臺灣博碩士論文加值系統

聚乳酸(polylactic acid, PLA) 為一種生物可降解材料，微生物分解PLA之能力可能與其蛋白酶和酯解酶活性有關。以含0.5% PLA 之海水及土壤培養基(PLAsBM 及PLAmBM)，分別自海水及土壤樣品篩選出19 株潛在具有降解PLA 能力之菌株。在基礎培養基中添加1% 酵母抽出物，可進一步篩選出蛋白酶及酯解酶活性較高的5 株分離株，其中蛋白酶與酯解酶活性分別提高至12.33 ± 0.97 ~ 47.56 ± 0.01 U/L 與 7.38 ± 0.19 ~ 36.56 ± 1.09 U/L，由此5 株分離株所得培養液降解PLA 薄膜，得水解力最強之分離株為PM25-6 (23.75%)。起始pH 值6.0-8.0 對菌株生長無顯著影響，但可顯著影響蛋白酶活性，其最適pH 值為7.0，溫度顯著影響菌株生長與酵素活性，30oC 所得蛋白酶與酯解酶活性明顯大於25oC 與37oC 所得。PM25-6 在添加1% 酵母抽出物之海水培養基於30oC 培養三天，所得蛋白酶與酯解酶活性分別為126.06 ± 3.07 U/L 與 212.70 ± 0.16 U/L，其濾液於30oC 作用PLA 薄膜五天所得水解率為36.01%。溫度及pH 值顯著影響利用粗酵素液水解PLA 活性，最適水解PLA 的溫度及pH 值分別為60oC 及pH 9.0，且於60oC 下水解率最高為85.25%。並利用RSM 所得最適化條件生產PLA 降解酵素，經五天水解PLA 薄膜後，水解率達99.13 %。
關鍵字：聚乳酸，生物降解，蛋白酶，酯解酶

壹、前言 1
貳、文獻整理 3
一、分解性塑膠 3
1. 光分解性塑膠 (Photodegradable plastics) 3
2. 熱崩解性塑膠 (Thermal degradable plastics) 3
3. 生物可分解性塑膠 (Biodegradable Plastics) 3
二、生物可分解塑料 4
1. 生物可分解塑料定義及分類 4
1.1 化學合成 4
1.2 生化合成 4
1.3 天然聚合物 5
2. 聚乳酸 5
2.1 原料來源 (乳酸) 5
2.2 聚乳酸製程方式 6
2.3 聚乳酸應用 7
三、生物可分解塑料降解原理 7
1. PLA 於環境降解情形 7
1.1實際堆肥觀察 7
1.2 掩埋過程中分子量變化 8
1.3 PLA 模擬堆肥測試 8
2. PLA 之生物性降解 10
2.1 可降解 PLA 菌株及其分泌酵素 10
2.2 不同添加物對酵素生產探討 10
四、PLA之廢棄物處理 11
1. 傳統塑料廢棄物處理 11
2. PLA 廢棄物處理 11
2.1 直接掩埋處理 12
2.2 以微生物或酵素水解處理廢棄物 12
五、反應曲面法 (Response Surface Methodology , RSM) 12
1. 反應曲面法原理及設計 13
1.1 品質因子階層 13
1.2 實驗設計 13
1.3 極值點區域逼近及決定 14
1.4 反應曲面模式建立及各因子影響效應分析 14
1.5 數學模式適切性檢驗 14
參、實驗設計 16
肆、實驗材料與方法 17
一、實驗材料 17
1. 實驗菌株 17
2. 材料 17
3. 化學藥品 17
4. 菌株培養培養基 17
5. 油脂 18
二、儀器設備 19
三、實驗方法 20
1. 菌株篩選 20
1.1 海水菌株篩選 20
1.2 土壤菌株篩選 20
1.3 篩選菌株純化、保存及活化 20
2. 分離菌株初步生長測試 21
3. 分離菌株生產酵素能力測試 21
4. 影響酵素生產因子探討 22
4.1 外加物質之影響探討 22
4.2 培養基起始 pH 值之影響探討 22
4.3 培養基溫度之影響探討 22
4.4 轉速之影響探討 22
5. 以 RSM 法探討最適酵素生產條件 23
6. 酵素水解 PLA 薄膜條件探討 23
6.1 PLA 薄膜製備 23
6.2 粗酵素液製備 23
6.3 溫度預處理 PLA 薄膜對水解率影響 24
6.4 不同 pH 值之粗酵素液對 PLA 薄膜水解率影響 24
6.5 不同溫度下粗酵素液之安定性及對 PLA 水解率影響 24
7. RSM 最適發酵條件確認及 PLA 水解率 24
8. 分析方法 25
8.1 微生物分析方法 25
8.2 Protease 活性測試 26
8.3 Esterase 活性測試 26
8.4 PLA 水解率測試 27
8.5統計分析方法 27
伍、結果與討論 28
一、菌株篩選 28
1. 海水及土壤菌株篩選 28
2. 分離菌株初步生長篩選 28
二、PLA薄膜 30
三、不同添加物影響酵素生產之探討 30
