嗜酸嗜熱細菌之分離及其酯解酵素性質之研究
從酸性的溫泉(約pH 3、55℃)附近已分離出數種細菌。依細菌的特性，其中一種鑑定為Bacillus acidocaldarius，編號為菌株A1，可生長於pH 3~4及45~65℃的培養條件下，生長最適pH及溫度分別是pH 4及55℃。以對硝基酚脂(p-nitrophenyl ester)為受質，可於培養細菌的上清液中偵測到酯解酵素(esterase)的活性。橄欖油的添加雖然可促進A1菌株生長，但是對於酯解酵素的分泌似乎並沒有明顯的影響。此酯解酵素作用之最適pH值及溫度分別為中性(pH 7)及75℃。此酯解酵素可分解長碳鏈的酯質受質(pNPC12-pNPC16)，亦具有相當之熱穩定性，於75℃需處理30分鐘才能去掉一半之活性，而以95℃處理3小時後仍保留有18﹪的活性。也測試了一些鹽類、清潔劑及金屬螯合劑EDTA對此酵素的影響，尚未找到可以促進該酯解酵素活性的離子；然而，清潔劑及某些離子則對酵素活性具有抑制的作用；另外，EDTA的添加不對酵素活性有所抑制。

ABSTRAT
Partial characterization of a novel extracellular thermostable esterase from newly isolated thermoacidophilic Bacillus acidocaldarius strain A1
Several moderate thermophilic bacterial strains were isolated from hot acid spring about pH 3 and 55 degree C by enrichment methods. One of those was identified as Bacillus acidocaldarius strain A1. This strain grew under a narrow pH (pH 3~4) and moderate high temperature (45~65 degree C) condition and was optimal at pH 4 and 55 degree C. A thermostable esterase activity was detected in the culture medium supernatant when assayed by using the p-nitrophenyl ester. Adding olive oil to culture medium promoted bacterial growth, but had no effects on the esterase activity. The pH and temperature optima for the esterase activity were pH 7 and 75 degree C, respectively. This crude esterase activity was found capable of hydrolyzing long-chain fatty acid esters (pNPC12 to pNPC16) and exhibited thermal stability. It showed a half-life of 30 min at 75 degree C, and after 3 h incubation at 95 degree C, 18﹪of activity still remained. The effects of several kinds of salts, detergent, and chelating agent, EDTA were also examined. No ions used could promote the esterase activity. However, some detergents and ions had inhibitory effects on the enzyme, and EDTA had no inhibitory effects on it.

陸、參考文獻
Andree, H., W. R. Muller, and R. D. Schmid. 1980. Lipases as detergent components. J. Appl. Biochem. 2：218-229.
Bjorkling, F., S. E. Godtfredsen, and O. Kirk. 1991. The future impact of industrial lipases. Trends Biotechnol. 9：360-363.
Blow, D. 1991. Enzymology. Lipases reach the surface. Nature(England) 351：444-445.
Bott, K. F. 1971. Acrylamide gel electrophoresis of intracellular proteins during early stages of sporulation in Bacillus subtilis. J. Bacteriol. 108：720-732.
Brock, T. D. 1985. Life at high temperatures. Science 230：132-138.
Brockman, H. L. 1984. in “Lipases,” ed. By Borgstorm B. and Brockman, H. L., Elsevier, Amsterdam, pp. 3-4.
Chung, G. H., Y. P. Phil, G. H. Jeohn, O. J. Yoo, and J. S. Rhee. 1991. Cloning and nucleotide sequence of thermostable lipase gene from Pseudomonas fluorescens SIK W1. Agric. Biol. Chem. 55：2539-2365.
Claudia, S. D., S. Helena, S. Walter, M. Ulrich, and D. S. Rolf. 1994. Screening, purification and properties of a thermophilic lipase from Bacillus thermocatenulatus. Biochim. Biophys. Acta. 1214：43-53.
Claudia, S. D., M. L. Rua, A. Haruyuki, and D. S. Rolf. 1996. Thermoalkalophilic lipase of Bacillus thermocatenulatus. I. Molecular cloning, nucleotide sequence, purification and some properties. Biochim. Biophys. Acta. 1301：105-114.
Derewenda, U., A. M. Brzozowski, D. M. Lawson, and Z. Derewenda. 1992. Catalysis at the interface: The anatomy of a conformational change in a triglyceride lipase. Biochemistry(Washington) 31：1532-1541.
