基因重組大腸桿菌所生產的藍藻蛋白(Cyanophycin granule polypeptide, CGP)結構上多了離胺酸(Lysine)，物性上則具有生物可降解性、溫度敏感性與低毒性的特點。因此，具有當藥物載體的潛力。然而大多藥物傳遞需要在中性中進行，但非水溶性藍藻在中性條件的低溶解度成為發展的瓶頸。
本研究利用Schiff base (imine) formation將非水溶性藍藻蛋白中離胺酸上的一級胺，與糖酸和糖胺上的醛基進行反應；也利用葡萄糖作為交聯劑，使非水溶性藍藻蛋白與PEI進行交聯反應。並藉由改變純化方式、溶劑、pH值與濃度探討非水溶性藍藻蛋白接枝物的溫度應答變化。除此之外，分別將各帶有正負電荷的非水溶性藍藻蛋白接枝物，以不同比例混合，來觀察非水溶性藍藻蛋白對於表面電荷改變的影響。
結果顯示非水溶性藍藻蛋白接枝糖酸與糖胺皆能改善原本在中性無法溶解的缺點，其相轉移溫度皆接近人體溫度37℃，而接枝物的官能基和碳鏈長短為影響其相轉移溫度高低和粒徑大小的因素。此外，不同的溶劑和純化方式也會導致同種接枝物的粒徑有所差異。帶有正負電荷的非水溶性藍藻蛋白接枝物混合後依然保有溫敏性高分子的特性其相轉移溫度為33-35℃，在TEM下觀察，未混合前的無定型狀成為core-shell的微胞，尤其在混合比例為1:1時結構較為穩定。
非水溶性藍藻蛋白接枝糖酸與糖胺，或正負電混合的非水溶性藍藻蛋白接枝物都具有相當好的溫敏性，且透過調控溶劑、純化方式和電荷比例能得到理想的粒徑與結構，因此利於未來應用在藥物傳遞上。

Cyanophycin granule polypeptide consist of lysine and arginine as the side chains, and is biodegradable, temperature responsive and low toxicity. Thus it has a potential in biomedical applications, such as drug delivery. However, the water insoluble cyanophycin cannot be dissolved in neutral aqueous solution which to become a limitation to its current developments.
This study focused on using sugar acid and sugar amine to modify insoluble cyanophycin, and also on using glucose to crosslink the insoluble cyanophycin with PEI. In order to investigate the physical properties of the grafted polymer physical properties, we used different purification methods, solvents, pHs and concentrations. The grafted polymers with positive and negative charges were mixed at different ratios to examine the changes in the surface charges.
The results showed the grafting of sugar acid and sugar amine could enhance the solubility of insoluble cyanophycin, and the phase transition temperatures to around 37℃. The functional groups and carbon chain lengths affected the phase transition temperatures and the particle sizes. Different solvents and purification methods could result in differences in the particle sizes for the same grafting. The grafting with positive and negative charges still retained the characteristic of the temperature responsive property.

DEMBINSKA, M.E. and ALLEN, M.M., Cyanophycin granule size variation in Aphanocapsa. Microbiology, 1988. 134(2): 295-298.
2. Sallam, A. and Steinbüchel, A., Cyanophycin‐degrading bacteria in digestive tracts of mammals, birds and fish and consequences for possible applications of cyanophycin and its dipeptides in nutrition and therapy. Journal of Applied Microbiology, 2009. 107(2): 474-484.
3. Neumann, K., Stephan, D.P., Ziegler, K., Hühns, M., Broer, I., Lockau, W., and Pistorius, E.K., Production of cyanophycin, a suitable source for the biodegradable polymer polyaspartate, in transgenic plants. Plant Biotechnology Journal, 2005. 3(2): 249-258.
4. Mooibroek, H., Oosterhuis, N., Giuseppin, M., Toonen, M., Franssen, H., Scott, E., Sanders, J., and Steinbüchel, A., Assessment of technological options and economical feasibility for cyanophycin biopolymer and high-value amino acid production. Applied Microbiology and Biotechnology, 2007. 77(2): 257-267.
