臺灣博碩士論文加值系統

表面表現蛋白質時，目標蛋白在誘導期間長時間暴露於培養環境中，在高轉速環境下，環境中的剪力可能造成蛋白質變性。本研究探討在不同培養條件下，培養轉速、培養的體積、容器的樣式等因素對於表面表現目標蛋白(surface-displayed Candida antarctica lipase A, sdCALA)之比活性影響，同時以動力學角度分析不同轉速對其動力學特性之影響，最後以最佳培養條件培養表現sdCALA，並分析其熱穩定性及有機溶劑耐受性。
實驗結果發現，在轉速為50 rpm，其比活性受氧氣濃度限制，比活性不高；在100 rpm以上，其比活性主要受剪力影響；在培養體積研究方面，Erlenmeyer型搖瓶培養體積對剪率的影響較小，體積主要影響質傳係數KLa，因此也影響比活性表現；Hinton型搖瓶與Erlenmeyer型搖瓶狀況類似，但以50 ml培養體積表現較低的比活性，經計算後發現應是受剪率影響。Hinton培養比Erlenmeyer培養有較佳的菌量生長，以100 ml培養體積於500 ml Hinton培養sdCALA有最佳比活性(0.243±0.020 U/mg)。在最適溫度及pH的研究方面，其最適溫度及pH為60˚C及pH 6，且培養轉速對sdCALA之最適溫度與pH無影響。在動力學分析方面，以最佳培養條件培養之sdCALA較以200 rpm培養之sdCALA以及胞內表現之CALA有更高之VMax及較低之Km，分別為0.070 μmole ml-1 min-1及0.087 μmole ml-1。在熱穩定性方面，sdCALA在70˚C下培養30分鐘後仍殘餘80 %以上活性，在80˚C以上活性迅速受溫度影響而下降；在有機溶劑耐受性方面，在四種親水性有機溶劑中，以總濃度30 %乙醇有最高之活性為0.67 U/ml；以異丙醇為溶劑，其結果與乙醇類似，在20%濃度下有最高之活性(0.51 U/ml)；以乙腈、丙酮為溶劑，不論何種濃度下其活性皆不高。

For a surface-displayed protein, the target protein is exposing directly to the culture environment during the induction period. The shaking speed of culture condition may cause the shear-denaturation. In this study the culture conditions (shaking speed, working volume, flask types) were tested to observe their influence on the surface- displayed protein (i.e., Candida antarctica lipase A, CALA).
The specific activities of sdCALA cultivated under 50 rpm were limited by the dissolved oxygen level regardless the flask types. When the shaking rate was above 100 rpm, the specific activities were mainly influenced by the shear stress. For Erlenmeyer flasks, the working volume significantly affects oxygen mass transfer rate (KLa), which in turn influences the specific activities of sdCALA. The results of Hinton flasks were similar to Erlenmeyer except the 50 ml sample, which showed a lower specific activity. This result was probably caused by the high shear rate based on our calculation. The optimal culture condition was found as follows: 500 ml Hinton flask containing 100 ml of medium with the shaking rate of 100 rpm, where the optimal sdCALA specific activity was 0.243±0.020 U/mg. For the temperature and pH effect, the optimal activity was observed at 60˚C and pH 6. The sdCALA obtained under various shaking rate gave the same temperature and pH profiles. The kinetic study revealed that the sdCALA obtained under optimal condition gave a higher VMax (0.070 μmole ml-1min-1) and a lower Km (0.087 μmole ml-1) than that of the sdCALA obtained under 200 rpm and the free CALA. For the thermal stability test, the sdCALA retains over 80 % of activity at 70˚C for 30 minutes and the activity was rapidly decreased when the temperature is over 80˚C. Four different hydrophilic organic solvents were used for the organic solvents tolerance test. The highest activity (0.67 U/ml) was obtained when the ethanol concentration was set at 30%. For isopropanol, the result was quite similar to that of ethanol. sdCALA performed best under 20% concentration (0.51 U/ml). When acetonitrile and acetone are present, sdCALA performed worse regardless of the concentrations.

