自陽明山國家公園溫泉中，分離出數株有能力水解三丁酸甘油酯（tributyrin）的細菌，取水解能力最佳者，作為研究之對象。經16S rDNA定序分析，與部分生理生化測試，將此菌暫定為Geobacillus uzenensis。
本研究利用細菌之外泌酵素粗萃取液，偵測此酯解酵素（esterase）之特性。在受質特異性（substrate specificity）上，相較於長鏈酯質（p-nitrophenyl myristate，pNPC14 、 p-nitrophenyl palmitate，pNPC16），此酵素偏好水解短鏈酯質（p-nitrophenyl acetate，pNPC2）。酵素反應之最適溫度為60~70℃，能在60℃下受熱2小時，保有完整活性；但在70℃下受熱15分鐘，則酵素活性盡失。在p-nitrophenyl acetate測試及酯解酵素活性染色結果中，此酵素能在pH 2～8下，具有活性作用，其最大活性表現在pH 3與6～7。當酵素液與pH 3～9緩衝溶液混合， 於4℃靜置24小時後，仍可保留100％活性。酵素對鉀離子（K+）的存在，有活性下降之情形發生；在化學試劑測試中，phenylmethylsulphonyl fluoride（PMSF）明顯抑制酵素活性，erhylenediamine tetraacetic acid（EDTA）、dithiothreitol（DTT）則不影響酵素活性；在清潔劑（detergent）測試中，Sodium dodecyl sulfate（SDS）、Triton X-100、Tween 20對酵素活性具抑制作用，Tween 80、CHAPS（3-[(3-cholamidopropyl)- dimethylammonio]
-1-propanesulfonate）則無影響。此酯解酵素對15%（v/v）乙醇（ethanol）、丙酮（acetone）等有機溶劑，其耐受度差，但在15%（v/v）DMSO（Dimethyl sulfoxide）存在下，仍有80%的活性。總之，本研究發現一耐熱、耐酸的酯解酵素，且此酵素能在酸性環境下（pH 2～3）有不錯活性，實為其他脂解酵素或酯解酵素少有的特性。

Several bacteria isolated from a hot spring in Yang Ming Mountain, National Park of Taiwan have capability to degrade tributyrin. The bacterium selected for this study showed the biggest clear zone on the tributyrin agar among all samples. This thermophilic esterase producing bacterium was analyzed using 16S rDNA and partial physiological characterization; and provisionally classified as Geobacillus uzenensis. In this study, the extracellular crude preparation was used to determine the characterization of esterase activity. The substrate specificity of the enzyme showed a preference for p-nitrophenyl acetate as compared with p-nitrophenyl myristate and p-nitrophenyl palmitate. The optimum temperature for the esterase was 60~70 oC. The enzyme was retained its original activity at 60 oC for 2 h incubation period. However, enzyme activity was fully lost at 70 oC for 15 min incubation period. In addition, the p-nitrophenyl acetate assay and esterase activity staining (zymogram) showed the enzyme was active over a pH range from pH 2 to 8; with two maximal activities peaked at pH 3 and 6~7 respectively. After the crude enzyme extract was mixed with the buffer (pH 3~9) and incubated for 24h at 4 oC, it was stable with a ratio of 100% compared to its original activity. Among the various metal ions influence tests, esterase activity was significantly inhibited only by K+. The chemical reagent, PMSF could caused a significant inhibition in the enzyme activity, whereas the esterase was unaffected by EDTA and DTT. The activity of the enzyme was also inhibited by SDS, Triton X-100 and Tween 20. However, Tween 80 and CHAPS had no effect. Last, the enzyme was inhibited with 15% (v/v) concentration of ethanol and acetone and it retained 80% activity in presence of DMSO at the same concentration of 15% (v/v) . Briefly, the esterase was stable in the acidic pH region. These properties support the interest of further research on its production and characterization.

Adams, M.W., Perler, F.B., Kelly, R.M., 1995. Extremozymes: expanding the limits of biocatalysis. Biotechnology (N Y), 13, 662-8.

Alvarez-Macarie, E., Augier-Magro, V., Baratti, J., 1999. Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis. Biosci Biotechnol Biochem, 63, 1865-70.

Antranikian, G., Vorgias, C.E., Bertoldo, C., 2005. Extreme environments as a resource for microorganisms and novel biocatalysts. Adv Biochem Eng Biotechnol, 96, 219-62.

Arpigny, J.L., Jaeger, K.E., 1999. Bacterial lipolytic enzymes: classification and properties. Biochem J, 343 Pt 1, 177-83.

Bornscheuer, U.T., 2002. Microbial carboxyl esterases: classification, properties and application in biocatalysis. FEMS Microbiol Rev, 26, 73-81.

Bornscheuer, U.T., Kazlauskas, R.J., 2006. Hydrolases in organic synthesis - Regio- and stereoselective biotransformations. 2nd ed. WILEY-VCH Verlag GmbH & Co. KGaA.

