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Effect of additives on freeze-drying and storage of Yarrowia lipolytica lipase
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Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
icantly higher than those obtained with nitrogen (22.9%), carbon (19.4%) and phosphorus (23%) limitations. The highest lipid/protein ratio was 0.89 obtained under magnesium limitation at 0.09 h-1 . While the amount of lipid is not as high (24.1%) the protein does not significantly diminish its value (27%). In conclusion, it is possible to manipulate cell composition in order to obtain high lipid to protein ratio. doi:10.1016/j.jbiotec.2010.09.394 [P-I.29] Recombinant Protein Purification Using Complementary Peptides as Affinity Tags María C. Martínez Ceron, Alexandra M. Targovnik, Nicolás Urtasun, Osvaldo Cascone ∗ , María V. Miranda, Silvia A. Camperi University of Buenos Aires, Argentina Keywords: Recombinant protein; Purification; Peptides; Affinity tags Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair LSAAIIIPFLLH and KNYPKKKMEKRF have been demonstrated by other authors. In this work we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilised on a chromatographic matrix using the green fluorescent protein (GFP) expressed in E. coli as the model. The peptide LSAAIIIPFLLH was synthesised by solid phase using the Fmoc chemistry and immobilised in NHS-agarose. The GFP was expressed either with a fusion tag KNYPKKKMEKRF on the carboxy terminus or without a fusion tag. After cell disruption, the extract was directly applied to a LSAAIIIPFLLH-agarose column using 20 mM sodium phosphate buffer, pH 7.0, for the adsorption step. The GFP fused with the peptide tag KNYPKKKMEKRF was adsorbed and then eluted specifically with 1 M Tris. The yield was 98% and the purification factor 4.6. On the other hand, GFP without tag pass through without interacting with the peptide-agarose column. The method designed can be applied for the purification of other recombinant proteins. doi:10.1016/j.jbiotec.2010.09.395
[P-I.30] Effect of additives on freeze-drying and storage of Yarrowia lipolytica lipase F. Darvishi 1,∗ , J. Destain 2 , I. Nahvi 1 , P. Thonart 2 , H. ZarkeshEsfahani 1 1
University of Isfahan, Iran, Islamic Republic of CWBI, University of Liège, Belgium Keywords: Yarrowia lipolytica lipase; Freeze-drying; Additives; Storage 2
The extracellular lipase of Yarrowia lipolytica is an enzyme that presents numerous potentialities for biotechnological applications, particularly in the food, pharmacology and chemical industries. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freezedrying. Lipase was produced by Yarrowia lipolytica U6 mutant strain in 20 L bioreactor. After centrifugation, non-concentrated culture supernatant samples (211,460 U/L) to frezze drying were supplemented with different concentration (0.5-1%) of maltodextrin and glycerol as additives. Effect of additives, temperature and storage time on lipase powders were determined by lipase activity. Maltodextrin gave the best protection of lipase during dehydration treatment and its freeze-drying yield (73-76%) is better than glycerol. Lipase powder with glycerol resembled glue and therefore glycerol is not good for lipase freeze-drying. Powders produced from enzyme were stored for 20 weeks at 4 or 20 ◦ C without loss of activity. All the powders obtained by freeze-drying were stable during storage. For the first time, maltodextrin and glycerol used as additives to freeze-drying of Y. lipolytica lipase. In all cases, additives used to dehydrate and stabilize lipase allowed for longer periods of storage. The formulation carried out with maltodextrin was better than glycerol for lipase downstream processing. The present study deals with stability of lipase powder during storage in order to use it in several applications. This study suggests that the use of additives and freeze-drying should therefore be considered as an efficient method of enzyme preservation. doi:10.1016/j.jbiotec.2010.09.396 [P-I.31] Nucleotide Dependency in Thermal Protection Activities of Chaperonins from Hyperthermophilic Archaeum Aeropyrum pernix K1 Soo-Wan Nam 1,2,∗ , Jeong-Hwan Kim 1 1
Department of Biomaterial Control (BK21 Program), Dong-Eui University, Korea, Republic of 2 Department of Biotechnology and Bioengineering, Dong-Eui University, Korea, Republic of Keywords: Chaperonin; Aeropyrum pernix; Thermal Protection; Nucleotide Dependency In the present study, nucleotide dependency in thermal protection activities of Aeropyrum pernix chaperonins (ApCpnA and ApCpnB) was estimated using citrate synthase (CS) from porcine heart, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, and malate dehydrogenase (MDH) from Thermus flavus as foreign proteins. The molecular chaperonins, ApCpnA and ApCpnB, were overexpressed in E. coli and purified by heat treatment at 80◦ C for 20 min and HiTrap Q anion-exchange chromatography. The addi-
