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1 pp. Organic solvent-tolerant enzymes Hiroyasu Ogino Osaka Prefecture University, Gakuencho, Naka-ku, Sakai, Osaka, Japan

2 Pseudomonas aeruginosa PST- Pseudomonas aeruginosa LST- P. aeruginosa PST- Pseudomonas P. aeruginosa LST- P. aeruginosa PST- P. aeruginosa LST- LST- DMSO

3 C P. putida; Pseudomonas putida, P. fluorescencs; Pseudomonas fluorescencs, P. chlororaphis; Pseudomonas chlororaphis, P. flagi; Pseudomonas flagi, E. coli; Escherichia coli, M. luteus; Micrococcus luteus, B. subtilis; Bacillus subtilis. PST- α DMSO - tert- - DMF - -

4 P. aeruginosa LST- DNA Lip Lip LST- P. aeruginosa PST- PST- PST- C α- CD PST- CD CD α- CD CD CD CD α CD β CD PST- α β PST- PST- PST- C Cys- Cys- PST- N Cys- Cys- PST- Cys- Cys- PST- PST- PST- PST- α- CD C CD

5 CD C CD LST- DMSO LST- PST- DMSO PST- DMSO Cbz -Arg Leu-NH Cbz-Arg-Leu-NH PST- Cbz-Arg-Leu-NH DMSO PST- PST- PST- PST-

6 1 1 1 4 生化学 第8 1巻 第1 2号 図5 PST-0 1プロテアーゼとサーモライ シンの立体構造 全体構造をリボンモデル 活性中心とジ スルフィド結合を形成するアミノ酸を ボールアンドスティックモデル 酵素に 配位している亜鉛イオン 小 とカルシ ウムイオン 大 を充填モデルで示す 図6 有機溶媒耐性が変化した変異 PST-0 1プロテアーゼの変異箇所 全体構造をワイヤーモデル 変異したアミノ酸残基を充填モデルで示す

7 LST- LST- DMSO LST--R LST--R SK TF SR SL GR GV SR SW DN LST--R SL LST--R LH PST- PST- Cbz-Arg-Leu-NH mm Cbz-Arg mm Leu-NH ph C PST- mm Cbz-Asp mm Phe-OMe ph C

8 PST- subsite PST- mm Cbz-Asp mm Phe-OMe ph C Ogino, H.in Protein Adaptation in Extremophiles Siddiqui, K.S. & Thomas, T. eds., pp., Nova Science Publishers, Inc., New York. Ogino, H. & Ishikawa, H.J. Biosci. Bioeng.,,. Zaks, A. & Klibanov, A.M.Proc. Natl. Acad. Sci. USA,,. pp. pp. Ogino, H., Miyamoto, K., & Ishikawa, H.Appl. Environ. Microbiol.,,. Ogino, H., Yasui, K., Shiotani, T., Ishihara, T., & Ishikawa, H. Appl. Environ. Microbiol.,,. Ogino, H., Yasui, K., Watanabe, F., & Ishikawa, H. Ann. NY. Acad. Sci.,,. Ogino, H., Miyamoto, K., Yasuda, M., Ishimi, K., & Ishikawa, H.Biochem. Eng. J.,,. Ito, T., Kikuta, H., Nagamori, E., Honda, H., Ogino, H., Ishikawa, H., & Kobayashi, T.J. Biosci. Bioeng.,,. Ogino, H., Nakagawa, S., Shinya, K., Muto, T., Fujimura, N., Yasuda, M., & Ishikawa, H.J. Biosci. Bioeng.,,.