De Simone, G., Menchise, V., Alterio, V., Mandrich, L., Rossi, M., Manco, G., Pedone, C., 2004. The crystal structure of an EST2 mutant unveils structural insights on the H group of the carboxylesterase/lipase family. J Mol Biol, 343, 137-46.
Degrassi, G., Uotila, L., Klima, R., Venturi, V., 1999. Purification and properties of an esterase from the yeast Saccharomyces cerevisiae and identification of the encoding gene. Appl Environ Microbiol, 65, 3470-2.

Deive, F.J., Costas, M., Longo, M.A., 2003. Production of a thermostable extracellular lipase by Kluyveromyces marxianus. Biotechnol Lett, 25, 1403-6.

Demirjian, D.C., Moris-Varas, F., Cassidy, C.S., 2001. Enzymes from extremophiles. Curr Opin Chem Biol, 5, 144-51.

Drauz, K., Waldmann, H., 2002. Enzyme catalysis in organic synthesis - A comprehensive handbook 2nd ed. Wiley-VCH Verlag GmbH, Weinheim.

Egorova, K., Antranikian, G., 2005. Industrial relevance of thermophilic Archaea. Curr Opin Microbiol, 8, 649-55.

Eichler, J., 2001. Biotechnological uses of archaeal extremozymes. Biotechnol Adv, 19, 261-78.

Ewis, H.E., Abdelal, A.T., Lu, C.D., 2004. Molecular cloning and characterization of two thermostable carboxyl esterases from Geobacillus stearothermophilus. Gene, 329, 187-95.

Faiz, O., Colak, A., Saglam, N., Canakci, S., Belduz, A.O., 2007. Determination and characterization of thermostable esterolytic activity from a novel thermophilic bacterium Anoxybacillus gonensis A4. J Biochem Mol Biol, 40, 588-94.

Fojan, P., Jonson, P.H., Petersen, M.T.N., Petersen, S.B., 2000. What distinguishes an esterase from a lipase: A novel structural approach. Biochimie, 82, 1033-1041.

Gilham, D., Lehner, R., 2005. Techniques to measure lipase and esterase activity in vitro. Methods, 36, 139-47.

Hotta, Y., Ezaki, S., Atomi, H., Imanaka, T., 2002. Extremely stable and versatile carboxylesterase from a hyperthermophilic archaeon. Appl Environ Microbiol, 68, 3925-31.

Hough, D.W., Danson, M.J., 1999. Extremozymes. Curr Opin Chem Biol, 3, 39-46.

Ikeda, M., Clark, D.S., 1998. Molecular cloning of extremely thermostable esterase gene from hyperthermophilic archaeon Pyrococcus furiosus in Escherichia coli. Biotechnol Bioeng, 57, 624-9.

Isobe, K., Wakao, N., 1997. Purification and some properties of a thermostable acid esterase from Acidiphilium sp. AIU 409. J Gen Appl Microbiol, 43, 151-156.

Kademi, A., Ait-Abdelkader, N., Fakhreddine, L., Baratti, J., 2000. Purification and characterization of a thermostable esterase from the moderate thermophile Bacillus circulans. Appl Microbiol Biotechnol, 54, 173-9.
Kadmi, A., Ait-Avdelkader, N., Fakhreddine, L., Baratti, J.C., 1999. A thermostable esterase activity from newly isolated moderate thermophilic bacterial strains. Enzyme and microbial technology 24, 332-338.

Kakugawa, S., Fushinobu, S., Wakagi, T., Shoun, H., 2007. Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima. Appl Microbiol Biotechnol, 74, 585-91.

Kim, S., Lee, S.B., 2004. Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound. Biosci Biotechnol Biochem, 68, 2289-98.

Leow, T.C., Rahman, R.N., Basri, M., Salleh, A.B., 2004. High level expression of thermostable lipase from Geobacillus sp. strain T1. Biosci Biotechnol Biochem, 68, 96-103.

Manco, G., Adinolfi, E., Pisani, F.M., Ottolina, G., Carrea, G., Rossi, M., 1998. Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily. Biochem J, 332 ( Pt 1), 203-12.

Manco, G., Di Gennaro, S., De Rosa, M., Rossi, M., 1994. Purification and characterization of a thermostable carboxylesterase from the thermoacidophilic eubacterium Bacillus acidocaldarius. Eur J Biochem, 221, 965-72.
Manco, G., Febbraio, F., Adinolfi, E., Rossi, M., 1999. Homology modeling and active-site residues probing of the thermophilic Alicyclobacillus acidocaldarius esterase 2. Protein Sci, 8, 1789-96.

Metin, K., Burcu Bakir Ateslier, Z., Basbulbul, G., Halil Biyik, H., 2006. Characterization of esterase activity in Geobacillus sp. HBB-4. J Basic Microbiol, 46, 400-9.

Monti, D., Ferrandi, E.E., Righi, M., Romano, D., Molinari, F., 2008. Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553. J Biotechnol, 133, 65-72.

Morana, A., Di Prizito, N., Aurilia, V., Rossi, M., Cannio, R., 2002. A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein. Gene, 283, 107-15.