中文摘要 i
Abstract ii
圖目錄 v
表目錄 vi
縮寫字對照表 vii
第一章、緒論 1
1.1 前言 1
1.2研究動機 2
第二章、文獻回顧 3
2.1 脂肪酶(Lipase) 3
2.1.1 簡介 3
2.1.2 Candida antarctica lipase A 5
2.2細胞表面表現系統(Cell surface display system) 7
2.3 冰核蛋白(Ice nucleation protein) 10
2.4 攪拌及溶氧等相關計算 11
第三章、實驗材料與方法 13
3.1實驗材料 13
3.1.1 實驗儀器設備 13
3.1.2實驗藥品 15
3.1.3 實驗用溶液 16
3.2實驗流程 19
3.3實驗方法 21
3.3.1 菌株與質體 21
3.3.2菌種保存與培養方法 25
3.3.3 破菌方法 26
3.3.4 CALA活性分析 (Activity assay) 26
3.3.5 溫度效應 28
3.3.6 pH效應 28
3.3.7 動力學研究(kinetic study) 29
3.3.8 溫度穩定性測試 29
3.3.9 有機溶劑耐受性測試 29
3.3.10 黏度測量 30
第四章、結果與討論 31
4.1 培養轉速對比活性之影響 31
4.2 培養體積對比活性之影響 35
4.3 CALA及sdCALA最適溫度探討 38
4.4 CALA及sdCALA最適pH探討 40
4.5 CALA及sdCALA動力學分析 42
4.6 sdCALA熱穩定性分析 46
4.7 sdCALA耐有機溶劑分析 47
第五章、結論與未來展望 49
5.1 結論 49
5.2 未來展望 50
參考文獻 51

[1] Dewan, S.S. Global Markets for Enzymes in Industrial Applications. 2017; Available from: https://www.bccresearch.com/marketresearch/biotechnology/enzymes-industrial-applications-report-bio030j.html.
[2] Hasan, F., Shah, A.A. and Hameed, A., "Industrial applications of microbial lipases", Enzyme and Microbial Technology, (2006) 39, 235-251.
[3] Pfeffer, J., Richter, S., Nieveler, J., Hansen, C.E., Rhlid, R.B., Schmid, R.D. and Rusnak, M., "High yield expression of lipase A from Candida antarctica in the methylotrophic yeast Pichia pastoris and its purification and characterisation", Applied Microbiology and Biotechnology, (2006) 72, 931-938.
[4] Joseph, B., Ramteke, P.W. and Thomas, G., "Cold active microbial lipases: Some hot issues and recent developments", Biotechnology Advances, (2008) 26, 457-470.
[5] Magadum, D.B. and Yadav, G.D., "Enantioselective resolution of (R,S)-alpha-methyl-4-pyridinemethanol using immobilized biocatalyst: Optimization and kinetic modeling", Biochemical Engineering Journal, (2017) 122, 152-158.
[6] Gilani, S.L., Najafpour, G.D., Heydarzadeh, H.D. and Moghadamnia, A., "Enantioselective synthesis of (S)-naproxen using immobilized lipase on chitosan beads", Chirality, (2017) 29, 304-314.
[7] Ali, C.H., Qureshi, A.S., Mbadinga, S.M., Liu, J.F., Yang, S.Z. and Mu, B.Z., "Biodiesel production from waste cooking oil using onsite produced purified lipase from Pseudomonas aeruginosa FW_SH-1: Central composite design approach", Renewable Energy, (2017) 109, 93-100.
[8] Liu, X.Y., Yu, X.J., Lv, J.S., Xu, J.X., Xia, J., Wu, Z., Zhang, T. and Deng, Y.F., "A cost-effective process for the coproduction of erythritol and lipase with Yarrowia lipolytica M53 from waste cooking oil", Food and Bioproducts Processing, (2017) 103, 86-94.
[9] Veteikyte, A., Siekstele, R., Tvaska, B. and Matijosyte, I., "Sequential application of waste whey as a medium component for Kluyveromyces lactis cultivation and a co-feeder for lipase immobilization by CLEA method", Applied Microbiology and Biotechnology, (2017) 101, 3617-3626.
