本研究探討 52 株具纖維素分解能力之嗜高溫微生物的生理特性及演化分類。根據 16S rDNA 序列之分析，3 株來自生技中心和 4 株分離自南台灣的細菌分別近似於菌屬 Bacillus 和 Geobacillus。新屬 Geobacillus 的四株菌中，菌株 T4 為可移動、有氧生長且可形成孢子的革蘭氏陰性桿菌，能分泌熱穩定的內切型纖維素分解酵素。當菌株 T4 生長於含 carboxymethylcellulose 的液體培養基時，其在培養懸浮液中的纖維素分解酵素活性可耐受 70℃ 的高溫。根據 16S rDNA 序列、DNA G+C 含量、生理生化特徵以及 DNA-DNA 雜交反應的分析，菌株 T4 可被鑑定為 Geobacillus thermoleovorans。此菌種已分別存放德國與中華民國菌種中心，其編號分別為 DSM 14791 及 CCRC 17200。此外，也根據熱穩定纖維素分解酵素之氨基酸序列構築一個含 20 個相關微生物的親緣演化樹。本研究結果建議這些屬於醣苷水解酵素大家族中的熱穩定纖維素分解酵素可分成 4 個亞族：GH family 12 subgroup TC-1、bacterial subgroup TC-2、bacterial subgroup TC-3 以及 GH family 1 subgroup TC-4。再結合以 16S rDNA 序列將菌株 T4 和 10 株相關微生物進行的分析，菌株 T4 近似於亞族 subfamily TC-4 但與亞族 subfamily TC-1 在演化關係上較遠。

Fifty two cellulolytic thermophilic microorganisms were analyzed for their physiological characterization and phylogenetic systematics. Based on 16S rDNA sequence analysis, 3 strains from DCB and 4 novel isolates from southern Taiwan are close related to the genera of Bacillus and Geobacillus respectively. Among 4 new Geobacillus strains, strain T4, a Gram negative, motile, aerobically growing sporulating rod, can secrete thermostable endoglucanase. When strain T4 was grown in CMC medium, the cellulolytic enzyme activity in culture supernatants was stable up to 70°C. Based on 16S rDNA sequence analysis, DNA G+C content, phenotypic and physiological characteristics, as well as DNA-DNA hybridization, strain T4 was classified as Geobacillus thermoleovorans T4 (DSM 14791 = CCRC 17200). Furthermore, a phylogenetic tree of 20 related microorganisms was also constructed based on their thermostable cellulase amino acid sequences. Our sequence analysis shows that cellulases belonging to the large family of glycoside hydrolases (GHs) can be divided into four subfamilies: TC-1 (GH family 12 group), TC-2 (bacterial group Ι in which fungal species Thermoascus aurantiacus fits), TC-3 (bacterial group ΙΙ), and TC-4 (GH family 1 group). Together with the 16S rDNA sequence analysis of strain T4 and 10 related microorganisms, strain T4 has a close phylogenetic relationship with subfamily TC-4 but far from subfamily TC-1.