Nachlas, M.M., Blackburn, R., 1958. The colorimetric determination of urinary lipase. J Biol Chem, 230, 1051-61.

Nawani, N., Kaur, J.K.a.J., 2006. A thermostable lipolytic enzyme from a thermophilic Bacillus sp.: Purification and characterization. Molecular and Cellular Biochemistry, 290, 17-22.

Nazina, T.N., Sokolova, D., Shestakova, N.M., Grigor'ian, A.A., Mikhailova, E.M., Babich, T.L., Lysenko, A.M., Turova, T.P., Poltaraus, A.B., Feng, T., Ni, F., Beliaev, S.S., 2005. The phylogenetic diversity of aerobic organotrophic bacteria from the Dagan high-temperature oil field. Mikrobiologiia, 74, 401-9.

Nazina, T.N., Tourova, T.P., Poltaraus, A.B., Novikova, E.V., Grigoryan, A.A., Ivanova, A.E., Lysenko, A.M., Petrunyaka, V.V., Osipov, G.A., Belyaev, S.S., Ivanov, M.V., 2001. Taxonomic study of aerobic thermophilic bacilli: descriptions of Geobacillus subterraneus gen. nov., sp. nov. and Geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of Bacillus stearothermophilus, Bacillus thermocatenulatus, Bacillus thermoleovorans, Bacillus kaustophilus, Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. th. Int J Syst Evol Microbiol, 51, 433-46.

Ollis, D.L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S.M., Harel, M., Remington, S.J., Silman, I., Schrag, J., et al., 1992. The alpha/beta hydrolase fold. Protein Eng, 5, 197-211.

Owusu, R.K., Cowan, D.A., 1989. Correlation between microbial protein thermostability and resistance to denaturation in aqueous: organic solvent two-phase systems. Enzyme and microbial technology 11, 568-574

Rhee, J.K., Ahn, D.G., Kim, Y.G., Oh, J.W., 2005. New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library. Appl Environ Microbiol, 71, 817-25.

Schmidt, M., Bornscheuer, U.T., 2005. High-throughput assays for lipases and esterases. Biomol Eng, 22, 51-6.
Schutte, M., Fetzner, S., 2007. EstA from Arthrobacter nitroguajacolicus Ru61a, a thermo- and solvent-tolerant carboxylesterase related to class C beta-lactamases. Curr Microbiol, 54, 230-6.

Sharma, A.K., Tiwari, R.P., Hoondal, G.S., 2001. Properties of a thermostable and solvent stable extracellular lipase from a Pseudomonas sp. AG-8. J Basic Microbiol, 41, 363-6.

Sugihara, A., Shimada, Y., Nagao, T., Iizumi, T., Nakamura, K., Fukase, T., Tominaga, Y., 1994. Purification and Characterization of a Carboxylesterase from Pseudomonas sp. KWI-56. Biosci Biotech Biochem , 58, 752-755.

Sugihara, A., Tani, T., Tominaga, Y., 1991. Purification and characterization of a novel thermostable lipase from Bacillus sp. J Biochem, 109, 211-6.

Sugihara, A., Ueshima, M., Shimada, Y., Tsunasawa, S., Tominaga, Y., 1992. Purification and characterization of a novel thermostable lipase from Pseudomonas cepacia. J Biochem, 112, 598-603.

Suzuki, Y., Miyamoto, K., Ohta, H., 2004. A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. FEMS Microbiol Lett, 236, 97-102.

Tirawongsaroj, P., Sriprang, R., Harnpicharnchai, P., Thongaram, T., Champreda, V., Tanapongpipat, S., Pootanakit, K., Eurwilaichitr, L., 2008. Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library. J Biotechnol, 133, 42-9.
Torres, M., Dolcet, M.M., Sala, N., Canela, R., 2003. Endophytic fungi associated with Mediterranean plants as a source of mycelium-bound lipases. J Agric Food Chem, 51, 3328-33.

Wang, B., Lu, D., Gao, R., Yang, Z., Cao, S., Feng, Y., 2004. A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1. Protein Expr Purif, 35, 199-205.

Welsh, F.W., Williams, R.E., 1989. Lipase Mediated Production of Flavor and Fragrance Esters from Fusel Oil. J Food Sci, 54, 1565-1568.

Wood, A.N., Fernandez-Lafuente, R., Cowan, D.A., 1995. Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity. Enzyme Microb Technol, 17, 816-25.

Yadav, R.P., Saxena, R.K., Gupta, R., Davidson, W.S., 1998. Purification and characterization of a regiospecific lipase from Aspergillus terreus. Biotechnol Appl Biochem, 28 ( Pt 3), 243-9.

Zhang, J., Liu, J., Zhou, J., Ren, Y., Dai, X., Xiang, H., 2003. Thermostable esterase from Thermoanaerobacter tengcongensis: high-level expression, purification and characterization. Biotechnol Lett, 25, 1463-7.

陳懋彥, 2002. 台灣地熱區嗜熱性細菌之研究. 國立臺灣大學植物學研究所博士論文.