Acinetobacter radioresistens可生產胞外之鹼性脂肪酵素，其活性在pH 10.0有最佳值。此酵素在清潔劑工業的應用上具有極高的發展潛力；考慮其量產所需的經濟程序是有其必要性的。本研究利用Tween 80當作碳源生產A. radioresistens脂肪酵素。與橄欖油相比，Tween 80的水解速率很慢，它在培養過程中以控制釋放的模式，提供油酸作為細胞生長及脂肪酵素生產之用，於是，有效地避免了油酸抑制的問題。在以0.3 % (v/v)的Tween 80為碳源的批式培養中，可於醱酵第6小時，得到25 U/mL的脂肪酵素最佳產量。Tween 80添加濃度最佳值的存在，乃細胞生長與酵素合成受抑制，兩者之間妥協的結果。高脂肪酵素產率可得自pH-stat控制的饋料批式醱酵；在醱酵第16小時，可得120 U/mL的酵素產量。不同的醱酵策略也在本研究中被比較。
本研究建立了生產Acinetobacter radioresistens脂肪酵素的饋料批式醱酵程序。以3 mL/L的Tween 80為碳源，利用一系列不控制pH值的批式醱酵，添加不同濃度的酵母萃取物（Yeast extract）和（或）消化蛋白（Tryptone）為氮源，以尋找一個均衡的培養基配方及pH設定值。以3 mL/L的Tween 80為碳源，7.5 g/L的酵母萃取物為氮源時，脂肪酵素產量為24 U/mL，此為批式醱酵生產A. radioresistens脂肪酵素的最佳培養基。醱酵液最終pH值（細胞進入生長停滯期時之pH值）為7.1，適於作為pH-stat進料之pH設定值。將此一培養基濃縮10倍，作為饋料批式醱酵的進料培養基，進行pH-stat饋料批次醱酵，此操作可使細胞及酵素的產量係數保持不變。在進料600 mL的培養基之後，可得138 U/mL的脂肪酵素產量。從兩種控制方式的饋料批式醱酵（DO-stat與pH-stat）結果發現，細胞比生長速率（μ）是影響脂肪酵素合成效率的真正因素。脂肪酵素比產率在μ為0.1 h�{1時有最大值；而酵素比生產速率的最佳值則落在μ= 0.2 h�{1附近。利用生長關連型模式模擬脂肪酵素的生產，可成功地預測饋料批式醱酵之結果。
另外，吾人可利用以Tween 80為碳源的均衡培養基進行重覆饋料批式醱酵生產A. radioresistens脂肪酵素；DO-stat與pH-stat的饋料策略在此被探討。維持固定的細胞濃度有助於重覆饋料次數的增加；而適當的細胞生長速率則是得到高酵素產量的關鍵。pH-stat進料類似指數進料，過高的細胞生長速率不利於脂肪酵素之生產；另一方面，DO-stat進料則類似於恆定流速的進料模式，以DO-stat為控制方式的重覆饋料批式醱酵提供了適當的細胞生長速率。在2.5-L的醱酵槽操作中，脂肪酵素生產速率可達42,000 U h�{1。

Acinetobacter radioresistens produces an extracellular alkaline lipase, which has an optimal pH of 10.0. This enzyme has a potential application in the detergent industry, and the development of an economical process for mass production is necessary. Lipase of Acinetobacter radioresistens was produced using Tween 80 as the carbon source. Compared with olive oil, the hydrolysis rate of Tween 80 in the fermentation broth was much slower. Taking advantage of the slow hydrolysis rate, Tween 80 could provide oleic acid for cell growth and lipase production through a mode of controlled release, and the known repression of lipase synthesis by oleic acid could be avoided. The optimal lipase yield obtained with batch culture in a 2.5-L tank fermentor was 25 U/mL with a fermentation time of 6 h, when using 0.3 % (v/v) Tween 80. The existence of an optimal yield with respect to concentration of Tween 80 was a result of the compromise between cell growth and the repression of lipase synthesis. To further the lipase productivity, a fed-batch culture with the pH-stat feeding method was employed, and the lipase productivity achieved was 120 U/mL in 16 h. Various strategies for production of A. radioresistens lipase were compared.