四、影響酵素生產之物理因子探討 31
1. 起始 pH 探討 31
2. 培養溫度探討 32
3. 震盪速率探討 33
五、RSM 實驗設計 34
六、PLA 酵素水解條件探討 36
1. 不同溫度預處理 PLA 薄膜對水解率之影響 36
2. 不同 pH 值之粗酵素液對 PLA 薄膜水解率影響 37
3. 不同溫度下粗酵素液之安定性及 PLA 水解率影響 37
七、RSM 最適發酵條件確認及PLA 水解率 39
陸、結論 42
柒、參考文獻 43
捌、圖表 52

行政院環境保護署。廢棄物處理。http://edw.epa.gov.tw/eng/reportStatisticEN.aspx?StatDataID=21. 2010。台北。台灣
李國源。2007。生物可分解性聚乳酸之特性、應用及分解。大同大學 生物工程研究所碩士論文。台北。台灣。
林晉毅。2006。聚乳酸微生物分解性之探討。屏東科技大學食品科學系碩士學位論文。屏東。台灣。
陳幸臣。2003。食品微生物學實驗法。華香園出版社。台北。台灣。
林姿君。2008。具血栓分解酶活性之海藻納豆菌發酵液之製備。國立
臺灣海洋大學食品科學系碩士學位論文。基隆。台灣。
陳映方。2005。鹿角塗面複方與四種中草藥天門冬、白朮、白芷、白蘞萃取物之抗老化與美白有效性評估。國立臺灣海洋大學食品科學系碩士學位論文。基隆。台灣。
陳映竹。2007。液態乳酸分子進行凝態電漿聚合的反應機制探討。台灣清華大學材料科學工程學系學位論文。新竹。台灣。
葉怡成。2009。高等實驗計畫法。五南圖書出版公司。台北。台灣。
張明堯。2001。液態培養生產靈芝菌絲體與靈芝多醣最適化之研究。台灣海洋大學食品科學研究所碩士論文。基隆。台灣。
黃培鈞。2004。選殖乳酸菌暨發酵乳清生產乳酸之研究。東海大學畜產學系碩士學位論文。台中。台灣。
蘇南維。Aspergillus sp. FC-10 （ATCC MYA-2469）鹼性蛋白酶之研究。國立臺灣大學農業化學研究所碩士論文。台北。台灣。
黎中正。1998。 實驗設與分析。高立圖書有限公司，台北市。
ASTM. D6400-04, 1999. Standard Specification for Compostable Plastics. West Conshohocken, PA, ASTM, pp 999-1001
ASTM. D6400-99e1, 1999. Standard specifications for compostable plastics1. West Conshohocken, PA, ASTM, 999- 1001
Auras, R., Harte, B. and Selke, S., 2004. Effect of Water on the Oxygen Barrier Properties of Poly(ethylene terephthalate) and Polylactide Films. Journal of Applied Polymer Science, 92: 1790-1803.
Box, G.E.P. and Wilson, K.B., 1951. On the experimental attainment optimum conditions. Journal of the Royal Statistical Society Series, 13: 1-45.
Chandra, R. and Rustgi, R., 1997. Biodegradation of maleated linear low-density polyethylene and starch blends. Polymer Degradation and Stability, 56: 185-202.
Chiarini, L., Mara, L. and Tabacchioni, S., 1992. Influence of growth supplements on lactic acid production in whey ultrafiltrate by Lactobacillus helveticus. Applied Microbiology and Biotechnology, 36: 461-464.
Doi, Y., 1990. Microbial Polyesters. New York: VCH Publishers.
Drumright, R.E., Gruber, P.R. and Henton, D.E., 2000. Polylactic Acid Technology, Advanced Materials, 12: 1841-1846.
