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Biohydrogen production in Escherichiacoli BL21 using H2 producing ability of recombinant E. coli NiFe-hydrogenase 1
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S165–S176 The odor learning of mice was carried out to mixed odor (isoamyl acetate + citral + linalool solved mineral oil) by the Y-maze assay. Learned mixed odor, unexperienced mixed odor (geraniol + ethyl butyrate + methyl caproate solved mineral oil) and mineral oil were exposed to the mice before or after the learning, respectively. The brain was isolated and divided in seven parts. The sample was dissolved by sonication and phosphorylation ratio of MAPK in each part was measured by dot blotting. Before learning, the phosphorylation ratios in olfactory bulb of the mice exposed to these three odors were not significantly different. Through the odor learning, those of the mice exposed to the learned mixed odor and mineral oil were elevated, whereas the phosphorylation ratio of the mice exposed to the unexperienced mixed odor was not elevated. Mineral oil was the solvent of learned mixed odor and thought to be experienced odor. That is, phosphorylation ratios were change after odor learning by exposure to only experienced odors. These results indicate that the modification to form the selective attention might be occurred in the initial stage of olfactory information processing system and that this modification does not influence on the discrimination of unexperienced odor. doi:10.1016/j.jbiosc.2009.08.457
SB-P1 Biohydrogen production in Escherichiacoli BL21 using H2 producing ability of recombinant E. coli NiFe-hydrogenase 1 Jaoon Y.H. Kim,1 Byung Hoon Jo,2 Young Hwa Cho,1 and Hyung Joon Cha1 Pohang University of Science and Technology, Department of Chemical Engineering, Pohang, Republic of Korea 1 and Pohang University of Science and Technology, School of Interdisciplinary Bioscience and Bioengineering, Pohang, Republic of Korea 2 By homologous expression of hyaA, hyaB genes respectively encoding small and large subunit of Escherichia coli NiFe-hydrogenase 1 core enzyme, the possibility of the enzyme for biological hydrogen production was reinvestigated. E. coli BL21 strain where H2 producing ability is impaired was used as a recombinant host. The recombinant BL21 overexpressing hydrogenase 1 showed active hydrogen production in contrast to wild type BL21 which did not produce. Largely different than notoriously oxygen sensitive FeFehydrogenase, the recombinant strain produced hydrogen gas at about 12.5 mL H2/h·L culture in 400 ml glucose minimal media under microaerobic condition. Through cell fractionation and protein localization analysis, we found that most of the recombinant protein was produced aggregated as inclusion body and it seems that only a tiny fraction was translocated to the membrane fraction. In terms of in vitro enzyme activity, however, the membrane fraction showed high H2 producing activity comparable to the total fraction based on unit protein. The enzyme was purified using 6 His-tag under ambient aerobic condition and it showed relatively oxygen-tolerant activity of 12.2 nmol H2/min·mg protein. Formate effect on H2 production was also studied. Addition of formate into culture media had high positive effect on the amount of hydrogen production by recombinant BL21, but it did not exert any effect on wild type BL21. This means that the formate effect is not directly related to FHL complex. Finally, effects of metal contents on in vivo/ in vitro H2 production were analyzed. We could find out that the enzyme has a considerable activity even when there is only iron or nickel in the media although the activity level remains 10–20k of
S169
that of when both Ni, Fe exist. This could imply one of the metals can substitute for the other to a certain extent. doi:10.1016/j.jbiosc.2009.08.458
SB-P2 High constitutive trehalose is required for resistance to multiple stresses in Saccharomyces cerevisiae Siraje Arif Mahmud, Takashi Hirasawa, and Hiroshi Shimizu Osaka University, Suita, Osaka, Japan Trehalose is believed to play an important role in the stress tolerance in the yeast Saccharomyces cerevisiae. Our previous study (1) indicated that trehalose accumulation before saline stress gives growth advantage in yeast. In this research, the responses to various other environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses were investigated in the triple deletion strain of NTH1, NTH2 and ATH1, the genes encoding trehalases that are involved in the trehalose degradation and the triple deletion strain overexpressing TPS1 or TPS2 encoding trehalose biosynthesis enzymes in S. cerevisiae. The level of constitutive trehalose accumulation in the TPS1- or TPS2-overexpressed triple deletion strain was higher than that in the original triple deletion strain. For heat, ethanol or oxidative stress conditions, yeast cells were grown up to the mid-log phase and then were subjected to these stresses. The response to these stresses in the yeast strains was evaluated by the specific growth rate after stress provocation. For freezing stress, cells collected at the mid-log phase were kept at -20°C for 1 week and the surviving cells were counted against the initial population to evaluate growth. The TPS2-overexpressed triple deletion strain showed high growth activity under ethanol stress condition. Moreover, the TPS1- and TPS2-overexpressed triple deletion strains also showed tolerance to high temperatures. In case of freezing stress, all the recombinant strains with high trehalose content showed high tolerance. On the other hand, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicate that high accumulation of trehalose can make yeast cells resistant to multiple stresses except for oxidative stress. Reference 1. Mahmud, S.A., Nagahisa, H., Hirasawa, T., Yoshikawa, K., Ashitani, K., and Shimizu H.: Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae. Yeast, 26, 17-30 (2009).