A fed-batch process for production of Acinetobacter radioresistens lipase was developed. Using Tween 80 as the carbon source, a series of pH-uncontrolled batch cultures with supplementation of yeast extract and/or tryptone were carried out to search for the balanced medium as well as the set point of pH to be used in the fed-batch phase. It was found that a medium containing 3 mL/L Tween 80 and 7.5 g/L yeast extract was optimal for the lipase production in batch culture (having a lipase yield of 24 U/mL), and the final culture pH, 7.1 (the pH at the beginning of the stationary phase), was a suitable set point for the pH-stat feeding. Using a medium of 10-fold concentration in the fed-batch phase, the culture was able to proceed with consistent yield coefficients of cell growth and lipase production, and a lipase yield of 138 U/mL was achieved after 600 mL of the medium was fed. Data obtained from fed-batch cultures with DO- and pH-stat feedings showed that specific growth rate μ was the intrinsic factor that determined the efficiency of lipase synthesis. The specific lipase productivity was maximal at μ= 0.1 h�{1, while the optimal lipase production rate occurred around μ= 0.2 h�{1. Based on a growth-associated pattern for lipase formation, the production of lipase with fed-batch culture could be simulated satisfactorily.
In addition, Acinetobacter radioresistens lipase was produced with repeated fed-batch culture using a balanced medium based on Tween 80 as the carbon source. The feeding strategies examined were pH-stat and DO-stat methods. A constant cell concentration was shown to be a prerequisite to extend the number of repeated cycles, and an adequate cell growth rate was critical for a high lipase yield. With the pH-stat feeding, which was found to approximate an exponential feeding, the cell growth rate was too high to effect the lipase production. On the other hand, the DO-stat feeding, which approximated a constant-rate feeding, could provide an adequate cell growth rate for efficient lipase production. The lipase production rate obtained in a 2.5-L fermentor could reach 42,000 U h�{1.

Antonian, E., “Recent advances in the purification, characterization and structure determination of lipases.” Lipids, 23, 1101–1106 (1988).
Breuil, C., D. B. Shindler, J. S. Sijher, and D. J. Kushner, “Stimulation of lipase production during bacterial growth on alkanes,” J. Bacteriol., 133, 601–606 (1978).
Brzozowski, A. M., U. Derewenda, Z. S. Derewenda, G. G. Godson, D. M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S. A Patkar, and L. Thim, “A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex,” Nature, 351, 491–494 (1991).
Chen, J. Y., C. M. Wen, and T. L. Chen, “Effect of oxygen transfer on lipase production by Acinetobacter radioresistens,” Biotechnol. Bioeng., 62, 311–316 (1999a).
Chen, S. J., C. Y. Cheng, and T. L. Chen, “A strategy for enhancing lipase production by Acinetobacter radioresistens from n-hexadecane,” J. Chin. Inst. Chem. Engrs., 30, 283–288 (1999b).
Chen, S. J., C. Y. Cheng, and T. L. Chen, “Production of an alkaline lipase by A. radioresistens,” J. Ferment. Bioeng., 86, 308–312 (1998).
Cheng, L. C., J. Y. Wu, and T. L. Chen, “A pseudo-exponential feeding method for control of specific growth rate in fed-batch cultures,” Biochem. Eng. J., 10, 227–232 (2002).
Cheng, L. C., L. I. Hor, J. Y. Wu, and T. L. Chen, “Effect of specific growth rate on the production of a recombinant nuclease by Escherichia coli,” Biochem. Eng. J., 14, 101–107 (2003).
Dalmau, E., J. L. Montesinos, M. Lotti, and C. Casas, “Effect of different carbon sources on lipase production by Candida rugosa,” Enz. Microb. Technol., 26, 657–663 (2000).