Ertunga, N.S., Cakmak, U., Colak, A., Faiz, O. and Sesli, E., 2009. Characterisation of esterolytic activity from two wild mushroom species, Amanita aginata var. vaginata and Tricholoma terreum. Food Chemistry, 115: 1486–1490.
Fukuzaki. H., Yoshida. M., Asano. M, and Kumakura. M., 1989. Synthesis of copoly (D, L-lactic acid) with relative low molecular weight and in vitro degradation. European Polymer Journal, 25: 1019-26.
Gilmore, D.F., Fuller, R.C. and Lenz, R., 1990. Biodegradation of poly(beta-hydroxyalkanoates). In: Barenberg SA, Brash JL, Narayan R, Redpath AE, editors. Degradable Materials: Perspectives, Issues and Opportunities. Bocan Raton, FL: CRC Press.
Giovanni, M., 1983. Response surface methodolgy and product optimization. Food Technology, 37: 11-41.
Gross, R.A. and Kalra, B., 2002. Biodegradable polymers for the environment. Green Chemistry, 297: 803-807.
Gruber, P.R., Hall, E.S., Kolstad, J.H., Iwen, M.L., Benson, R.D. and Borchardt, R.L., 1992. Continuous process for manufacture of lactide polymers with controlled optical purity. United States of America Patent and Trademark Office, 5: 142-023.
Gu, J.D., Ford, T.E., Mitton, D.B. and Mitchell, R., 2000. Microbial corrosion of metals. In: Review, editor. The Uhlig corrosion handbook, 2nd edn. Wiley, New York, pp 915-927
Gu, J.D., Ford, T.E., Mitton, D.B. and Mitchell, R., 2006. Microbial degradation and deterioration of polymeric materials. In: Revie W, editor. The Uhlig Corrosion Handbook. 2nd Edition. Wiley, New York, pp 439-60.
Gugumus, F., In: Pospisil, J. and Klemchuk, P.P., editors., 1990. Photooxidation of polymers and its inhibition. Oxidation inhibition in organic materials CRC press, 29-162.
Hofvendahl, K. and Hahn-Hagerdal, B., 2000. Factors affecting the fermentative lactic acid production from renewable resources. Enzyme and Microbial Technology, 26: 87-107.
Ikura, Y. and Kudo, T., 1999. Isolation of a microorganism capable of degrading poly-(L-lactide). Journal of General and Applied Microbiology, 45: 247-251.
ISO. 14852 , 1999. Determination of the ultimate aerobic biodegradability of plastic materials in an aqueous medium — Method by analysis of evolved carbon dioxide. London, UK.
ISO/DIS 14855-1, 1999. Determination of the Ultimate Aerobic Biodegradability and Disintegration of Plastic Materials under Controlled Composting Conditions—Method by Analysis of Evolved Carbon dioxide.
Jarerat, A. and Tokiwa, Y., 2001. Degradation of poly(L-lactide) by fungus. Macromolecular Bioscience, 1:136-140.
Jarerat, A. and Tokiwa, Y., 2003a. Poly(L-lactide) degradation by Saccharotrix waywayandensis. Biotechnology Letters, 25:401-404.
Jarerat, A. and Tokiwa, Y., 2003b. Poly(L-lactide) degradation by Kibdelosporangium aridum. Biotechnology Letters, 25:2035-2038.
Jarerat, A., Tokiwa, Y. and Tanaka, H., 2006. Production of poly(L-lactide)-degrading enzyme by Amycolatopsis orientalis for biological recycling of poly(L-lactide). Applied Microbiology and Biotechnology, 72:726-731.
Kale, G., Auras, R. and Singh, S.P., 2006. Degradation of Commercial Biodegradable Packages under Real Composting and Ambient Exposure Conditions. Journal of Polymers and the Environment, 14: 317-334.
Kale, G., Kijchavengkul, T., Auras, R., Rubino, M., Selke, S.E. and Singh, S.P., 2007a. An overview of compostability of bioplastic packaging materials, Macromolecular Bioscience,7:255-277.
Kale, G., Auras, R., Singh, S.P. and Narayan, R., 2007b. Biodegradability of polylactide bottles in real and simulated composting conditions. Polymer Testing, 26:1049-1061.
Kammoun, R., Chouayekh, H., Abid, H., Naili, B. and Bejar, S., 2009. Purification of CBS 819.72 -amylase by aqueous two-phase systems: Modelling using Response Surface Methodology. Biochemical Engineering Journal, 46: 306-312.