[10] Chen, G.Y., Liu, J., Yao, J.G., Qi, Y. and Yan, B.B., "Biodiesel production from waste cooking oil in a. magnetically fluidized bed reactor using whole-cell biocatalysts", Energy Conversion and Management, (2017) 138, 556-564.
[11] Akanbi, T.O. and Barrow, C.J., "Candida antarctica lipase A effectively concentrates DHA from fish and thraustochytrid oils", Food Chem, (2017) 229, 509-516.
[12] Panpipat, W., Xu, X. and Guo, Z., "Improved acylation of phytosterols catalyzed by Candida antarctica lipase A with superior catalytic activity", Biochemical Engineering Journal, (2013) 70, 55-62.
[13] Nyyssölä, A., Miettinen, H., Kontkanen, H., Lille, M., Partanen, R., Rokka, S., Järvenpää, E., Lantto, R. and Kruus, K., "Treatment of milk fat with sn-2 specific Pseudozyma antarctica lipase A for targeted hydrolysis of saturated medium and long-chain fatty acids", International Dairy Journal, (2015) 41, 16-22.
[14] Zengin, G., Uysal, A., Aktumsek, A., Mocan, A., Mollica, A., Locatelli, M., Custodio, L., Neng, N.R., Nogueira, J.M.F., Aumeeruddy-Elalfi, Z. and Mahomoodally, M.F., "Euphorbia denticulata Lam.: A promising source of phyto-pharmaceuticals for the development of novel functional formulations", Biomedicine & Pharmacotherapy, (2017) 87, 27-36.
[15] Ahmad, J., Singhal, M., Amin, S., Rizwanullah, M., Akhter, S., Kamal, M.A., Haider, N., Midoux, P. and Pichon, C., "Bile Salt Stabilized Vesicles (Bilosomes): A Novel Nano-Pharmaceutical Design for Oral Delivery of Proteins and Peptides", Current Pharmaceutical Design, (2017) 23, 1575-1588.
[16] Rezvani, M., Najafpour, G.D., Mohammadi, M. and Zare, H., "Amperometric biosensor for detection of triglyceride tributyrin based on zero point charge of activated carbon", Turkish Journal of Biology, (2017) 41, 268-277.
[17] Narwal, V. and Pundir, C.S., "An improved amperometric triglyceride biosensor based on co-immobilization of nanoparticles of lipase, glycerol kinase and glycerol 3-phosphate oxidase onto pencil graphite electrode", Enzyme and Microbial Technology, (2017) 100, 11-16.
[18] Kirk, O. and Christensen, M.W., "Lipases from Candida antarctica: Unique biocatalysts from a unique origin", Organic Process Research & Development, (2002) 6, 446-451.
[19] Widmann, M., Juhl, P.B. and Pleiss, J., "Structural classification by the Lipase Engineering Database: a case study of Candida antarctica lipase A", Bmc Genomics, (2010) 11.
[20] Wikmark, Y., Svedendahl Humble, M. and Backvall, J.E., "Combinatorial library based engineering of Candida antarctica lipase A for enantioselective transacylation of sec-alcohols in organic solvent", Angewandte Chemie International Edition in English, (2015) 54, 4284-4288.
[21] Jan, A.H., Subileau, M., Deyrieux, C., Perrier, V. and Dubreucq, E., "Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design", Biochimica Et Biophysica Acta-Proteins and Proteomics, (2016) 1864, 187-194.
[22] Smith, G.P., "Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface", Science, (1985) 228, 1315-1317.
[23] Lee, S.Y., Choi, J.H. and Xu, Z.H., "Microbial cell-surface display", Trends in Biotechnology, (2003) 21, 45-52.
[24] Hirano, S.S. and Upper, C.D., "Bacteria in the leaf ecosystem with emphasis on Pseudomonas syringae - a pathogen, ice nucleus, and epiphyte", Microbiology and Molecular Biology Reviews, (2000) 64, 624-+.
[25] Li, Q., Yan, Q., Chen, J., He, Y., Wang, J., Zhang, H., Yu, Z. and Li, L., "Molecular characterization of an ice nucleation protein variant (inaQ) from Pseudomonas syringae and the analysis of its transmembrane transport activity in Escherichia coli", Int J Biol Sci, (2012) 8, 1097-1108.