Dherbomez, M., J. L. Lacrampe, and J. Larouquere, “Contribution a l’etude de la lipase de Candida lipolytica,” Rev. Fr. Corps Gras, 22, 147 (1975).
Fakhreddine, L., A. Kademi, N. Aït-Abdelkader, and J. C. Baatti, “Microbial growth and lipolytic activities of moderate thermophilic bacterial strains,” Biotechnol. Lett., 20, 879–883 (1998).
Frost, G. M., and D. A. Moss, “Production of enzymes by fermentation,” in Biotechnology, vol. 7a, ed. J.F. Kennedy, VCH Publishers, New York, 113–121 (1987).
Fukumoto, J., Y. Tsujisaka, and M. Iwai, “Preparation of a stable lipase composition and purification thereof,” U. S. Patent 3262863 (1966).
Gaspar, M. L., M. Cunningham, R. Pollero, and M. Cabello, “Occurrence and properties of an extracellular lipase in Mortierella vinacea,” Mycologia, 91, 108–113 (1999).
Gordillo, M. A., A. Sanz, A. Sánchez, F. Valero, J. L. Montesinos, J. Lafuente, and C. Solà, “Enhancement of Candida rugosa lipase production by using different control fed-batch operational strategies,” Biotechnol. Bioeng., 60, 156–168 (1998).
Hellmuth, K., D. J. Korz, E. A. Sanders, and W. D. Deckwer, “Effect of growth rate on stability and gene expression of recombinant plasmids during continuous and high cell density cultivation of Escherichia coli TG1,” J. Biotechnol., 32, 289–298 (1994).
Hsu, C. H., and S. W. Tsai, “Improvements of Acinetobacter radioresistens lipase adsorption on Celite 535 by adding salts,” Tamkang J. of Sci. and Eng., 4, 133–139 (2001).
Iwai, M., Y. Tsujisaka, Y. Okamoto, and J. Fukumoto, “Lipid requirement for the lipase production by Geotrichum candidum link,” Agric. Biol. Chem., 37, 929 (1973).
Jaeger, K. E., and T. Eggert, “Lipases for biotechnology,” Curr. Opin. Biotechnol., 13, 390–397 (2002).
Jonsson, U., and B. G. Snygg, “Lipase production and activity as a function of incubation time, pH and temperature of four lipolytic micro organisms,” J. Appl. Bacteriol., 37, 571 (1974).
Khalilzadeh, R., S. A. Shojaosadati, N. Maghsoudi, J. Mohammadian-Mosaabadi, M. R. Mohammadi, A. Bahrami, N. Maleksabet, M. A. Nassiri-Khalilli, M. Ebrahimi, and H. Naderimanesh, “Process development for production of recombinant human interferon-� expressed in Escherichia coli,” J. Ind. Microbiol. Biotechnol., 31, 63–69 (2004).
Kim, S. S., E. K. Kim, and J. S. Rhee, “Effects of growth rate on the production of Pseudomonas fluorescens lipase during the fed-batch cultivation of Escherichia coli,” Biotechnol. Prog., 12, 718–722 (1996).
Kirk, O., T. V. Borchert, and C. C. Fuglsang, “Industrial enzyme applications,” Curr. Opin. Biotechnol., 13, 345–351 (2002).
Kok, R. G., C.B. Nudel, R. H. Gonzalez, I. M. Nugteren-Roodzant, and K. J. Hellingwerf, “Physiological factors affecting production of extracellular lipase (LipA) in Acinetobacter calcoaceticus BD413: fatty acid repression of lipA expression and degradation of LipA,” J. Bacteriol., 178, 6025–6035 (1996).
Korz, D. J., U. Rinas, K. Hellmuth, E. A. Sanders, and W. D. Deckwer, “Simple fed-batch technique for high cell density cultivation of Escherichia coli,” J. Biotechnol. 39, 59–65 (1995).