Brzozowski, A. M., U. Derewenda, Z. S. Derewenda, G. G. Dodson, D. M. Lawson, J. P. Turkenberg, F. Bjorkling, B. Huge-Jensen, S. A. Patkar, and L. Thim. 1991. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature 351：491-494.
Duncombe, W. G. 1963. The colorimetric micro-determination of long chain fatty acids. Biochem. J. 88：7-10.
Gilbert, E. J., A. Cornish, and C. W. Jones. 1991. Purification and properties of extracellular lipase from Pseudomonas aeruginosa EF2. J. Gen. Microbiol. 137：2223-2229.
Giuseppe, M., D. G. Spartaco, D. R. Mario, and R. Mose. 1994. Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius. Eur. J. Biochem. 221：965-972.
Giuseppe, M., A. Elena, F. M. Pisani, O. Gianluca, C. Giacomo, and R. Mose. 1998. Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase. Biochem. J. 332：203-212.
Giuseppe, M., Ferdinando., A. Elena, and R. Mose. 1999. Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2. Protein Science 8：1789-1796.
Giver, L., A. Gershenson, R. O. Freskgrad, and F. H. Amold. 1998. Directed evolution of a thermostable esterase. Proc. Natl Acad. Sci. USA 95：12809-12813.
Goldberg, M. I. and J. S. Fruton. 1969. Beef liver esterase as a catalyst of acyl transfer to amino acid esters. Biochemistry 8：86-97.
Harwood, J. 1989. The versatility of lipase for industrial uses. Trends Biochem. Sci. 14：125-126.
Herbert, R. A. 1992. A perspective on the biotechnological potential of extremophiles. TIBTECH 10：395-402.
Hespell, R. B. and R. J. O’Bryan-Shan. 1988. Esterase activities in Butyrivibrio fibrisolvens strains. Appl. Environ. Microb. Aug：1917-1922.
Higerd, T. B. and J. Spizizen. 1973. Isolation of two acetyl esterase from extracts of Bacillus sbtilis. J. Bacteriol. 114：1184-1192.
Huddleston, S., C. A. Yallo, and B. M. Charalambous. 1995. The identification and partial characterization of a novel inducible extracellular thermostable esterase from the archaeon Sulfolobus shibatae. Biochem. Biophys. Res. Commun. 216：495-500.
Janssen, P. H., C. R. Monk, and H. W. Morgan. 1994. A thermophilic, lipolytic Bacillus sp., and continuous assay of its p-nitrophenyl-palmitate esterase activity. FEMS Microb. Lett. 120：195-200.
Jinichi, T., A. Yukihiko, K. Kimio, F. Mikio, and S. Junichi. 1998. Purification and characterization of triacylglycerol lipase from Aspergillus oryaze. Biosci. Biotech. Biochem. 62：759-763.
Juan A., B. R. A. Leandro, and N. German. 1993. Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from and Aeromonas hydrophila Human isolate. Appl. Environ. Microb. 59：2411-2417.
Kademi, A., A. A. Nadra, F. Loubna, and J. C. Baratti. 1999. A thermostable esterase activity from newly isolated moderate thermophilic bacterial strains. Enzyme Microb. Technol. 24：332-338.
Kim, H. K., M. H. Sung, H. M. Kim, and T. K. Oh. 1994. Occurrence of thermostable lipase in thermophilic Bacillus sp. strain 398. Biosci. Biotech. Biochem. 58：961-962.
Kim, H. K., S. Y. Park, J. K. Lee, and T. K. Oh. 1998. Gene cloning and characterization of thermostable lipase from Bacillus stearothermophilus L1. Biosci. Biotechnol. Biochem. 62：66-71.
Kugimiya, W., Y. Otani, Y. Hashimoto, and Y. Takagi. 1986. Molecular cloning and nucleotide sequence of the lipase gene from Pseudomonas fragi. Biochem. Biophys. Res. Commun. 14：185-190.
Kwon, D. Y. and J. S. Rhee. 1986. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil Chem. Soc. 63：89-92.
Lin, S. F., C. M. Chiou, C. M. Yeh, and Y. C. Tasi. 1996. Purification and partial characterization of an alkaline lipase from Pseudomonas pseudoalcaligenes F-111. Appl. Environ. Microb. 62：1093-1095.
Laurence, C., R. Jerome, R. J. Luc, D. Jacques, and B. Georges. 1998. Thermostable esterase screened on hyperthermophilic archaeal and bacterial strains isolated from deep-sea hydrothermal vents: Characterization of esterase activityh of a hyperthermophilic archaeum, Pyrococcus abvssi. J. Marine Biotech. 6：104-110.