doi:10.1016/j.jbiosc.2009.08.459
SB-P3 Dynamic modeling of the main metabolism of Escherichia coli for the specific gene knockouts in batch and continuous culture Tuty Asmawaty Abdul Kadir,1 and Kazuyuki Shimizu1,2 Dept. of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan, 1 and Institute for Advanced Bioscience, Keio University, Yamagata, Japan, Japan 2
SB-P1 Biohydrogen production in Escherichiacoli BL21 using H2 producing ability of recombinant E. coli NiFe-hydrogenase 1 Jaoon Y.H. Kim,1 Byung Hoon Jo,2 Young Hwa Cho,1 and Hyung Joon Cha1 Pohang University of Science and Technology, Department of Chemical Engineering, Pohang, Republic of Korea 1 and Pohang University of Science and Technology, School of Interdisciplinary Bioscience and Bioengineering, Pohang, Republic of Korea 2 By homologous expression of hyaA, hyaB genes respectively encoding small and large subunit of Escherichia coli NiFe-hydrogenase 1 core enzyme, the possibility of the enzyme for biological hydrogen production was reinvestigated. E. coli BL21 strain where H2 producing ability is impaired was used as a recombinant host. The recombinant BL21 overexpressing hydrogenase 1 showed active hydrogen production in contrast to wild type BL21 which did not produce. Largely different than notoriously oxygen sensitive FeFehydrogenase, the recombinant strain produced hydrogen gas at about 12.5 mL H2/h·L culture in 400 ml glucose minimal media under microaerobic condition. Through cell fractionation and protein localization analysis, we found that most of the recombinant protein was produced aggregated as inclusion body and it seems that only a tiny fraction was translocated to the membrane fraction. In terms of in vitro enzyme activity, however, the membrane fraction showed high H2 producing activity comparable to the total fraction based on unit protein. The enzyme was purified using 6 His-tag under ambient aerobic condition and it showed relatively oxygen-tolerant activity of 12.2 nmol H2/min·mg protein. Formate effect on H2 production was also studied. Addition of formate into culture media had high positive effect on the amount of hydrogen production by recombinant BL21, but it did not exert any effect on wild type BL21. This means that the formate effect is not directly related to FHL complex. Finally, effects of metal contents on in vivo/ in vitro H2 production were analyzed. We could find out that the enzyme has a considerable activity even when there is only iron or nickel in the media although the activity level remains 10–20k of
S169
that of when both Ni, Fe exist. This could imply one of the metals can substitute for the other to a certain extent. doi:10.1016/j.jbiosc.2009.08.458
SB-P2 High constitutive trehalose is required for resistance to multiple stresses in Saccharomyces cerevisiae Siraje Arif Mahmud, Takashi Hirasawa, and Hiroshi Shimizu Osaka University, Suita, Osaka, Japan Trehalose is believed to play an important role in the stress tolerance in the yeast Saccharomyces cerevisiae. Our previous study (1) indicated that trehalose accumulation before saline stress gives growth advantage in yeast. In this research, the responses to various other environmental stresses, such as high ethanol concentration, heat, oxidative, and freezing stresses were investigated in the triple deletion strain of NTH1, NTH2 and ATH1, the genes encoding trehalases that are involved in the trehalose degradation and the triple deletion strain overexpressing TPS1 or TPS2 encoding trehalose biosynthesis enzymes in S. cerevisiae. The level of constitutive trehalose accumulation in the TPS1- or TPS2-overexpressed triple deletion strain was higher than that in the original triple deletion strain. For heat, ethanol or oxidative stress conditions, yeast cells were grown up to the mid-log phase and then were subjected to these stresses. The response to these stresses in the yeast strains was evaluated by the specific growth rate after stress provocation. For freezing stress, cells collected at the mid-log phase were kept at -20°C for 1 week and the surviving cells were counted against the initial population to evaluate growth. The TPS2-overexpressed triple deletion strain showed high growth activity under ethanol stress condition. Moreover, the TPS1- and TPS2-overexpressed triple deletion strains also showed tolerance to high temperatures. In case of freezing stress, all the recombinant strains with high trehalose content showed high tolerance. On the other hand, in case of oxidative stress, trehalose accumulation could not make the yeast cells tolerant. Our results indicate that high accumulation of trehalose can make yeast cells resistant to multiple stresses except for oxidative stress. Reference 1. Mahmud, S.A., Nagahisa, H., Hirasawa, T., Yoshikawa, K., Ashitani, K., and Shimizu H.: Effect of trehalose accumulation on response to saline stress in Saccharomyces cerevisiae. Yeast, 26, 17-30 (2009).
doi:10.1016/j.jbiosc.2009.08.459
SB-P3 Dynamic modeling of the main metabolism of Escherichia coli for the specific gene knockouts in batch and continuous culture Tuty Asmawaty Abdul Kadir,1 and Kazuyuki Shimizu1,2 Dept. of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Fukuoka, Japan, 1 and Institute for Advanced Bioscience, Keio University, Yamagata, Japan, Japan 2