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Production and two phase purification of catalase from Phanerochaete chrysosporium
New Biotechnology · Volume 29S · September 2012
Poster 1.2.08 Cellooligosaccharide assimilating E. coli by displaying active BGL on the cell surface using novel anchor protein Tsutomu Kondo
Tanaka ∗ , Yuuki
Hirata, Hitomi
Kawabata, Akihiko
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan We demonstrated direct assimilation of cellobiose using betaglucosidase (BGL)-displaying E. coli. After screening active BGLs, Tfu0937, which is a BGL from Thermobifida fusca YX, was successfully displayed on the E. coli cell surface and directly assimilated cellobiose as a carbon source. Then, we screened for a suitable anchor protein for BGL cell-surface display and found a novel anchor protein, Blc, which allowed for both N- and C-terminal fusion of Tfu0937. The pNPG activity of Tfu-0937 using Blc as an anchor protein was significantly improved, up to 7535 U/O.D. 600/ml, which is approximately 65-fold higher than using PgsA as the anchor protein. Finally, Tfu0937-displaying E. coli JCM20137 was successfully grown on 0.2% cellobiose, and the O.D. 600 was 1.05 after 20 h, which is almost the same rate of growth on 0.2% glucose after 16 h. Tfu0937-displaying E. coli JCM20137 was also successfully grown on 0.2% cellotriose, cellotetraose, cellopentaose and cellohexaose. http://dx.doi.org/10.1016/j.nbt.2012.08.140 Poster 1.2.09 Production and two phase purification of catalase from Phanerochaete chrysosporium Berna Kavakc¸ıo˘ glu ∗ , Leman Tarhan Dokuz Eylul University, Science Faculty, Chemistry Department, Turkey Catalase (EC 1.11.1.6) is an enzyme primarily found in the peroxisomes of nearly all aerobic and certain anaerobic cells, and serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without production of free radicals [1]. Catalase has useful applications in various fields such as food, textile and even medicine [2]. For these reasons, production and purification of catalase with cheap and simple ways is significant to meet the demand. Within the context of this study, the optimization of the culture conditions of white rot fungus Phanerochaete chrysosporium to obtain maximum catalase activity and then purification of the enzyme by using aqueous two phase systems (ATPSs) were carried out. For the optimization studies, five separate solid and two different liquid mediums were investigated. Maximum catalase activity as 238.56 U/mg was observed in the extract obtained by using PDA as a solid medium with the combination of carbon-nitrogen restricted liquid medium. As for that the purification procedure, various polyethylene glycol (PEG)-phosphate salt (K2 HPO4 ) ATPSs were evaluated in terms of partition parameters including partition coefficient of the target enzyme (Ke ) and the total protein (Kp ), purification fold (PF), yield (Y%) and recovery (R%). The best results were obtained in the PEG 1000 18%–K2 HPO4 12% (pH 8.2)
system. For this system, Ke and Kp values were determined as 10.55 and 0.21, respectively while %Y was found as 91.95% and the purity of the enzyme was increased to 5.72 with the %R of 81.96%. Keywords: Phanerochaete chrysosporium; Catalase; Aqueous two phase systems
References 1. Ran J, et al. A facile method for improving the covalent crosslinking adsorption process of catalase immobilization. Bioresour Technol 2010;101:6285–90. 2. Akertek E, Tarhan L. Characterization of immobilized catalase and their application in pasteurization of milk with H2 O2 . Appl Biochem Biotechnol 1995;50:291–303.
http://dx.doi.org/10.1016/j.nbt.2012.08.141 Poster 1.2.10 The effect of ATP and NADH induced by acetic acid as cosubstrate in the 4S route of DBT biodesulphurization by Pseudomonas putida CECT 5279 with mixture of resting whole cells with different age I. Martinez, A. Alcon, V.E. Santos, F. Garcia-Ochoa ∗ Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Avenida Complutense s/n, 28040 Madrid, Spain Biodesulphurization (BDS) is a promise process to remove sulphur from organic compounds present in fuel oils which are recalcitrant to traditional hydrodesulfurization. Pseudomonas putida CECT 5279 is a GMO able to carry out the 4S route, a metabolic pathway for dibenzothiophene (DBT, model compound) desulfurization. Some of the steps of the 4S route needs reducing equivalents (FMN), which are obtained by the cells from NADH; also ATP is needed for the cell metabolism. The aim of this work is to study the effect of ATP and NADH induced by acetic acid, used as co-substrate in the 4S route. The addition of organic acids can regenerate cofactors as NADH and increases the ATP production. Cells were cultured in a 2 L bioreactor (30◦ C, 200 rpm, 2 L/min aeration) using BSM medium, being collected at different growth times. BDS experiments were conducted by resting whole cells in an orbital shaker (30◦ C, 200 rpm, 150 mL flasks containing 16 mL of HEPES medium and 15 g/L of acetic acid). In some runs, several additions of DBT were carried out in fed batch operation. Analytical methods have been described elsewhere. ATP was measured using an enzymatic kit and NADH was extracted using heated Tris–EDTA buffer and quantified by in situ fluorescence. Results show that acetic acid used as co-substrate provides higher values of ATP and NADH concentration during the BDS process, and better BDS yields were reached, mainly in fed-batch operation. http://dx.doi.org/10.1016/j.nbt.2012.08.142