前言 1
（一）纖維素 1
一、纖維素的組成 1
二、纖維素生物能源之開發利用 2
（二）纖維素分解酵素 3
一、內切型纖維素分解酵素 3
二、外切型纖維素分解酵素 4
三、纖維二醣酵素 4
（三）嗜高溫纖維素分解菌及菌屬 Geobacillus 5
一、嗜高溫生物 5
二、具纖維素分解能力之嗜高溫菌 6
三、纖維素複合體 7
四、嗜高溫菌屬 Geobacillus 8
（四）酵素熱穩定性 10
（五）醣苷水解酵素中的纖維素分解酵素 11
一、醣苷水解酵素 11
二、熱穩定纖維素分解酵素之分類 12
（六）纖維素分解酵素的應用 13
實驗目的 16
材料與方法 17
（一）實驗材料 17
一、菌株 17
二、培養基 17
三、試劑 18
四、纖維素分解酵素作用之基質 19
（二）實驗方法 19
一、菌種之分離與純化 19
二、菌種的培養與保存 20
三、纖維素分解酵素活性之初步測定 20
四、菌種的形態與生理生化之測試 20
五、菌種 16S rDNA 序列之定序分析 21
六、親緣演化樹的建立 24
七、核酸和氨基酸序列之編號 25
八、菌種之鑑定 25
九、纖維素分解酵素活性的測定 26
結果與討論 30
（一）菌種之生長溫度 30
（二）剛果紅測定纖維素分解酵素之活性 30
（三）菌種 16S rDNA 序列之定序分析 31
（四）菌株 b5 和 b6 之纖維素分解酵素活性的測定 32
（五）菌株 T4 的形態與生理生化之測試 34
（六）菌株 T4 與菌屬 Geobacillus 等相關細菌之親緣演化樹 36
（七）菌株 T4 之鑑定 37
（八）菌株 T4 與相關菌種之纖維素分解酵素的特性 38
（九）菌株 T4 與具熱穩定纖維素分解酵素菌種的親緣演化分析 42
（十）熱穩定纖維素分解酵素之親緣演化關係 42
（十一）熱穩定纖維素分解酵素之氨基酸組成及其熱穩定的探討 44
結論與建議 46
參考文獻 48
圖表 65
附錄一 89
Modified chemically defined CMC medium 89
附錄二 90
（一）16S rDNA sequences of strain b5 90
（二）16S rDNA sequences of strain b6 91
（三）16S rDNA sequences of strain b7 92
（四）16S rDNA sequences of strain T1 93
（五）16S rDNA sequences of strain T2 94
（六）16S rDNA sequences of strain T3 95
（七）16S rDNA sequences of strain T4 96
附錄三（第一篇英文 paper，已被期刊 Extremophiles 接受刊登）
Isolation and characterization of a cellulolytic Geobacillus thermoleovorans T4 strain from sugar refinery wastewater 97

附錄四（第二篇中文 paper，已被期刊「生物科學」接受刊登 ）
熱穩定性纖維素分解細菌 112
附錄五（第三篇英文 paper，已投稿至期刊 Archives of Microbiology，審查中） 139
Phylogenetic characteristics and classification of Geobacillus thermoleovorans T4 with 10 other related thermostable cellulolytic microorganisms 139

王正仁、陳孟伶、林畢修平、陳啟祥, 1999. 水解酵素在工業上的利用. 生物產業. 10:1-11.
王鳳英, 1994. 利用固態發酵以玉米穗軸為基質生產纖維素分解酵素. 博士論文. 國立台灣大學農業化學研究所, 台北市, pp. 231.
谷口肇, 1989. 纖維雙醣的生產技術. 日農化會誌. 63(6):1133-1135.
李振漢, 1999. 黑黴菌屬 β-葡萄糖甘酵素 II 基因之選殖及其序列分析. 碩士論文. 私立大同工學院化學工程研究所, 台北市, pp. 64.
林彥行, 1995. 耐高溫放線菌之分離及應用. 碩士論文. 國立台灣大學農業化學研究所, 台北市, pp. 89.
林貝珊, 1999. 火鶴花細菌性葉枯病病原菌核酸探針及 PCR 引子之研發與應用. 碩士論文. 國立台灣大學植物病蟲害學研究所, 台北市, pp. 89.
葉丁源, 1997. 嗜高溫放線菌纖維素分解酵素之探討. 碩士論文. 國立台灣大學農業化學研究所, 台北市, pp. 125.
Acebal, C., M. P. Castillon, P. Estrada, I. Mata, E. Costa, J. Aguado, D. Romero, and F. Jimenez. 1986. Enhanced cellulase production from Trichoderma reesei QM 9414 on physically treated wheat straw. Appl. Microbiol. Biotechnol. 24:218-223.
Almeida, L., D. Bishop, and A. C. Paulo. 1996. Cellulase activities and finishing effects. Textile Chemist & Colorist. 28:28-32.
