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O microemulsion and influence of cholesterol addition
Abstracts / Journal of Bioscience and Bioengineering 108 (2009) S96–S113 through the whole tank, this result is due to geometrical similarity of both the tanks. References 1. Bornscheuer, U.T.: Enzymes in lipid modification, Wiley-VCH Publication, (2000). 2. Lius, K.: Soybeans: Chemistry, technology and utilization, Kluwer Academic Publisher (1997). 3. Patill, N.S., Ghadge, R. S., Sawant, S. B., and Joshi, J. B.: Lipase deactivation at gasliquid interface and its subsequent reactivation. AIChE J. 6, 46, 1280-1283 (2000).
doi:10.1016/j.jbiosc.2009.08.311
EP-P24 Hydrophobicity of micro water pool of lecithin based W/O microemulsion and influence of cholesterol addition Yoshie Shirakawa, Masanao Imai, and Isao Suzuki Nihon University, Graduate School of Bioresource Sciences, Course in Bioresource Utilization Sciences, 1866 Kameino, Fujisawa, Kanagawa-pref., Japan Lecithin based W/O microemulsion has a high potential to develop a biocompatible drag delivery system, release control carrier and enzyme reaction media of hydrophobic substrate. This paper demonstrated that the effect of water content of lecithin based W/O microemulsion on the hydrophobicity of micro water pool and the fluidity of micro-interface based on fluorometric measurement. The water content was referred as W value. It indicated the molar ratio of water to lecithin. It was almost constant in lower additive cholesterol concentration less than 0.776 mM. In higher concentration range, the water content was gradually decayed with increasing cholesterol concentration. The hydrophobicity of micro water pool was evaluated from the shift of maximum wavelength of Coumarin 343. In less than 10 of W value, the hydrophobicity of micro water pool was increased by small amount of additive cholesterol (0.026 mM), while the lecithin concentration was set at 4.6 mM. The hydrophobicity of micro water pool was almost constant in more than 10 of W value. In this range of W, the effect of additive cholesterol was negligible small. The fluidity of micro-interface was estimated from fluorescence polarity of TMA-DPH. It was decayed with increasing of water content. This trend was commonly appeared in both cholesterol presence system or not. At a same water concentration system, the fluidity was slightly elevated by additive cholesterol. Additive cholesterol was a key factor to govern the formation of lecithin based W/O microemulsion and the control of hydrophobicity of water pool. doi:10.1016/j.jbiosc.2009.08.312
EP-P25 Enzymatic synthesis of peptides in ionic liquids Hidetaka Noritomi,1 Katsuyuki Suzuki,1 Manabu Kikuta,2 and Satoru Kato1 Tokyo Metropolitan University, Hachioji, Tokyo, Japan 1 and Dai-Ichi Kogyo Seiyaku Co., Ltd., Minami-ku, Kyoto, Japan 2
S107
The peptide is one of the most important biomaterials in the pharmaceutical and food fields. Peptide synthesis is carried out by several technologies such as chemical synthesis, enzymatic synthesis, and recombinant DNA technology. On the other hand, the biotransformation has recently become more attractive accompanying with an increase in the importance of the development of environmentallybenign processes to the organic synthesis, since enzymes are biodegradable and non-toxic catalysts, and exhibit high activity and specificity. Proteases have been used as a biocatalyst in order to carry out peptide synthesis in nonaqueous media. In the present work, peptides were synthesized from N-acetyl-L-tryptophan ethyl ester and glycyl glycinamide by α-chymotrypsin in ionic liquids. The initial rates of peptide synthesis and hydrolysis were strongly influenced by the water content when 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) was used as a solvent. The activity of αchymotrypsin in ionic liquids changed with a kind of anions consisting of the same cation. The peptide synthesis in [emim][FSI] was much superior to that in conventional organic solvents such as acetonitrile and tetrahydrofuran. The reactivity in ionic liquids was influenced by the reaction temperature, and was similar to that in organic solvents, and tended to decrease with an increase in temperature. However, the activity of α-chymotrypsin in ionic liquids was highly retained compared to that in organic solvents at high temperatures, and the productivity of peptides in [emim][FSI] and [emim][PF6] was observed even at 80 °C. doi:10.1016/j.jbiosc.2009.08.313
EP-P26 Site-specific protein modification with functional molecule using novel enzyme Ryosuke Takase,1 Tsutomu Tanaka,2 and Akihiko Kondo1 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan 1 and Organization of Advanced Science and Technology, Kobe University, Kobe, Japan 2 Proteins have been widely used for various kinds of field. However, we cannot always get our desired proteins having our desired functions. Therefore, it is significantly important to develop novel protein engineering method to improve protein function. Enzymatic protein modification method is one of the widely used ones for protein engineering. The substrate sequence of enzyme is introduced target protein as a tag, and functional molecule can be modified sitespecifically due to the substrate specificity of enzyme. This strategy has advantage that we can modify functional molecule to target protein without impairing protein function. For example, SortaseA from Staphylococcus aureus recognizes LPXTG substrate sequence, and cleaves between T and G. Then SortaseA conjugates between Cterminal Thr and N-terminal triglycine sequence. However, there are only a few enzymes useful for protein modification, hence we screened novel enzymes useful for protein modification. In this study, we expressed Sortase (Sortase-LPW) from Lactobacillus plantarum NCIMB8826 using E. coli as a host. We use LPQTSEQtagged EGFP as a model protein and we used Amine-PEO2-Biotin that has amino group at its N-terminal as a model functional molecule. We mixed tagged GFP, Amine-PEO2-biotin and sortase-LPW then incubated at 37 °C overnight. Then we identified conjugated biotin by western blotting. Amine-biotin was successfully modified with the tagged EGFP, on the other hand, GFP without tag sequence and biotin without amino group were not modified. From the results, we succeed