www.elsevier.com/locate/nbt S51
Poster 1.2.08 Cellooligosaccharide assimilating E. coli by displaying active BGL on the cell surface using novel anchor protein Tsutomu Kondo
Tanaka ∗ , Yuuki
Hirata, Hitomi
Kawabata, Akihiko
Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan We demonstrated direct assimilation of cellobiose using betaglucosidase (BGL)-displaying E. coli. After screening active BGLs, Tfu0937, which is a BGL from Thermobifida fusca YX, was successfully displayed on the E. coli cell surface and directly assimilated cellobiose as a carbon source. Then, we screened for a suitable anchor protein for BGL cell-surface display and found a novel anchor protein, Blc, which allowed for both N- and C-terminal fusion of Tfu0937. The pNPG activity of Tfu-0937 using Blc as an anchor protein was significantly improved, up to 7535 U/O.D. 600/ml, which is approximately 65-fold higher than using PgsA as the anchor protein. Finally, Tfu0937-displaying E. coli JCM20137 was successfully grown on 0.2% cellobiose, and the O.D. 600 was 1.05 after 20 h, which is almost the same rate of growth on 0.2% glucose after 16 h. Tfu0937-displaying E. coli JCM20137 was also successfully grown on 0.2% cellotriose, cellotetraose, cellopentaose and cellohexaose. http://dx.doi.org/10.1016/j.nbt.2012.08.140 Poster 1.2.09 Production and two phase purification of catalase from Phanerochaete chrysosporium Berna Kavakc¸ıo˘ glu ∗ , Leman Tarhan Dokuz Eylul University, Science Faculty, Chemistry Department, Turkey Catalase (EC 1.11.1.6) is an enzyme primarily found in the peroxisomes of nearly all aerobic and certain anaerobic cells, and serves to protect the cell from the toxic effects of hydrogen peroxide by catalyzing its decomposition into molecular oxygen and water without production of free radicals [1]. Catalase has useful applications in various fields such as food, textile and even medicine [2]. For these reasons, production and purification of catalase with cheap and simple ways is significant to meet the demand. Within the context of this study, the optimization of the culture conditions of white rot fungus Phanerochaete chrysosporium to obtain maximum catalase activity and then purification of the enzyme by using aqueous two phase systems (ATPSs) were carried out. For the optimization studies, five separate solid and two different liquid mediums were investigated. Maximum catalase activity as 238.56 U/mg was observed in the extract obtained by using PDA as a solid medium with the combination of carbon-nitrogen restricted liquid medium. As for that the purification procedure, various polyethylene glycol (PEG)-phosphate salt (K2 HPO4 ) ATPSs were evaluated in terms of partition parameters including partition coefficient of the target enzyme (Ke ) and the total protein (Kp ), purification fold (PF), yield (Y%) and recovery (R%). The best results were obtained in the PEG 1000 18%–K2 HPO4 12% (pH 8.2)
system. For this system, Ke and Kp values were determined as 10.55 and 0.21, respectively while %Y was found as 91.95% and the purity of the enzyme was increased to 5.72 with the %R of 81.96%. Keywords: Phanerochaete chrysosporium; Catalase; Aqueous two phase systems
References 1. Ran J, et al. A facile method for improving the covalent crosslinking adsorption process of catalase immobilization. Bioresour Technol 2010;101:6285–90. 2. Akertek E, Tarhan L. Characterization of immobilized catalase and their application in pasteurization of milk with H2 O2 . Appl Biochem Biotechnol 1995;50:291–303.
http://dx.doi.org/10.1016/j.nbt.2012.08.141 Poster 1.2.10 The effect of ATP and NADH induced by acetic acid as cosubstrate in the 4S route of DBT biodesulphurization by Pseudomonas putida CECT 5279 with mixture of resting whole cells with different age I. Martinez, A. Alcon, V.E. Santos, F. Garcia-Ochoa ∗ Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Avenida Complutense s/n, 28040 Madrid, Spain Biodesulphurization (BDS) is a promise process to remove sulphur from organic compounds present in fuel oils which are recalcitrant to traditional hydrodesulfurization. Pseudomonas putida CECT 5279 is a GMO able to carry out the 4S route, a metabolic pathway for dibenzothiophene (DBT, model compound) desulfurization. Some of the steps of the 4S route needs reducing equivalents (FMN), which are obtained by the cells from NADH; also ATP is needed for the cell metabolism. The aim of this work is to study the effect of ATP and NADH induced by acetic acid, used as co-substrate in the 4S route. The addition of organic acids can regenerate cofactors as NADH and increases the ATP production. Cells were cultured in a 2 L bioreactor (30◦ C, 200 rpm, 2 L/min aeration) using BSM medium, being collected at different growth times. BDS experiments were conducted by resting whole cells in an orbital shaker (30◦ C, 200 rpm, 150 mL flasks containing 16 mL of HEPES medium and 15 g/L of acetic acid). In some runs, several additions of DBT were carried out in fed batch operation. Analytical methods have been described elsewhere. ATP was measured using an enzymatic kit and NADH was extracted using heated Tris–EDTA buffer and quantified by in situ fluorescence. Results show that acetic acid used as co-substrate provides higher values of ATP and NADH concentration during the BDS process, and better BDS yields were reached, mainly in fed-batch operation. http://dx.doi.org/10.1016/j.nbt.2012.08.142
www.elsevier.com/locate/nbt S51