Andren, R. T., M. Mandels, and J. E. Modeiros. 1976. Production of sugar from waste cellulose by enzymatic hydrolysis: primary evolution of substrates. Process Biochem. 11:2-11.
Argos, P., M. G. Rossmann, U. M. Grau, H. Zuber, G. Frank, and J. D. Tratschin. 1979. Thermal stability and protein structure. Biochemistry 18:5698–5703.
Ash, C., J. A. E. Farrow, S. Wallbanks, and M. D. Collins. 1991. Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett. Appl. Microbiol. 13:202-206.
Atalla, R. H., J. M. Hackney, I. Uhlin, and N. S. Thompson. 1993. Hemicelluloses as structure regulators in the aggregation of native cellulose. Int. J. Biol. Macromol. 15:109-112.
Bauer, M. W., L. E. Driskill, W. Callen, M. A. Snead, E. J. Mathur, and R. M. Kelly. 1999. An endoglucanase, EglA, from the hyperthermophilic archaeon Pyrococcus furiosus hydrolyzes β-1,4 bonds in mixed-linkage (1→3),(1→4)-β-D-glucans and cellulose. J. Bacteriol. 181:284-290.
Béguin, P. 1987. Cloning of cellulase gene. Crit. Rev. Biotechnol. 6:129-162.
Béguin, P., and J.-P. Aubert. 1994. The biological degradation of cellulose. FEMS Microbiol. Rev. 13:25-58.
Béguin, P., and M. Lemaire. 1996. The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation. Crit. Rev. Biochem. Mol. Biol. 31:201-236.
Beltrame, P. L., P. Carniti, A. Visciglio, B. Focher, and A. Marzetti. 1992. Fractionation and bioconversion of steam-exploded wheat straw. Bioresour. Technol. 39:165-171.
Bhat, M. K., and S. Bhat. 1997. Cellulose degrading enzymes and their potential industrial applications. Biotechnol. Adv. 15:583-620.
Bisaria, V. S., and T. K. Ghose. 1981. Biodegradation of cellulostic materials: substrates, microorganisms, enzymes and products. Enzyme Microb. Technol. 3:90-104.
Bisaria, V. S., and S. Mishra. 1989. Regulatory aspects of cellulase biosynthesis and secretion. Crit. Rev. Biotechnol. 9:61-103.
Bok, J.-D., D. A. Yernool, and D. E. Eveleigh. 1998. Purification, characterization, and molecular analysis of thermostable cellulases CelA and CelB from Thermotoga neapolitana. Appl. Environ. Microbiol. 64:4774-4781.
Brama, T. 1994. The detergent market. Household & Personal Products Industry. Jan. pp.36-48.
Breves, R., K. Bronnenmeier, N. Wild, F. Lottspeich, W. L. Staudenbauer, and J. Hofemeister. 1997. Genes encoding two different β-glucosidases of Thermoanaerobacter brockii are clustered in a common operon. Appl. Environ. Microbiol. 63:3902-3910.
Brock, T. D. 1986. Introduction: an overview of the thermophiles, pp. 1-16. In T. D. Brock (ed.), Thermophiles: general, molecular, and applied microbiology. John Wiley & Sons, New York.
Bronnenmeier, K., K. Kundt, K. Riedel, W. H. Schwarz, and W. L. Staudenbauer. 1997. Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-β-glucanase Avicelase II. Microbiology 143:891-898.
Brown, R. M., Jr., and I. M. Saxena. 2000. Cellulose biosynthesis: a model for understanding the assembly of biopolymers. Plant Physiol. Biochem. 38:57-67.
Chahal, D. S. 1985. Solid-state fermentation with Trichoderma reesei for cellulase production. J. Appl. Environ. Microbiol. 49:205-210.
Claus, D., and R. C. W. Berkeley. 1986. Genus Bacillus Cohn 1872, pp. 1105-1139. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (eds.), Bergey's manual of systematic bacteriology, vol II. Williams and Wilkins, Baltimore.