5. Allen, M.M., Cyanobacterial cell inclusions. Annual Reviews in Microbiology, 1984. 38(1): 1-25.
6. Aboulmagd, E., Oppermann-Sanio, F.B., and Steinbüchel, A., Molecular characterization of the cyanophycin synthetase from Synechocystis sp. strain PCC6308. Archives of Microbiology, 2000. 174(5): 297-306.
7. Hannig, G. and Makrides, S.C., Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnology, 1998. 16(2): 54-60.
8. Berg, H., Ziegler, K., Piotukh, K., Baier, K., Lockau, W., and Volkmer‐Engert, R., Biosynthesis of the cyanobacterial reserve polymer multi‐L‐arginyl‐poly‐L‐aspartic acid (cyanophycin) Mechanism of the cyanophycin synthetase reaction studied with synthetic primers. European Journal of Biochemistry, 2000. 267(17): 5561-5570.
9. Voss, I., Diniz, S.C., Aboulmagd, E., and Steinbüchel, A., Identification of the Anabaena sp. Strain PCC7120 Cyanophycin Synthetase as Suitable Enzyme for Production of Cyanophycin in Gram-Negative Bacteria Like Pseudomonas p utida and Ralstonia e utropha. Biomacromolecules, 2004. 5(4): 1588-1595.
10. Frey, K.M., Oppermann-Sanio, F.B., Schmidt, H., and Steinbüchel, A., Technical-scale production of cyanophycin with recombinant strains of Escherichia coli. Applied and environmental microbiology, 2002. 68(7): 3377-3384.
11. Kroll, J., Klinter, S., and Steinbüchel, A., A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium. Applied Microbiology and Biotechnology, 2011. 89(3): 593-604.
12. Oppermann-Sanio, F. and Steinbüchel, A., Occurrence, functions and biosynthesis of polyamides in microorganisms and biotechnological production. Naturwissenschaften, 2002. 89(1): 11-22.
13. Sallam, A. and Steinbüchel, A., Biotechnological production of cyanophycin dipeptides. 2018, Google Patents.
14. Schwamborn, M., Chemical synthesis of polyaspartates: a
biodegradable alternative to currently used polycarboxylate homo- and copolymers. Polymer Degradation and Stability, 1998. 59(1-3): 39-45.
15. Sallam, A. and Steinbüchel, A., Dipeptides in nutrition and therapy: cyanophycin-derived dipeptides as natural alternatives and their biotechnological production. Applied Microbiology and Biotechnology, 2010. 87(3): 815-828.
16. Roseman, S. and Comb, D.G., The hexosamine moiety of N-acetylneuraminic acid (sialic acid). Journal of the American Chemical Society, 1958. 80(12): 3166-3167.
17. Strominger, J.L., Park, J.T., and Thompson, R.E., Composition of the cell wall of Staphylococcus aureus: its relation to the mechanism of action of penicillin. The Journal of Biological Chemistry, 1959. 234(12): 3263-3268.
18. Howard, J., Preparation of pyruvic acid. Organic Syntheses, 1932. 1: 475-480.
19. Izumi, Y., Matsumura, Y., Tani, Y., and Yamada, H., Pyruvic acid production from 1, 2-propanediol by thiamin-requiring Acinetobacter sp. 80-M. Agricultural and Biological Chemistry, 1982. 46(11): 2673-2679.
20. Burdick, B.A. and Schaeffer, J.R., Co-immobilized coupled enzyme systems on nylon mesh capable of gluconic and pyruvic acid production. Biotechnology Letters, 1987. 9(4): 253-258.
21. Yonehara, T., Miyata, R., Matsuno, H., Goto, M., and Yahanda, S., Development of fermentative production of pyruvate by metabolic control-monograph. Seibutsu-Kogaku Kaishi, 2000. 78(2): 56-62.
22. Pal, D., Keshav, A., Mazumdar, B., Kumar, A., and Uslu, H., Production and Recovery of Pyruvic Acid: Recent Advances. Journal of The Institution of Engineers (India): Series E, 2017. 98(2): 165-175.