9 Pseudomonas aeruginosa LST- Cryptococcus sp. S- Rhizopus oryzae Burkholderia cepacia AKU- Streptomyces rimosus R -W Kurtzmanomyces sp. I- Bacillus sphaericus y Pseudomonas sp. S Bacillus megaterium CCOC-P Bacillus thermoleovorans CCR Penicillium chrysogenum Pseudomonas aeruginosa MTCC Bacillus sp. Pseudomonas aeruginosa san-ai Pyrobaculum calidifontis VA Bacillus licheniformis S- Bacillus sp. Sulfolobus solfataricus P Pseudomonas aeruginosa PST- Lumbricus rubellus Bacillus cereus BG Pseudomonas aeruginosa strain K Pseudomonas aeruginosa san-ai Bacillus licheniformis RSP-- Bacillus sp. APR- Pseudomonas aeruginosa PD Pseudomonas aeruginosa PseA a gamma-proteobacterium DGII Pseudomonas aeruginosa B- Bacillus cereus Aquifex aeolicus L Fasciola hepatica α- Haloarcula sp. S- Brachybacterium sp. strain LB Rhodococcus ruber DSM α- Streptomyces thermocyaneoviolaceus IFO Burkholderia cepacia ST- Chromobacterium sp. DS- Elaies guineensis Streptomyces sp. REN- Staphylococcus warneri ISK- Pyrococcus furiosus Paenibacillus illinoisensis ST- K Bacillus sp. BC Ogino, H., Katou, Y., Akagi, R., Mimitsuka, T., Hiroshima, S., Gemba, Y., Doukyu, N., Yasuda, M., Ishimi, K., & Ishikawa, H.Extremophiles,,. Ogino, H., Inoue, S., Akagi, R., Yasuda, M., Doukyu, N., & Ishimi, K.Biochem. Eng. J.,,. Ogino, H., Hiroshima, S., Hirose, S., Yasuda, M., Ishimi, K., & Ishikawa, H.Mol. Genet. Genomics,,. Ogino, H., Mimitsuka, T., Muto, T., Matsumura, M., Yasuda, M., Ishimi, K., & Ishikawa, H.J. Mol. Microbiol. Biotechnol.,,. Ogino, H., Watanabe, F., Yamada, M., Nakagawa, S., Hirose, T., Noguchi, A., Yasuda, M., & Ishikawa, H.J. Biosci. Bioeng.,,. Ogino, H., Gemba, Y., Yutori, Y., Doukyu, N., Ishimi, K., & Ishikawa, H.Biotechnol. Prog.,,. Ogino, H., Yokoo, J., Watanabe, F., & Ishikawa, H. Biochem. Eng. J.,,. Ogino, H., Uchiho, T., Yokoo, J., Kobayashi, R., Ichise, R., & Ishikawa, H.Appl. Environ. Microbiol.,,. Ogino, H., Uchiho, T., Doukyu, N., Yasuda, M., Ishimi, K., & Ishikawa, H.Biochem. Biophys. Res. Commun.,,. Kawata, T. & Ogino, H.Biotechnol. Prog., in press. Ogino, H., Gemba, Y., Yamada, M., Shizuka, M., Yasuda, M., & Ishikawa, H.Biochem. Eng. J.,,. Ogino, H., Yamada, M., Watanabe, F., Ichinose, H., Yasuda,