Coughlan, M. P. 1985a. Cellulases: Production properties and applications. Biochem. Soc. Trans. 13:405-406.
Coughlan, M. P. 1985b. The properties of fungal and bacterial cellulases with comment on their production and application, pp. 39-109. In G. E. Russell (ed.), Biotechnology and genetic engineering reviews, vol 3. Interscience, Newcastle-upon-Tyne.
Demharter, W., and R. Hensel. 1989. Bacillus thermocloaceae, sp. nov., a new thermophilic species from sewage sludge. Syst. Appl. Microbiol. 11:272–276.
Devereux, J., P. Haeberli, and O. Smithies. 1984. A comprehensive set of sequence analysis programs for the VAX. Nucleic. Acids. Res. 12:387–395.
Doi, R. H., and A. Kosugi. 2004. Cellulosomes: plant-cell-wall-degrading enzyme complexes. Nat. Rev. Microbiol. 2:541-551.
Ezaki, T., Y. Hashimoto, and E. Yabuuchi. 1989. Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int. J. Syst. Bacteriol. 39:224-229.
Felsenstein, J. 1989. Phylip - Phylogeny inference package. Cladistics. 5:164-166.
Fontes, C. M. G. A., J. H. Clarke, G. P. Hazlewood, T. H. Fernandes, H. J. Gilbert, and L. M. A. Ferreira. 1997. Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus. Appl. Microbiol. Biotechnol. 48:473-479.
Fortina, M. G., D. Mora, P. Schumann, C. Parini, P. L. Manachini, and E. Stackebrandt. 2001. Reclassification of Saccharococcus caldoxylosilyticus as Geobacillus caldoxylosilyticus (Ahmad et al. 2000) comb. nov. Int. J. Syst. Evol. Microbiol. 51:2063-2071.
Galbe, M., and G. Zacchi. 2002. A review of the production of ethanol from softwood. Appl. Microbiol. Biotechnol. 59:618-628.
Gamal, R. F. 1985. Effect of substrate pretreatment on microbiol protein production. Egypt. J. Microbiol. (Spec. Issue) 81-89.
Gehin, A., C. Cailliez, E. Petitdemange, and L. Benoit. 1996. Studies of Clostridium cellulolyticum ATCC 35319 under dialysis and co-culture conditions. Lett. Appl. Microbiol. 23:208-212.
Gilkes, N. R., B. Henrissat, D. G. Kilburn, R. C. Miller, Jr., and R. A. J. Warren. 1991. Domains in microbial β-1,4-glycanases: sequence conservation, function, and enzyme families. Microbiol. Rev. 55:303-315.
Goedegebuur, F., T. Fowler, J. Phillips, P. van der Kley, P. van Solingen, L. Dankmeyer, and S. D. Power. 2002. Cloning and relational analysis of 15 novel fungal endoglucanases from family 12 glycosyl hydrolase. Curr. Genet. 41:89-98.
Goyal, A. K., and D. E. Eveleigh. 1996. Cloning, sequencing and analysis of the ggh-A gene encoding a 1,4-β-D-glucan glucohydrolase from Microbispora bispora. Gene 172:93-98.
Grabnitz, F., M. Seiss, K. P. Rucknagel, and W. L. Staudenbauer. 1991. Structure of the β-glucosidase gene bglA of Clostridium thermocellum. Sequence analysis reveals a superfamily of cellulases and β-glycosidases including human lactase/phlorizin hydrolase. Eur. J. Biochem. 200:301-309.
Gupta, N., V. S. Reddy, S. Maiti, and A. Ghosh. 2000. Cloning, expression, and sequence analysis of the gene encoding the alkali-stable, thermostable endoxylanase from alkalophilic, mesophilic Bacillus sp. strain NG-27. Appl. Environ. Microbiol. 66:2631-2635.
Hakamada, Y., Y. Hatada, K. Koike, T. Yoshimatsu, S. Kawai, T. Kobayashi, and S. Ito. 2000. Deduced amino acid sequence and possible catalytic residues of a thermostable, alkaline cellulase from an alkaliphilic bacillus strain. Biosci. Biotechnol. Biochem. 64:2281-2289.