23. Roufs, J., Pyruvate: does it amp endurance and burn more fat. Muscle Fitness, 1996. 57(2): 195-197.
24. Li, Y., Chen, J., and Lun, S.-Y., Biotechnological production of pyruvic acid. Applied Microbiology and Biotechnology, 2001. 57(4): 451-459.
25. Wu, N., Yang, M., Gaur, U., Xu, H., Yao, Y., and Li, D., Alpha-ketoglutarate: physiological functions and applications. Biomolecules & Therapeutics, 2016. 24(1): 1.
26. Hixt, U. and Müller, H., L-alanyl-glutamine–a glutamine dipeptide for paraenteral nutrition. Environ. Health Perspect, 1996. 2: 72-76.
27. Stoll, B., McNelly, S., Buscher, H.P., and Häussinger, D., Functional hepatocyte heterogeneity in glutamate, aspartate and α‐ketoglutarate uptake: A histoautoradiographical study. Hepatology, 1991. 13(2): 247-253.
28. Kristensen, N.B., Jungvid, H., Fernández, J.A., and Pierzynowski, S., Absorption and metabolism of α‐ketoglutarate in growing pigs. Journal of Animal Physiology and Animal Nutrition, 2002. 86(7‐8): 239-245.
29. Le Boucher, J., Coudray-Lucas, C., Lasnier, E., Jardel, A., Ekindjian, O.G., and Cynober, L.A., Enteral administration of ornithine alpha-ketoglutarate or arginine alpha-ketoglutarate: a comparative study of their effects on glutamine pools in burn-injured rats. Critical Care Medicine, 1997. 25(2): 293-298.
30. Otto, C., Yovkova, V., and Barth, G., Overproduction and secretion of α-ketoglutaric acid by microorganisms. Applied Microbiology and Biotechnology, 2011. 92(4): 689.
31. Mehltretter, C., The chemical synthesis of D-glucuronic acid, in Advances in Carbohydrate Chemistry. 1953, Elsevier.231-249.
32. Mehltretter, C., Alexander, B., Mellies, R., and Rist, C., A Practical Synthesis of D-Glucuronic Acid through the Catalytic Oxidation of 1, 2-Isopropylidene-D-glucose2. Journal of the American Chemical Society, 1951. 73(6): 2424-2427.
33. Bock, K.W. and Köhle, C., UDP‐glucuronosyltransferase 1A6: structural, functional, and regulatory aspects. Methods in Enzymology, 2005. 400: 57-75.
34. Blanc, P.J., Characterization of the tea fungus metabolites. Biotechnology Letters, 1996. 18(2): 139-142.
35. Houpt, J.B., Effect of glucosamine hydrochloride in the treatment of pain of osteoarthritis of the knee. Journal of Rheumatology, 1999. 26: 2423-2430.
36. Godbey, W., Wu, K.K., and Mikos, A.G., Poly (ethylenimine) and its role in gene delivery. Journal of Controlled Release, 1999. 60(2-3): 149-160.
37. Yue, Y. and Wu, C., Progress and perspectives in developing polymeric vectors for in vitro gene delivery. Biomaterials Science, 2013. 1(2): 152-170.
38. Dube, B., Rose, L., Sawant, K., and Uludag, H., Cholic acid modified 2 kDa polyethylenimine as efficient transfection agent. Biotechnology Progress, 2013. 29(5): 1337-1341.
39. Moghimi, S.M., Symonds, P., Murray, J.C., Hunter, A.C., Debska, G., and Szewczyk, A., A two-stage poly (ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy. Molecular Therapy, 2005. 11(6): 990-995.
40. Gosselin, M.A., Guo, W., and Lee, R.J., Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconjugate Chemistry, 2001. 12(6): 989-994.
41. Vancha, A.R., Govindaraju, S., Parsa, K.V., Jasti, M., González-García, M., and Ballestero, R.P., Use of polyethyleneimine polymer in cell culture as attachment factor and lipofection enhancer. BMC Biotechnology, 2004. 4(1): 23.
42. Schmaljohann, D., Thermo-and pH-responsive polymers in drug delivery. Advanced Drug Delivery Reviews, 2006. 58(15): 1655-1670.