Kosaric, N., J. E. Zajic, G. Aboue, T. Jack, and D. Gerson, “Lipase synthesis in hydrocarbon fermentation,” Biotechnol. Bioeng., 21, 1133–1149 (1979).
Lee, S. Y., “High cell-density culture of Escherichia coli,” Trends Biotechnol., 14, 98–105 (1996).
Li, S. C., J. Y. Wu, C. Y. Chen, and T. L. Chen, “Semicontinuous production of lipase by Acinetobacter radioresistens in presence of nonwoven fabric,” Appl. Biochem. Biotechnol., 87, 73–80 (2000).
Lin, S. F., “Production and stabilization of a solvent-tolerant alkaline lipase from Pseudomonas pseudoalcaligenes F-111,” J. Ferment. Bioeng., 82, 448–451 (1996).
Lin, Y. C., J. Y. Wu, and T. L. Chen, “Production of Acinetobacter radioresistens lipase with repeated batch culture in presence of nonwoven fabric,” Biotechnol. Bioeng., 76, 214–218 (2001).
Liu, I. L., and S. W. Tsai, “Improvement of lipase production and separation from Acinetobacter radioresistens in the presence of polypropylene powders filled with carbon sources,” Appl. Biochem. Biotech., Part A: Enzyme Eng. and Biotechnol., 104, 129–140 (2003).
Macrae, A. R., “Lipase-catalyzed interesterification of oils and fats,” J. Am. Oil. Chem. Soc., 60, 291–294 (1981).
Meito Sangyo Co. Ltd., “Manufacture of lipase,” Jap. Patent Appl. 58, 51889, Chem. Abstr., 99, 4086 (1983).
Montesinos, J. L., N. Obradors, M. A. Gordillo, F. Valero, J. Lafuente, and C. Sola, “Effect of nitrogen sources in batch and continuous cultures to lipase production by Candida rugosa,” Appl. Biochem. Biotech., 59, 25–37 �n(1996).
Ng, I. S., S. W. Tsai, and S. J. Chen, “Purification and Characterization of extracellular lipase from Acinetobacter radioresistens CMC-2,” J. Chin. Inst. Chem. Engrs., 30, 355–362 (1999).
Nor, Z. M., M. I. Tamer, J. M. Scharer, M. Moo-Young, and E. J. Jervis, “Automated fed-batch culture of Kluyveromyces fragilis based on novel method for on-line estimation of cell specific growth rate,” Biochem. Eng. J., 9, 221–231 (2001).
Oh, J. S., B. G. Kim, and T. H. Park, “Importance of specific growth rate for subtilisin expression in fed-batch cultivation of Bacillus subtilis spoIIG mutant,” Enz. Microb. Technol., 30, 747–751 (2002).
Paalme, T., K. Tiisma, A. Kahru, K. Vanatalu, and R. Vilu, “Glucose-limited fed-batch cultivation of Escherichia coli with computer-controlled fixed growth rate,” Biotechnol. Bioeng., 35, 312–319 (1990).
Pal, N., S. Das, and A. K. Kundu, “Influence of culture and nutritional conditions on the production of lipase by submerged culture of Aspergillus niger,” J. Ferment. Technol., 56, 593 (1978).
Palekar, A., A. Vasudevan, and P. T. Yan, “S. Purification of lipase: a review,” Biocat. Biotransf., 18, 177–200 (2000).
Plou, F. J., M. Ferrer, O. M. Nuero, M. V. Calvo, M. Alcalde, F. Reyes, and A. Ballesteros, “Analysis of Tween 80 as an esterase/lipase substrate for lipolytic activity.” Biotechnol. Tech., 12, 183–186 (1998).
Riesenberg, D., and R. Guthke, “High-cell-density cultivation of microorganism,” Appl. Microbiol. Biotechnol., 51, 422–430 (1999).