Haki, G. D., and S. K. Rakshit. 2003. Developments in industrially important thermostable enzymes: a review. Bioresour. Technol. 89:17-34.
Halldorsdottir, S., E. T. Thorolfsdottir, R. Spilliaert, M. Johansson, S. H. Thorbjarnardottir, A. Palsdottir, G. O. Hreggvidsson, J. K. Kristjansson, O. Holst, and G. Eggertsson. 1998. Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. Appl. Microbiol. Biotechnol. 49:277-284.
Heinen, U. J., and W. Heinen. 1972. Characteristics and properties of a caldoactive bacterium producing extracellular enzymes and two related strains. Arch. Mikrobiol. 82:1–23.
Henrissat, B., M. Claeyssens, P. Tomme, L. Lemesle, and J. P. Mornon. 1989. Cellulase families revealed by hydrophobic cluster analysis. Gene 81:83-95.
Henrissat, B. 1991. A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 280:309-316.
Henrissat, B., and A. Bairoch. 1993. New families in the classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem. J. 293:781-788.
Henrissat, B., and A. Bairoch. 1996. Updating the sequence-based classification of glycosyl hydrolases. Biochem. J. 316:695-696.
Henrissat, B., and G. J. Davies. 2000. Glycoside hydrolases and glycosyltransferases. Families, modules, and implications for genomics. Plant Physiol. 124:1515–1519.
Herbert, R. A., and T. J. Sharp. 1992. Molecular biology and biotechnology of extremophiles. Chapman and Hall, New York.
Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules, pp. 21-132. In H. N. Munro (ed.), Mammalian protein metabolism, vol. Ш. Academic, New York.
Kalogridou-Vassiliadou, D. 1992. Biochemical activities of Bacillus species isolated from flat sour evaporated milk. J. Dairy Sci. 75:2681-2686.
Kumar, S., and R. Nussinov. 2001. How do thermophilic proteins deal with heat? Cell. Mol. Life Sci. 58:1216–1233.
Kuzmanova, S., E. Vandeska, and A. Dimitrovski. 1991. Production of mycelial protein and cellulolytic enzymes from food wastes. J. Ind. Microbiol. 7:257-261.
Lamed, R., J. Naimark, E. Morgenstern, and E. A. Bayer. 1987. Specialized cell surface structures in cellulolytic bacteria. J. Bacteriol. 169:3792-3800.
Liebl, W., P. Ruile, K. Bronnenmeier, K. Riedel, F. Lottspeich, and L. Greif. 1996. Analysis of a Thermotoga maritima DNA fragment encoding two similar thermostable cellulases, CelA and CelB, and characterization of the recombinant enzymes. Microbiology. 142:2533-2542.
Liou, R.-F. 2001. Northern hybridization, pp. 43-63. In C. Y. Tsai, and R. H. Juang, (eds.), Biotechnology core techniques, methods in biotechnology, vol. 1(in Chinese). Biotechnology Research Center, National Taiwan University, Taipei, Taiwan.
Logan, N. A., and R. C. W. Berkeley. 1984. Identification of Bacillus strains using the API system. J. Gen. Microbiol. 130:1871-1882.
Lopez, G. 1999. DNA supercoiling and temperature adaptation: a clue to early diversification of life. J. Mol. Evol. 46:439–452.
Lutzen, N. W., M. H. Nielsen, K. M. Oxenboell, M. Schulein, and B. Stentebjerg-Olesen. 1983. Cellulases and their application in the conversion of lignocellulose to fermentable sugars. Phil. Trans. R. Soc. Lond. B. 300:283-291.
Lynd, L. R., P. J. Weimer, W. H. van Zyl, and I. S. Pretorius. 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. 66:506-577.
Maheshwari, R., G. Bharadwaj, and M. K. Bhat. 2000. Thermophilic fungi: their physiology and enzymes. Microbiol. Mol. Biol. Rev. 64:461-488.