43. Reineke, T.M., Stimuli-responsive polymers for biological detection and delivery. 2015, ACS Publications.
44. Hoogenboom, R., Temperature-responsive polymers: properties, synthesis and applications. 2014: 15-44.
45. Schultz, A.R. and Flory, P.J., Phase equilibria in polymer-solvent systems. Journal of the American Chemical Society 1952. 74: 4760-4767.
46. Cowie, J.M. and McEwen, I.J., Influence of microstructure on the upper and lower critical solution temperatures of poly (methylmethacrylate) solutions. Journal of the Chemical Society, Faraday Transactions 1: Physical Chemistry in Condensed Phases, 1976. 72: 526-533.
47. Van Assche, G., Van Mele, B., Li, T., and Nies, E., Adjacent UCST phase behavior in aqueous solutions of Poly (vinyl methyl ether): Detection of a narrow low temperature UCST in the lower concentration range. Macromolecules, 2011. 44(4): 993-998.
48. Okhapkin, I.M., Makhaeva, E.E., and Khokhlov, A.R., Water solutions of amphiphilic polymers: nanostructure formation and possibilities for catalysis, in Conformation-Dependent Design of Sequences in Copolymers I. 2006, Springer.177-210.
49. Zhang, G. and Wu, C., Folding and formation of mesoglobules in dilute copolymer solutions, in Conformation-dependent Design of Sequences in copolymers I. 2006, Springer.101-176.
50. Wu, C. and Wang, X., Globule-to-coil transition of a single homopolymer chain in solution. Physical Review Letters, 1998. 80(18): 4092.
51. Kotsuchibashi, Y., Ebara, M., Aoyagi, T., and Narain, R., Recent advances in dual temperature responsive block copolymers and their potential as biomedical applications. Polymers, 2016. 8(11): 380.
52. Käfer, F., Liu, F., Stahlschmidt, U., Jérôme, V.r., Freitag, R., Karg, M., and Agarwal, S., LCST and UCST in One: Double Thermoresponsive Behavior of Block Copolymers of Poly (ethylene glycol) and Poly (acrylamide-co-acrylonitrile). Langmuir, 2015. 31(32): 8940-8946.
53. Roth, P.J., Davis, T.P., and Lowe, A.B., UCST-driven self-assembly and crosslinking of diblock copolymer micelles. Polymer Chemistry, 2012. 3(8): 2228-2235.
54. Kim, B., Hong, D., and Chang, W.V., LCST and UCST double-phase transitions of poly (N-isopropylacrylamide-co-2-acrylamidoglycolic acid)/poly (dimethylaminoethyl methacrylate) complex. Colloid and Polymer Science, 2015. 293(3): 699-708.
55. Wingard, L.L., Miller, S.R., Sellker, J.M., Stenn, E., Allen, M.M., and Wood, A.M., Cyanophycin production in a phycoerythrin-containing marine Synechococcus strain of unusual phylogenetic affinity. Applied and Environmental Microbiology, 2002. 68(4): 1772-1777.
56. Dobrynin, A.V., Colby, R.H., and Rubinstein, M., Polyampholytes. Journal of Polymer Science Part B: Polymer Physics, 2004. 42(19): 3513-3538.
57. Huggins, M.L., Solutions of long chain compounds. The Journal of Chemical Physics, 1941. 9(5): 440-440.
58. Flory, P.J., Thermodynamics of high polymer solutions. The Journal of Chemical Physics, 1942. 10(1): 51-61.
59. Seuring, J. and Agarwal, S., Polymers with upper critical solution temperature in aqueous solution. Macromolecular Rapid Communications, 2012. 33(22): 1898-1920.
60. Quiroz, F.G. and Chilkoti, A., Sequence heuristics to encode phase behaviour in intrinsically disordered protein polymers. Nature Materials, 2015. 14(11): 1164.
61. Klouda, L. and Mikos, A.G., Thermoresponsive hydrogels in biomedical applications. European Journal of Pharmaceutics and Biopharmaceutics, 2008. 68(1): 34-45.