Riesenberg, D., K. Menzel, V. Schulz, K. Schumann, G. Veith, G. Zuber, and W. A. Knorre, “High cell density fermentation of recombinant Escherichia coli expressing human interferon alpha 1,” Appl. Microbiol. Biotechnol., 34, 77–82 (1990).
Schmidt, A., F. Hollmann, J. B. Park, and B. Buhler, “The use of enzyme in the chemical industry in Europe,” Curr. Opin. Biotechnol., 13, 359–366 (2002).
Shen, C. C., J.Y. Wu, C. Y. Chen, and T. L. Chen, “Lipase production by Acinetobacter radioresistens in the presence of a nonwoven fabric,” Biotechnol. Prog., 15, 919–922 (1999).
Shin, C. S., M. S. Hong, D. Y. Kim, H. C. Shin, and J. Lee, “Growth-associated synthesis of recombinant human glucagons and human growth hormone in high-cell-density cultures of Escherichia coli,” Appl. Microbiol. Biotechnol., 49, 364–370 (1998).
Sidhu, P., R. Sharma, S. K. Soni, and J. K. Gupta, “Production of extracellular alkaline lipase by a new thermophilic Bacillus sp.,” Folia Microbiol., 43, 51–54 (1998).
Sitajer, H., and I. Maliszewska, “Production of exogenous lipase by bacteria, fungi and acitomycetes,” Enzyme Microb. Technol., 10, 492–497 (1988).
Suzuki, T., Y. Mushiga, T. Yamane, and S. Shimizu, “Mass production of lipase by fed-batch culture of Pseudomonas fluorescens,” Appl. Microbiol. Biotechnol., 27, 417–422 (1988).
Towner, K. J., E. Bergogne-Bérézin, and C. A. Fewson, “Acinetobacter: portrait of a genus,” in The Biology of Acinetobacter, Plenum Press, New York, 1–24 (1991).
Vadehra, D. V., and L. G. Harmon, “Factors affecting production of Staphylococcal lipase,” J. Appl. Bacteriol., 32, 147 (1969).
Vadhera, D., “Staphylococcal lipases,” Lipid, 9, 158–165 (1974).
Walsh, G., and D. Headon, “Protein biotechnology,” John Wiley & Sons, New York, 302–336 (1994).
Wang, T. J., and T. L. Chen, “Lipase production by Acinetobacter radioresistens in a batch fill-and-draw culture,” Appl. Biochem. Biotechnol., 73, 185–194 (1998).
Watatnabe, N., Y. Ota, Y. Minoda, and K. Yamada, “Studies on alkaline lipase from Pseudomonas species. Part I. Isolation and identification of alkaline lipase producing microorganisms, cultural conditions and some properties of the crude enzymes,” Agric. Biol. Chem., 41, 365 (1977).
Yee, L., and H. W. Blanch, “Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli,” Biotechnology, 10, 1550–1556 (1992).
Yee, L., and H. W. Blanch, “Recombinant trypsin production in high cell density fed-batch cultures in Escherichia coli,” Biotechnol. Bioeng., 41, 781–790 (1993).
王惠民，“以正十六烷回收Acinetobacter radioresistens脂肪酵素之研究”，國立成功大學化學工程學系博士論文，(2003)。
吳意珣，“利用野生菌株Acinetobacter radioresistens生產耐鹼性脂肪酶及其特性探討”，國立成功大學化學工程學系碩士論文，(1998)。
洪明全，“Acinetobacter radioresistens耐鹼性脂肪酵素基因選殖及其蛋白質之表現”，國立成功大學碩士論文，(1999)。
陳樹人，“以Acinetobacter radioresistens生產耐鹼性脂肪酵素-醱酵生理之探討”，國立成功大學化學工程學系博士論文，(1999)。
許嘉慧，“利用吸附法濃縮野生菌株Acinetobacter radioresistens生產之鹼性脂肪酵素”，國立成功大學化學工程學系碩士論文，(1999)。