Maidak, B. L., J. R. Cole, T. G. Lilburn, C. T. Jr Parker, P. R. Saxman, R. J. Farris, G. M. Garrity, G. J. Olsen, T. M. Schmidt, and J. M. Tiedje. 2001. The RDP-II (Ribosomal Database Project). Nucleic Acids Res. 29:173-174.
Mandels, M. 1985. Applications of cellulases. Biochem. Soc. Trans. 13:414-415.
Mandels, M., R. Andreotti, and C. Roche. 1976. Measurement of saccharifying cellulose. Biotechnol. Bioeng. Symp. 6:21-33.
Mandels, M., and E. T. Reese. 1957. Induction of cellulase in Trichoderma Viride as influenced by carbon sources and metals. J. Bacteriol. 73:269-278.
Markossian, S., P. Becker, H. Märkl, and G. Antranikian. 2000. Isolation and characterization of lipid-degrading Bacillus thermoleovorans IHI-91 from an icelandic hot spring. Extremophiles 4:365-371.
Miller, G. L. 1959. Use of dinitrosalicylic as reagent for the determination of reducing sugars. Anal. Chem. 31:426-428.
Moo-Young, M., D. S. Chahal, J. E. Swan, and C. W. Robinson. 1977. SCP production by Chaetomium cellulolyticum, a new thermotolerant cellulolytic fungus. Biotechnol. Bioeng. 19:527-538.
Murai, T., M. Ueda, T. Kawaguchi, M. Arai, and A. Tanaka. 1998. Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing β-glucosidase and carboxymethylcellulase from Aspergillus aculeatus. Appl. Environ. Microbiol. 64:4857-4861.
Nazina, T. N., T. P. Tourova, A. B. Poltaraus, E. V. Novikova, A. A. Grigoryan, A. E. Ivanova, A. M. Lysenko, V. V. Petrunyaka, G. A. Osipov, S. S. Belyaev, and M. V. Ivanov. 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 thermoglucosidasius and Bacillus thermodenitrificans to Geobacillus as the new combinations G. stearothermophilus, G. thermocatenulatus, G. thermoleovorans, G. kaustophilus, G. thermoglucosidasius and G. thermodenitrificans. Int. J. Syst. Evol. Microbiol. 51:433-466.
Ng, T. K., and W. R. Kenealy. 1986. Industrial applications of thermostable enzymes, pp. 197-215. In T. D. Brock (ed.), Thermophiles: general, molecular, and applied microbiology. John Wiley & Sons, New York.
Ohtoko, K., M. Ohkuma, S. Moriya, T. Inoue, R. Usami, and T. Kudo. 2000. Diverse genes of cellulase homologues of glycosyl hydrolase family 45 from the symbiotic protists in the hindgut of the termite Reticulitermes speratus. Extremophiles 4:343-349.
Ooi, T., A. Shinmyo, H. Okada, S. Murao, T. Kawaguchi, and M. Arai. 1990. Complete nucleotide sequence of a gene coding for Aspergillus aculeatus cellulase (FI-CMCase). Nucleic. Acids. Res. 18:5884.
Ozturk, Z. N., and Z. B. Ogel. 2000. PCR with degenerate primers amplifies a subgenomic DNA fragment from the endoglucanase gene(s) of Torula thermophila, a thermophilic fungus. Mol. Biotechnol. 16:109-115.
Page, R. D. M. 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12:357-358.
Parry, N. J., D. E. Beever, E. Owen, W. Nerinckx, M. Claeyssens, J. Van Beeumen, and M. K. Bhat. 2002. Biochemical characterization and mode of action of a thermostable endoglucanase purified from Thermoascus aurantiacus. Arch. Biochem. Biophys. 404:243-253.