62. Gil, E. and Hudson, S., Stimuli-reponsive polymers and their bioconjugates. Progress in Polymer Science, 2004. 29(12): 1173-1222.
63. Kocak, G., Tuncer, C., and Bütün, V., pH-Responsive polymers. Polymer Chemistry, 2017. 8(1): 144-176.
64. Chaturvedi, K., Ganguly, K., Nadagouda, M.N., and Aminabhavi, T.M., Polymeric hydrogels for oral insulin delivery. Journal of Controlled Release, 2013. 165(2): 129-138.
65. Khlystov, N.A., Chan, W.Y., Kunjapur, A.M., Shi, W., Prather, K.L., and Olsen, B.D., Material properties of the cyanobacterial reserve polymer multi-l-arginyl-poly-l-aspartate (cyanophycin). Polymer, 2017. 109: 238-245.
66. Seuring, J., S. Agarwal, Polymers with upper critical solution temperature in aqueous solution. Macromol Rapid Commun, 2012. 33(22): 1898-1920.
67. Khan, I., Brimblecombe, P., and Clegg, S., Solubilities of pyruvic acid and the lower (C 1-C 6) carboxylic acids. Experimental determination of equilibrium vapour pressures above pure aqueous and salt solutions. Journal of Atmospheric Chemistry, 1995. 22(3): 285-302.
68. Chen, H., Laurent, S., Bédu, S., Ziarelli, F., Chen, H.-l., Cheng, Y., Zhang, C.-C., and Peng, L., Studying the signaling role of 2-oxoglutaric acid using analogs that mimic the ketone and ketal forms of 2-oxoglutaric acid. Chemistry & Biology, 2006. 13(8): 849-856.
69. Lee, J., Saha, B., and Vlachos, D.G., Pt catalysts for efficient aerobic oxidation of glucose to glucaric acid in water. Green Chemistry, 2016. 18(13): 3815-3822.
70. Bichsel, Y. and Von Gunten, U., Formation of iodo-trihalomethanes during disinfection and oxidation of iodide-containing waters. Environmental Science & Technology, 2000. 34(13): 2784-2791.
71. Gibney, K.A., Sovadinova, I., Lopez, A.I., Urban, M., Ridgway, Z., Caputo, G.A., and Kuroda, K., Poly (ethylene imine) s as antimicrobial agents with selective activity. Macromolecular Bioscience, 2012. 12(9): 1279-1289.
72. Miller-Chou, B.A. and Koenig, J.L., A review of polymer dissolution. Progress in Polymer Science, 2003. 28(8): 1223-1270.
73. Garidel, P., Hildebrand, A., Neubert, R., and Blume, A., Thermodynamic characterization of bile salt aggregation as a function of temperature and ionic strength using isothermal titration calorimetry. Langmuir, 2000. 16(12): 5267-5275.
74. Maiti, K., Mitra, D., Guha, S., and Moulik, S.P., Salt effect on self-aggregation of sodium dodecylsulfate (SDS) and tetradecyltrimethylammonium bromide (TTAB): physicochemical correlation and assessment in the light of Hofmeister (lyotropic) effect. Journal of Molecular Liquids, 2009. 146(1-2): 44-51.
75. Rekharsky, M., Inoue, Y., Tobey, S., Metzger, A., and Anslyn, E., Ion-pairing molecular recognition in water: Aggregation at low concentrations that is entropy-driven. Journal of the American Chemical Society, 2002. 124(50): 14959-14967.
76. Hung, Y.H., Characterization of (insoluble cyanophycin)- (polyethylene glycol methyl ether) conjugates and its application in drug encapsulation. 2017: 82.
77. Mayama, H., Iwataki, T., and Yoshikawa, K., Thermodynamics in the folding phase-transition of single T4DNA molecules in poly (ethylene glycol) solution. Chemical Physics Letters, 2000. 318(1-3): 113-117.
78. Liu, X., Yaszemski, M.J., and Lu, L., Expansile crosslinked polymersomes for pH sensitive delivery of doxorubicin. Biomaterials Science, 2016. 4(2): 245-249.