Kay, M.J., McCabe, R.W. and Morton, L.H.G., 1993. Chemical and Physical Changes Occurring in Polyester Polyurethane during Biodegradation. International Biodeterioration & Biodegradation, 31: 209-225
Khuri, A.I. and Cornell, J.A., 1987. Response Surfaces: Design and Analysis. Marcel Decker Inc., New York.
Krebsfa¨nger, N., Zocher, F., Altenbuchner, J. and Bornscheuer, U. T., 1998. Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens. Enzyme and Microbial Technology, 22:641-646.
Kyriacos, A., Athanasiou, Gabriele, G., Niederauer, and Mauli Agrawal, C., 1996. Sterilization, toxicity, biocompatibility and clinical applications of polylactic acid/ polyglycolic acid copolymers. Biomoterids, 17: 93-102.
Li, S. M., Garreau, H. and Vert, M., 1990. Structure-property relationships in the case of the degradation of massive aliphatique poly(a-hydroxy acids) in aqueous media. Part 1. Poly (DL-lactic acid). Journal of Materials Science: Materials in Medicine, 1: 123-130.
Li, F., Wang, S. Liu, W. and Chen, G., 2008. Purification and characterization of poly(L-lactic acid)-degrading enzymes from Amycolatopsis orientalis ssp.orientalis. FEMS Microbiol Lett, 282:52-58.
Litchfield, J.H., 1996. Microbiological production of lactic acid. Advances in Applied Microbiology, 42: 45-95.
Lu, L., Peter, S.J., Lyman, M.D., Lai, H.L., Leite, S.M., Tamada, J.A., Uyama, S., Vacanti, J.P., Langer, R. and Mikos, A.G., 2000. In vitro and in vivo degradation of porous poly(DL-lactic-co-glycolic acid) foams. Biomaterials, 21:1837-1845.
Lunt, J., 1998. Large-scale production properties and commercial application of polylactic acid polymer. Polymer Degradation and Stability, 59: 145-152.
Matsuda, E., Abe, N., Tamakawa, H., Kaneko, J. and Kamio, Y., 2005. Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1. The Journal of Bacteriology, 187: 7333-7340.
Masaki, K., Kamini, N.R., Ikeda, H. and Iefuji, H., 2005. Cutinase-like enzyme from the yeast Cryptococcus sp. strain S-2 hydrolyses polylactic acid and other biodegradable plastics. Applied and Environmental Microbiology, 7: 7548-7550.
McDonald, R.T., McCarthy, S. and Gross, R.A., 1996. Enzymatic degradability of poly(lactide): effects of chain stereochemistry and material crystallinity. Macromolecules, 29:7356-7361.
Moon, S.I., Urayama, H. and Kimura, Y., 2003. Structural characterization and degradability of poly(L-lactic acid)s incorporating phenylsubstituted alpha-hydroxy acids as comonomers. Macromolecular Bioscience, 3:301-309.
Mueller, R.J., 2003. Biodegradability of polymers: regulations and methods for testing. Steinbüchel A, editor. Biopolymers, 10: 366-388.
Nakamura, K., Tomita, T., Abe, N. and Kamio, Y., 2001. Purification and characterization of an extracellular poly(L-lactic acid) depolymerase from a soil isolate, Amycolatopsis sp. strain K104-1. Applied and Environmental Microbiology, 67:345-353.
Olayan H.B., Hamid H.S. and Owen E.D., 1996. Photochemical and thermal crosslinking of polymers. Journal of Macromolecular Science - Reviews in Macromolecular Chemistry & Physics , 36: 671-719.
Pauli, T. and Fitzpatrick, J.J., 2002. Malt combing nuts as a nutrient supplement to whey permeate for producing lactic by fermentation with Lactobacillus casei. Process Biochemistry, 38: 1-6.
Pospisil, J. and Nespurek, S., 1997. Highlights in chemistry and physics of polymer stabilization. Macromolecular Symposia, 115: 143-63.
Pranamuda, H., Tokiwa, Y. and Tanaka, H., 1997. Polylactide degradation by an Amycolatopsis sp.. Applied and Environmental Microbiology, 63:1637-1640.
Pranamuda, H., Tsuchii, A. and Tokiwa, Y., 2001. Poly(L-lactide)- degrading enzyme produced by Amycolatopsis sp. Macromolecular Bioscience, 1:25-29
Reeve, M.S., McCarthy, S.P., Downey, M.J. and Gross, R.A., 1994. Polylactide stereochemistry: effect on enzymatic degradability. Macromolecules, 27: 825-831.