[26] Jung, H.C., Kwon, S.J. and Pan, J.G., "Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis", Bmc Biotechnology, (2006) 6.
[27] Jung, H.C., Ko, S., Ju, S.J., Kim, E.J., Kim, M.K. and Pan, J.G., "Bacterial cell surface display of lipase and its randomly mutated library facilitates high-throughput screening of mutants showing higher specific activities", Journal of Molecular Catalysis B-Enzymatic, (2003) 26, 177-184.
[28] Gomez Sanchez, C.E., Martinez-Trujillo, A. and Aguilar Osorio, G., "Oxygen transfer coefficient and the kinetic parameters of exo-polygalacturonase production by Aspergillus flavipes FP-500 in shake flasks and bioreactor", Lett Appl Microbiol, (2012) 55, 444-452.
[29] Liu, Y.C., Chang, H. and I, Y.P., "A neural network model for estimating O-2 absorption coefficient in shake-flasks", Biotechnology Letters, (2000) 22, 1885-1888.
[30] Camacho, F.G., Rodriguez, J.J.G., Miron, A.S., Garcia, M.C.C., Belarbi, E.H. and Grima, E.M., "Determination of shear stress thresholds in toxic dinoflagellates cultured in shaken flasks - Implications in bioprocess engineering", Process Biochemistry, (2007) 42, 1506-1515.
[31] Li, C., Xia, J.-Y., Chu, J., Wang, Y.-H., Zhuang, Y.-P. and Zhang, S.-L., "CFD analysis of the turbulent flow in baffled shake flasks", Biochemical Engineering Journal, (2013) 70, 140-150.
[32] Schleif, R., "AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action", Fems Microbiology Reviews, (2010) 34, 779-796.
[33] Chiou, S.H. and Wu, W.T., "Immobilization of Candida rugosa lipase on chitosan with activation of the hydroxyl groups", Biomaterials, (2004) 25, 197-204.
[34] M.M. Shamel, K.B.R.a.M.H., "Operational Stability of Lipase Enzyme:Effect of Temperature and Shear", Developments in Chemical Engineering and Mineral Processing, (2005) 13 599-604.
[35] Tari, C., Parulekar, S.J., Stark, B.C. and Webster, D.A., "Synthesis and excretion of alpha-amylase in vgb(+) and vgb(-) recombinant Escherichia coli: A comparative study", Biotechnology and Bioengineering, (1998) 59, 673-678.
[36] Dimitrijevic, A., Velickovic, D., Bihelovic, F., Bezbradica, D., Jankov, R. and Milosavic, N., "One-step, inexpensive high yield strategy for Candida antarctica lipase A isolation using hydroxyapatite", Bioresour Technol, (2012) 107, 358-362.
[37] Zamost, B.L., Nielsen, H.K. and Starnes, R.L., "THERMOSTABLE ENZYMES FOR INDUSTRIAL APPLICATIONS", Journal of Industrial Microbiology, (1991) 8, 71-81.
[38] Yang, C., Wang, F., Lan, D., Whiteley, C., Yang, B. and Wang, Y., "Effects of organic solvents on activity and conformation of recombinant Candida antarctica lipase A produced by Pichia pastoris", Process Biochemistry, (2012) 47, 533-537.
[39] Juan L. Ramos, E.D., Jose´-Juan Rodrı´guez-Herva, P.G., Ali Haıぴdour, F.R. and Ferna´ndez-Barrero, a.A., "Mechanisms for Solvent Tolerance in Bacteria", THE JOURNAL OF BIOLOGICAL CHEMISTRY, (1997) 272, 3887–3890.
[40] Ana Segura, E.D., Gilberto Mosqueda, and Junker, J.L.R.a.F., "Multiple responses of Gram-negative bacteria to organic solvents", Environmental Microbiology, (1999) 1, 191–198.
[41] Jung, H.C., Kwon, S.J. and Pan, J.G., "Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis", BMC Biotechnol, (2006) 6, 23.
[42] Leon, R., Fernandes, P., Pinheiro, H.M. and Cabral, J.M.S., "Whole-cell biocatalysis in organic media", Enzyme and Microbial Technology, (1998) 23, 483-500.