymers: Metabolic engineering of polyhydroxyalkanoate biosynthesis pathways as a successful example. Macromolecular Chemistry and Physics 202, 1-24.
Steinbuchel, A., Aerts, K., Babel, W., Follner, C., Kiebergesell, M., Madkour, M. H., Mayer, F., Pieper-Furst, U., Pries, A., Valentin, H. E., and Wieczorek, R. (1995). Considerations on the structure and biochemistry of bacterial polyhydroxyalkanoic acid inclusions. Canadian Journal of Microbiology 41, 94-105.
Sturesson, S., Tjerneld, F., and Johansson, G. (1990). Partition of macromolecules and cell particles in aqueous two-phase systems based on hydroxypropyl starch and poly(ethylene glycol). Applied Biochemistry and Biotechnology 26, 281-295.
Sudesh, K., Abe, H., and Doi, Y. (2000). Synthesis, structure and properties of polyhydroxyalkanoates: Biological polyesters. Progress in Polymer Science (Oxford) 25, 1503-1555.
Tjerneld, F., Berner, S., Cajarville, A., and Johansson, G. (1986). New aqueous two-phase system based on hydroxypropyl starch useful in enzyme purification. Enzyme and Microbial Technology 8, 417-423.
Tsuge, T., Fukui, T., Matsusaki, H., Taguchi, S., Kobayashi, G., Ishizaki, A., and Doi, Y. (2000). Molecular cloning of two (R)-specific enoyl-CoA hydratase genes from Pseudomonas aeruginosa and their use for polyhydroxyalkanoate synthesis. FEMS Microbiology Letters 184, 193-198.
Velu, N., Divakar, K., Nandhinidevi, G., and Gautam, P. (2012). Lipase from Aeromonas caviae AU04: Isolation, purification and protein aggregation. Biocatalysis and Agricultural Biotechnology 1, 45-50.
Vernau, J., and Kula, M. R. (1990). Extraction of proteins from biological raw material using aqueous polyethylene glycol-citrate phase systems. Applied Biochemistry and Biotechnology 12, 397-404.
Verral, M. S. (1996). Downstream Processing of Natural Products: A Practical Handbook. Wiley &; Sons, New York.
Walter, H., Brook, D. E., and Fisher, D. (1985). Partitioning in aqueous two-phase system. Academic press, New York.
White, J. S., and White, D. C. (1997). Source Book of Enzyme. 1st edition. CRC Press, USA.
Wu, Y. T., Zhu, Z. Q., Lin, D. Q., and Li, M. (1999). Modeling of liquid-liquid equilibrium of polyethylene glycol-salt aqueous two-phase systems - The effect of partial dissociation of the salt. Fluid Phase Equilibria 154, 109-122.
Yang, S., Huang, Z., Jiang, Z., and Li, L. (2008). Partition and purification of a thermostable xylanase produced by Paecilomyces thermophila in solid-state fermentation using aqueous two-phase systems. Process Biochemistry 43, 56-61.
Zhang, S., Kamachi, M., Takagi, Y., Lenz, R. W., and Goodwin, S. (2001). Comparative study of the relationship between monomer structure and reactivity for two polyhydroxyalkanoate synthases. Applied Microbiology and Biotechnology 56, 131-136.
Zhang, S., Kolvek, S., Goodwin, S., and Lenz, R. W. (2004). Poly(hydroxyalkanoic acid) biosynthesis in Ectothiorhodospira shaposhnikovii: Characterization and reactivity of a type III PHA synthase. Biomacromolecules 5, 40-48.
Zhang, S., Yasuo, T., Lenz, R. W., and Goodwin, S. (2000). Kinetic and mechanistic characterization of the polyhydroxybutyrate synthase from Ralstonia eutropha. Biomacromolecules 1, 244-251.