Rhee, Y.H., Kim, Y.H. and Shin, K.S., 2006. Characterization of an extracellular poly(3-hydroxyoctanoate) depolymerase from the marine isolate, Pseudomonas luteola M13-4. Enzyme and Microbial Technology Technol, 38: 529-535.
Sakai, K., Kawano, H., Iwami, A., Nakamura, M. and Moriguchi, M., 2001. Isolation of a thermophilic poly-L-lactide degrading bacterium from compost and its enzymatic characterization. ournal of Bioscience and Bioengineering, 92:298-300.
Shah, A.A., Hasan, F., Hameed, A. and Ahmed, S., 2008. Biological degradation of plastics: a comprehensive review. Biotechnology Advances, 26: 246-65.
Shigeno, Y.A., Teeraphatpornchai, T., Teamtisong, K., Nomura, N., Uchiyama, H., Nakahara, T. and Kambe, T.N., 2003. Cloning and sequencing of a poly(DL-lactic acid) depolymerase gene from Paenibacillus amylolyticus strain TB-13 and its functional expression in Escherichia coli. Applied and Environmental Microbiology, 69:2498-2504
Silva-Santisteban, B.O.Y. and Maugeri, F., 2005. Agitation, aeration and shear stress as key factors in inulinase production by Kluyveromyces marxianus. Enzyme and Microbial Technology, 36: pp. 17-724.
Teeraphatpornchai, T., Nakajima-Kambe, T., Shigeno-Akutsu,Y., Nakayama, M., Nomura, N., Nakahara, T. and Uchiyama, H., 2003. Isolation and characterization of a bacterium that degrades various polyester-based biodegradable plastics. Biotechnology Letters, 25: 23-28.
Tokiwa, Y. and Calabia, B.P., 2006. Biodegradability and biodegradation of poly(lactide). Applied Microbiology and Biotechnology, 72:244-51.
Torres, A., Li, S.M., Roussos, S. and Vert, M. 1996. Screening of microorganisms for biodegradation of poly(lactic acid) and lactic acid-containing polymers. Applied and Environmental Microbiology, 62: 2393-2397.
Tokiwa, Y., Konno, M. and Nishida, H., 1999. Isolation of silk degrading microorganisms and its poly(L-lactide) degradability. Chemistry Letters, 28: 355-356.
Tomita, K., Kuroki, Y. and Nagai, K., 1999. Isolation of thermophiles degrading poly(L-lactic acid). Journal of Bioscience and Bioengineering, 87:752-755.
Tomita, K., Tsuji, H., Nakajima, T., Kikuchi, Y., Ikarashi, K. and Ikeda, N., 2003. Degradation of poly(D-lactic acid) by a thermophile. Polymer Degradation and Stability, 81:167-171.
Tomita, K., Nakajima, T., Kikuchi, Y. and Miwa, N., 2004. Degradation of poly(L-lactic acid) by a newly isolated thermophile. Polymer Degradation and Stability, 84:433-438.
Tsai, S.P., Coleman, R.D., Moon, S.H., Schneider, K.A. and Sanville Millard, C., 1993. Strain screening and development for industrial lactic acid fermentation. Applied Biochemistry and Biotechnology, 39: 323-335.
Urayama, H., Kanamori, T. and Kimura, Y., 2002. Properties and biodegradability of polymer blends of poly(l-lactide)s with different optical purity of the lactate units. Macromolecular Materials and Engineering, 287:116-121.
Vert, M., Li, S.M. and Garreau, H., 1991. More about the degradation of LA-GA derived matrices in aqueous media. Journal of Controlled Release, 16: 15-26.
Wang, Q., Hou , Y., Xu, Z., Miao, J. and Li, G., 2008. Optimization of cold-active protease production by the psychrophilic bacterium Colwellia sp. NJ341 with response surface methodology. Bioresource Technology, 99: 1926-1931.
Wee, Y.J., Kim J.N. and Ryu H.W., 2006. Biotechnological Production of Lactic Acid and Its Recent Applications. Food Technology and Biotechnology, 44: 163-172.
Williams, S.F. and Peoples, O.P., 1996. Biodegradable plastics from plants. Chemtech , 38: 38-44.
Williams, D.F., 1981. Enzymic hydrolysis of polylactic acid. Engineering in Medicine, 10: 5-7.