Perito, B., E. Hanhart, T. Irdani, M. Iqbal, A. J. McCarthy, and G. Mastromei. 1994. Characterization and sequence analysis of a Streptomyces rochei A2 endoglucanase-encoding gene. Gene 148:119-124
Peters, S., S. Koschinsky, F. Schwieger, and C. C. Tebbe. 2000. Succession of microbial communities during hot composting as detected by PCR-single-strand-conformation polymorphism-based genetic profiles of small-subunit rRNA genes. Appl. Environ. Microbiol. 66:930-936.
Philippidis, G. P., and C. Hatzis. 1997. Biochemical engineering analysis of critical process factors in the biomass-to-ethanol technology. Biotechnol. Prog. 13:222-231.
Poon, R., A. Yagminas, A. Singh, V. E. Valli, and I. Chu. 2001. Short-term oral toxicity of gasohol in female rats. J. Appl. Toxicol. 21:461-467.
Priest, F. G., M. Goodfellow, and C. Todd. 1988. A numerical classification of the genus Bacillus. J. Gen. Microbiol. 134:1847-1882.
Rixon, J. E., L. M. Ferreira, A. J. Durrant, J. I. Laurie, G. P. Hazelwood, and H. J. Gilbert. 1992. Characterization of the gene celD and its encoded product 1,4-beta-D-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa. Biochem. J. 285:947-955.
Russell, R. J., J. M. Ferguson, D. W. Hough, M. J. Danson, and G. L. Taylor. 1997. The crystal structure of citrate synthase from the hyperthermophilic archaeon Pyrococcus furiosus at 1.9 Å resolution. Biochemistry 36:9983–9994.
Ruttersmith, L. D., and R. M. Daniel. 1991. Thermostable cellobiohydrolase from the thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1. Purification and properties. Biochem. J. 277:887–890.
Ruttersmith, L. D., and R. M. Daniel. 1992. Cellulytic and hemicellulytic enzymes functional above 100 °C. Ann. NY Acad. Sci. 672:137–141.
Saarilahti, H. T., B. Henrissat, and E. T. Palva. 1990. CelS: a novel endoglucanase identified from Erwinia carotovora subsp. carotovora. Gene 90:9-14.
Sakamoto, S., G. Tamura, K. Ito, T. Ishikawa, K. Iwano, and N. Nishiya. 1995. Cloning and sequencing of cellulase cDNA from Aspergillus kawachii and its expression in Saccharomyces cerevisiae. Curr. Genet. 27:435-439.
San Martin, R., J. M. Aguilera, and A. I. Hohlberg. 1988. Effect of cellulase pretreatments on red algae agar extractability. Carbohydr. Polym. 8:33-43.
Schwarz, W. H. 2001. The cellulosome and cellulose degradation by anaerobic bacteria. Appl. Microbiol. Biotechnol. 56:634-649.
Siddiqui, K. S., A. A. Saqib, M. H. Rashid, and M. I. Rajoka. 2000. Carboxyl group modification significantly altered the kinetic properties of purified carboxymethylcellulase from Aspergillus niger. Enzyme Microb. Technol. 27:467-474.
Sneath, P. H. A. 1986. Endospore-forming gram-positive rods and cocci, pp. 1104-1207. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt (eds.), Bergey's manual of systematic determinative bacteriology, vol. 2. The Williams and Wilkins Co., Baltimore.
Sung, M.-H., H. Kim, J.-W. Bae, S.-K. Rhee, C. O. Jeon, K. Kim, J.-J. Kim, S.-P. Hong, S.-G. Lee, J.-H. Yoon, Y.-H. Park, and D.-H. Baek. 2002. Geobacillus toebii sp. nov., a novel thermophilic bacterium isolated from hay compost. Int. J. Syst. Evol. Microbiol. 52:2251-2255.
Sunna, A., S. Tokajian, J. Burghardt, F. Rainey, G. Antranikian, and F. Hashwa. 1997. Identification of Bacillus kaustophilus, Bacillus thermocatenulatus and Bacillus strain HSR as members of Bacillus thermoleovorans. Syst. Appl. Microbiol. 20:232-237.