10 M., & Ishikawa, H.J. Biosci. Bioeng.,,. Bobe, I.M., Abdelmoez, W., Ogino, H., Yasuda, M., Ishimi, K., & Ishikawa, H.Biotechnol. Bioeng.,,. Tsuchiyama, S., Doukyu, N., Yasuda, M., Ishimi, K., & Ogino, H.Biotechnol. Prog.,,. Kamini, N.R., Fujii, T., Kurosu, T., & Iefuji, H.Process Biochem.,,. Hiol, A., Jonzo, M.D., Rugani, N., Druet, D., Sarda, L., & Comeau, L.C.Enzyme Microb. Technol.,,. Ishimoto,R.,Sugimoto,M.,&Kawai,F.FEMS Microbiol. Lett.,,. Lescic, I., Vukelic, B.,Majeric -Elenkov, M., Saenger, W., & Abramic, M.Enzyme Microb. Technol.,,. Kakugawa, K., Shobayashi, M., Suzuki, O., & Miyakawa, T. Biosci. Biotechnol. Biochem.,,. Hun, C.J., Rahman, R.N.Z.A., Salleh, A.B., & Basri, M. Biochem. Eng. J.,,. Rahman, R.N.Z.A., Chin, J.H., Salleh, A.B., & Basri, M. Mol. Gen. Genomics,,. Baharum, S.N., Salleh, A.B., Razak, C.N.A., Basri, M., Rahman, M.B.A., & Rahman, R.N.Z.R.A.Ann. Microbiol.,,. Rahman, R.N.Z.R.A., Baharum, S.N., Basri, M., & Salleh, A.B. Anal. Biochem.,,. Lima, V.M.G., Krieger, N., Mitchell, D.A., Baratti, J.C., de Filippis, I., & Fontana, J.D.J. Mol. Catal B: Enzym.,,. Castro-Ochoa, L.D., Rodrl guez-gómez, C., Valerio-Alfaro, G., &Ros,R.O.Enzyme Microb. Technol.,,. Bancerz, R., Ginalska, G., Fiedurek, J., & Gromada, A. J. Ind. Microbiol. Biotechnol.,,. Singh, S. & Banerjee, U.C.J. Mol. Catal. B: Enzym.,,. Eltaweel, M.A., Rahman, R.N.Z.R.A., Salleh, A.B., & Basri, M.Ann. Microbiol.,,. Karadzic, I., Masui, A., Zivkovic, L.I., & Fujiwara, N. J. Biosci. Bioeng.,,. Hotta, Y., Ezaki, S., Atomi, H., & Imanaka, T.Appl. Environ. Microbiol.,,. Torres, S. & Castro, G.R.New horizons in biotechnology, pp., Kluwer Academic Publishers, Dordrecht. Torresa, S., Baigorl, M.D., & Castro, G.R.Process Biochem.,,. Karpushova, A., Brümmer, F., Barth, S., Lange, S., & Schmid, R.D.. Appl. Microbiol. Biotechnol.,,. Park, Y.-J., Choi, S.Y., & Lee, H.-B.Biochim. Biophys. Acta,,. Nakajima, N, Sugimoto, M, & Ishihara, K.J. Biosci. Bioeng.,,. Ghorbel, B., Sellami-Kamoun, A., & Nasri, M.Enzyme Microb. Technol.,,. Geok, L.P., Razak, C.N.A., Rahman, R.N.Z.A., Basri, M., & Salleh, A.B.Biochem. Eng. J.,,. Rahman, R.N.Z.R.A., Geok, L.P., Basri, M., & Salleh, A.B. Enzyme Microb. Technol.,,. Karadzic, I., Masui, A., & Fujiwara, N.J. Biosci. Bioeng.,,. Sareen, R., Bornscheuer, U.T., & Mishra, P.J. Mol. Catal. B: Enzym.,,. Sareen, R., Bornscheuer, U.T., & Mishra, P.Biotechnol. Lett.,,. Kumar, D. & Bhalla, T.C.Indian J. Exp. Biol.,,. Najafi, M.F., Deobagkar, D., & Deobagkar, D.Electr. J. Biotechnol.,,. Gupta, A., Roy, I., Khare, S.K., & Gupta, M.N.J. Chromatogr. A,,. Gupta, A. & Khare, S.K.Bioresource Technol.,,. Sana, B., Ghosh, D., Saha, M., & Mukherjee, J.Process Biochem.,,. Khan, S., Misra, A.K., Tripathi, C.K.M., Mishra, B.N., & Bihari, V.Indian J. Exp. Biol.,,. Shafee, N., Tan, C.-C., Mahamad, S., Rahman, R.N.Z.A., Basri, M., & Salleh, A.B.Ann. Microbiol.,,. Khan, A.R., Nirasawa, S., Kaneko, S., Shimonishi, T., & Hayashi, K.Enzyme Microb. Technol.,,. Dowd, A.J., Dooley, M., Fagain, C., & Dalton, J.P.Enzyme Microb. Technol.,,. Fukushima, T., Mizuki, T., Echigo, A., Inoue, A., & Usami, R. Extremophiles,,. Doukyu, N., Yamagishi, W., Kuwahara, H., Ogino, H., & Furuki, N.Extremophiles,,. Doukyu, N., Yamagishi, W., Kuwahara, H., & Ogino, H. Biosci. Biotechnol. Biochem.,,. Kosjek, B., Stampfer, W., Pogorevc, M., Goessler, W., Faber, K., & Kroutil, W.Biotechnol. Bioeng.,,. Ishihara, K., Yamaguchi, H., Hamada, H., Nakamura, K., & Nakajima, N.J. Mol. Catal. B-Enzym.,,. Doukyu, N. & Aono, R.Appl. Microbiol. Biotechnol.,,. Takeda, Y., Aono, R., & Doukyu, N.Extremophiles,,. Doukyu, N., Shibata, K., & Ogino, H.Appl. Microbiol. Biotechnol.,,. Doukyu, N., Shibata, K., Ogino, H., & Sagermann, M. Appl. Microbiol. Biotechnol.,,. Sakharov, I.Yu., Castillo, J., Areza, J.C., & Galaev, I.Yu. Bioseparation,,. Rodakiewicz-Nowak, J. & Ito, M.J. Chem. Technol. Biotechnol.,,. Mizuno, K., Fukuda, D., Kakihara, M., Kohno, M., Ha, T.L., Sonomoto, K., & Ishizaki, A.Food Sci. Technol. Res.,,. Cacciapuoti, G., Bertoldo, C., Brio, A., Zappia, V., & Porcelli, M.Extremophiles,,. Doukyu, N., Kuwahara, H., & Aono, R. Biosci. Biotechnol. Biochem.,,. Sardessai, Y. & Bhosle, S.Mar. Biotechnol.,,.

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