Macrae, A. R. 1981. Lipase-catalyzed interesterification of oils and fats. J. Am. Oil Chem. Soc. 60：291-294.
Makhzoum, A., R. K. Owusu, and J. S. Knapp. 1993. The conformational stability of a lipase from psychrotrophic Pseudomonas fluorescens. Food. Chem. 46：355-359.
Marcrae, A. R. and R. C. Hammond. 1985. Present and future applications of lipases. Biotechnol. Genet. Eng. Rev. 3：193-217.
Masato, I. and D. S. Clark. 1998. Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli. Biotech. Bioeng. 57：624-629.
Matsunaga, A., N. Koyama, and Y. Nosoh. 1974. Purification and properties of an esterase from Bacillus stearothermophilus. Arch. Biochem. Biophys. 160：504-513.
Mozhaev, V. V. 1993. Mechanism-based strategies for protein thermostabilization. Trends Biotechnol. 11：88-95.
Nakagawa, A., T. Tsujita, and H. Okuda. 1984. Purification and some properties of intracellular esterase from Pseudomonas fluorescens. J. Biochem.(Tokyo) 95：1047-1054.
Ohkawa, I., S. Shiga, and M. Kageyama. 1979. An esterase on the outer membrane of Pseudomonas aeruginosa for the hydrolysis of long chain acyl esters. J. Biochem. 86：643-656.
Okumura, S., M. Iwai, and Y. Tsujisaka. 1983. Purification of four esterases from Aspergillus niger NRRL 337. Agric Biol. Chem. 47：1865-1868.
Owusu, R. K. and D. A. Cowan. 1991. Isolation and partial characterization of a novel thermostable carboxylesterase from a thermophilic Bacillus. Enzyme Microb. Technol. 13：158-163.
Peter, H. A. S., S. M. Nicholas, M. E. Sharpe, and G. H. John. 1986. “Bergey’s Manual of Systematic Bacteriology,” Vol. 2., by Bunchanan R. E. and Gibbon N. E., Williams and Wilkins Co., Baltimore：pp. 1104-1139.
Shabati, Y. and N. Daya-Mishne. 1992. Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments. Appl. Environ. Microbiol. 58：147-180.
Shao W. and J. Wiegel. 1995. Purification and characterization of two thermostable acetyl xylan esterases from thermoanaerobacterium sp. strain JW/SL-YS485. Appl. Env. Microbiol. 61：729-733.
Shum, A. C. and A. J. Markovetz. 1974. Specificity and induction of undecyl acetate esterase from Pseudomonas cepacia grown on 2-tridecanone. J. Bacteriol. 118：890-897.
Simoes, D. D. C. M., D. McNeill, B. Kristiansen, and M. Mattey. 1997. Purification and partial characterization of a 1.57kDa thermostable esterase from Bacillus stearothermophilus. FEMS Microb. Letters. 147：151-156.
Sobek, H. and H. Gorisch. 1988. Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem. J. 250：453-458.
Sobek, H. and H. Gorisch. 1989. Further kinetic and molecular characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Biochem. J. 261：993-998.
Sonnleitner, B. 1983. Biotechnology of thermophilic bacteria: growth, products, and applications. Adv. Biochem. Eng. Biotech. 28：69-138.
Sugihara, A., T. Tani, and Y. Tominaga. 1991. Purification and characterization of a novel thermostable lipase from Bacillus sp. J. Biochem. 109：211-216.
Sugihara, A., M. Ueshima, Y. Shimada, S. Tsunesawa, and Y. Tominaga. 1992. Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia. J. Biochem. 112：598-603.
Ramos, T. G. M., H. P. Schaer, and O. Ghisalba. 1987. Application of microbes and microbial esterases to the preparation of optically active N-acetylindoline-2-carboxylic acid. Agric. Biol. Chem. 51：1833-1838.
Torossian, K. and A. W. Bell. 1991. Purification and characterization of and acid-resistant triacylglycerol lipase from Aspergillus niger. Biotech. Appl. Biochem. 13：205-211.
Winkler, U. K. and M. Stuckmann. 1979. Glycogen, hyaluronate, and some other polysaccharides greatly enhance the formation of exolipase by Serratia marcescens. J. Bacteriol. 138：663-670.
Young, P. L., H. C. Guk, and S. R. Joon. 1993. Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli. Biochim. Biophs. Acta 1169：156-164.
王孟群. 1983. 實用微生物學實驗. 九州圖書公司 pp. 60-124.