Tai, S.-K., H.-P. P. Lin, J. Kuo, and J. K. Liu. Isolation and characterization of a cellulolytic Geobacillus thermoleovorans T4 strain from sugar refinery wastewater. Extremophiles（in press）
Takase, M., and K. Horikoshi. 1988. A thermostable β-glucosidase isolated from a bacterial species of the genus Thermus. Appl. Microbiol. Biotechnol. 29:55-60.
Tamaoka, J., and K. Komagata. 1984. Determination of DNA-base composition by reversed-phase high-performance liquid-chromatography. FEMS Microbiol. Lett. 25:125-128.
Teather, R. M., and P. J. Wood. 1982. Use of Congo red-polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43:777-780.
Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25:4876-4882.
Vieille, C., and G. J. Zeikus. 2001. Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65:1-43.
Wang, X., X. He, S. Yang, X. An, W. Chang, and D. Liang. 2003. Structural basis for thermostability of β-glycosidase from the thermophilic eubacterium Thermus nonproteolyticus HG102. J. Bacteriol. 185:4248-4255.
Weerkamp, A., and W. Heinen. 1972. Effect of the temperature on the fatty acid composition of the extreme thermophiles B. caldolyticus and B. caldotenax. J. Bacteriol. 109:443–446.
Wheals, A. E., L. C. Basso, D. M. G. Alves, and H. V. Amorim. 1999. Fuel ethanol after 25 years. Trends Biotechnol. 17:482-487.
White, D., R. J. Sharp, and F. G. Priest. 1993. A polyphasic taxonomic study of thermophilic bacilli from a wide geographical area. Antonie Van Leeuwenhoek 64:357-386
Wilmotte, A., G. Van der Auwera, and R. De Wachter. 1993. Structure of the 16S ribosomal RNA of the thermophilic cynobacterium Chlorogloeopsis HTF ('Mastigocladus laminosus HTF') strain PCC7518, and phylogenetic analysis. FEBS Lett. 317:96-100.
Wilson, D. B. 1992. Biochemistry and genetics of actinomycete cellulases. Crit. Rev. Biotechnol. 12:45-63.
Wittmann, S., F. Shareck, D. Kluepfel, and R. Morosoli. 1994. Purification and characterization of the CelB endoglucanase from Streptomyces lividans 66 and DNA sequence of the encoding gene. Appl. Environ. Microbiol. 60:1701-1703.
Wood, T. M., and M. K. Bhat. 1988. Methods for measuring cellulase activities. Methods Enzymol. 160:87-112.
Wood, W. E., D. G. Neubauer, and F. J. Stutzenberger. 1984. Cyclic AMP levels during induction and repression of cellulase biosynthesis in Thermomonospora curvata. J. Bacteriol. 160:1047-1054.
Wright, R. M., M. D. Yablonsky, Z. P. Shalita, A. K. Goyal, and D. E. Eveleigh. 1992. Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable β-glucosidase expressed in Escherichia coli. Appl. Environ. Microbiol. 58:3455-3465.
Yeoman, K. H., and C. Edward. 1994. Protease production by Streptomyces thermovulgaris grown on rapemeal-derived media. J. Appl. Bacteriol. 77:164-270.
Yumoto, I., A. Nakamura, H. Iwata, K. Kojima, K. Kusumoto, Y. Nodasaka, and H. Matsuyama. 2002. Dietzia psychralcaliphila sp. nov., a novel, facultatively psychrophilic alkaliphile that grows on hydrocarbons. Int. J. Syst. Evol. Microbiol. 52:85-90.
Zahran, H. H., A. M. Moharram, and H. A. Mohammad. 1992. Some ecological and physiological studies on bacteria isolated from salt-affected soils of Egypt. J. Basic Microbiol. 32:405-413.
Zarilla, K., and J. J. Perry. 1987. Bacillus thermoleovorans, sp. nov., a species of obligately thermophilic hydrocarbon utilizing endospore-forming bacteria. Syst. Appl. Microbiol. 9:258-264.