doi:10.1016/j.jbiosc.2009.08.311
EP-P24 Hydrophobicity of micro water pool of lecithin based W/O microemulsion and influence of cholesterol addition Yoshie Shirakawa, Masanao Imai, and Isao Suzuki Nihon University, Graduate School of Bioresource Sciences, Course in Bioresource Utilization Sciences, 1866 Kameino, Fujisawa, Kanagawa-pref., Japan Lecithin based W/O microemulsion has a high potential to develop a biocompatible drag delivery system, release control carrier and enzyme reaction media of hydrophobic substrate. This paper demonstrated that the effect of water content of lecithin based W/O microemulsion on the hydrophobicity of micro water pool and the fluidity of micro-interface based on fluorometric measurement. The water content was referred as W value. It indicated the molar ratio of water to lecithin. It was almost constant in lower additive cholesterol concentration less than 0.776 mM. In higher concentration range, the water content was gradually decayed with increasing cholesterol concentration. The hydrophobicity of micro water pool was evaluated from the shift of maximum wavelength of Coumarin 343. In less than 10 of W value, the hydrophobicity of micro water pool was increased by small amount of additive cholesterol (0.026 mM), while the lecithin concentration was set at 4.6 mM. The hydrophobicity of micro water pool was almost constant in more than 10 of W value. In this range of W, the effect of additive cholesterol was negligible small. The fluidity of micro-interface was estimated from fluorescence polarity of TMA-DPH. It was decayed with increasing of water content. This trend was commonly appeared in both cholesterol presence system or not. At a same water concentration system, the fluidity was slightly elevated by additive cholesterol. Additive cholesterol was a key factor to govern the formation of lecithin based W/O microemulsion and the control of hydrophobicity of water pool. doi:10.1016/j.jbiosc.2009.08.312
EP-P25 Enzymatic synthesis of peptides in ionic liquids Hidetaka Noritomi,1 Katsuyuki Suzuki,1 Manabu Kikuta,2 and Satoru Kato1 Tokyo Metropolitan University, Hachioji, Tokyo, Japan 1 and Dai-Ichi Kogyo Seiyaku Co., Ltd., Minami-ku, Kyoto, Japan 2
S107
The peptide is one of the most important biomaterials in the pharmaceutical and food fields. Peptide synthesis is carried out by several technologies such as chemical synthesis, enzymatic synthesis, and recombinant DNA technology. On the other hand, the biotransformation has recently become more attractive accompanying with an increase in the importance of the development of environmentallybenign processes to the organic synthesis, since enzymes are biodegradable and non-toxic catalysts, and exhibit high activity and specificity. Proteases have been used as a biocatalyst in order to carry out peptide synthesis in nonaqueous media. In the present work, peptides were synthesized from N-acetyl-L-tryptophan ethyl ester and glycyl glycinamide by α-chymotrypsin in ionic liquids. The initial rates of peptide synthesis and hydrolysis were strongly influenced by the water content when 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) was used as a solvent. The activity of αchymotrypsin in ionic liquids changed with a kind of anions consisting of the same cation. The peptide synthesis in [emim][FSI] was much superior to that in conventional organic solvents such as acetonitrile and tetrahydrofuran. The reactivity in ionic liquids was influenced by the reaction temperature, and was similar to that in organic solvents, and tended to decrease with an increase in temperature. However, the activity of α-chymotrypsin in ionic liquids was highly retained compared to that in organic solvents at high temperatures, and the productivity of peptides in [emim][FSI] and [emim][PF6] was observed even at 80 °C. doi:10.1016/j.jbiosc.2009.08.313
EP-P26 Site-specific protein modification with functional molecule using novel enzyme Ryosuke Takase,1 Tsutomu Tanaka,2 and Akihiko Kondo1 Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, Kobe, Japan 1 and Organization of Advanced Science and Technology, Kobe University, Kobe, Japan 2 Proteins have been widely used for various kinds of field. However, we cannot always get our desired proteins having our desired functions. Therefore, it is significantly important to develop novel protein engineering method to improve protein function. Enzymatic protein modification method is one of the widely used ones for protein engineering. The substrate sequence of enzyme is introduced target protein as a tag, and functional molecule can be modified sitespecifically due to the substrate specificity of enzyme. This strategy has advantage that we can modify functional molecule to target protein without impairing protein function. For example, SortaseA from Staphylococcus aureus recognizes LPXTG substrate sequence, and cleaves between T and G. Then SortaseA conjugates between Cterminal Thr and N-terminal triglycine sequence. However, there are only a few enzymes useful for protein modification, hence we screened novel enzymes useful for protein modification. In this study, we expressed Sortase (Sortase-LPW) from Lactobacillus plantarum NCIMB8826 using E. coli as a host. We use LPQTSEQtagged EGFP as a model protein and we used Amine-PEO2-Biotin that has amino group at its N-terminal as a model functional molecule. We mixed tagged GFP, Amine-PEO2-biotin and sortase-LPW then incubated at 37 °C overnight. Then we identified conjugated biotin by western blotting. Amine-biotin was successfully modified with the tagged EGFP, on the other hand, GFP without tag sequence and biotin without amino group were not modified. From the results, we succeed