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
icantly higher than those obtained with nitrogen (22.9%), carbon (19.4%) and phosphorus (23%) limitations. The highest lipid/protein ratio was 0.89 obtained under magnesium limitation at 0.09 h-1 . While the amount of lipid is not as high (24.1%) the protein does not significantly diminish its value (27%). In conclusion, it is possible to manipulate cell composition in order to obtain high lipid to protein ratio. doi:10.1016/j.jbiotec.2010.09.394 [P-I.29] Recombinant Protein Purification Using Complementary Peptides as Affinity Tags María C. Martínez Ceron, Alexandra M. Targovnik, Nicolás Urtasun, Osvaldo Cascone ∗ , María V. Miranda, Silvia A. Camperi University of Buenos Aires, Argentina Keywords: Recombinant protein; Purification; Peptides; Affinity tags Affinity tags have become highly popular tools for purifying recombinant proteins from crude extracts. Besides, short peptides are excellent ligands for affinity chromatography, as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity. Hydropathically complementary peptides designed de novo show enough selectivity to be used successfully as peptide ligands for protein purification from crude extracts. Recognition specificity and selectivity in the interaction between the complementary peptide pair LSAAIIIPFLLH and KNYPKKKMEKRF have been demonstrated by other authors. In this work we designed a recombinant protein purification method using a peptide affinity tag that binds to a peptide-binding partner immobilised on a chromatographic matrix using the green fluorescent protein (GFP) expressed in E. coli as the model. The peptide LSAAIIIPFLLH was synthesised by solid phase using the Fmoc chemistry and immobilised in NHS-agarose. The GFP was expressed either with a fusion tag KNYPKKKMEKRF on the carboxy terminus or without a fusion tag. After cell disruption, the extract was directly applied to a LSAAIIIPFLLH-agarose column using 20 mM sodium phosphate buffer, pH 7.0, for the adsorption step. The GFP fused with the peptide tag KNYPKKKMEKRF was adsorbed and then eluted specifically with 1 M Tris. The yield was 98% and the purification factor 4.6. On the other hand, GFP without tag pass through without interacting with the peptide-agarose column. The method designed can be applied for the purification of other recombinant proteins. doi:10.1016/j.jbiotec.2010.09.395
[P-I.30] Effect of additives on freeze-drying and storage of Yarrowia lipolytica lipase F. Darvishi 1,∗ , J. Destain 2 , I. Nahvi 1 , P. Thonart 2 , H. ZarkeshEsfahani 1 1
University of Isfahan, Iran, Islamic Republic of CWBI, University of Liège, Belgium Keywords: Yarrowia lipolytica lipase; Freeze-drying; Additives; Storage 2
The extracellular lipase of Yarrowia lipolytica is an enzyme that presents numerous potentialities for biotechnological applications, particularly in the food, pharmacology and chemical industries. This work describes the development and storage of powders obtained from supernatants containing Y. lipolytica lipase by freezedrying. Lipase was produced by Yarrowia lipolytica U6 mutant strain in 20 L bioreactor. After centrifugation, non-concentrated culture supernatant samples (211,460 U/L) to frezze drying were supplemented with different concentration (0.5-1%) of maltodextrin and glycerol as additives. Effect of additives, temperature and storage time on lipase powders were determined by lipase activity. Maltodextrin gave the best protection of lipase during dehydration treatment and its freeze-drying yield (73-76%) is better than glycerol. Lipase powder with glycerol resembled glue and therefore glycerol is not good for lipase freeze-drying. Powders produced from enzyme were stored for 20 weeks at 4 or 20 ◦ C without loss of activity. All the powders obtained by freeze-drying were stable during storage. For the first time, maltodextrin and glycerol used as additives to freeze-drying of Y. lipolytica lipase. In all cases, additives used to dehydrate and stabilize lipase allowed for longer periods of storage. The formulation carried out with maltodextrin was better than glycerol for lipase downstream processing. The present study deals with stability of lipase powder during storage in order to use it in several applications. This study suggests that the use of additives and freeze-drying should therefore be considered as an efficient method of enzyme preservation. doi:10.1016/j.jbiotec.2010.09.396 [P-I.31] Nucleotide Dependency in Thermal Protection Activities of Chaperonins from Hyperthermophilic Archaeum Aeropyrum pernix K1 Soo-Wan Nam 1,2,∗ , Jeong-Hwan Kim 1 1
Department of Biomaterial Control (BK21 Program), Dong-Eui University, Korea, Republic of 2 Department of Biotechnology and Bioengineering, Dong-Eui University, Korea, Republic of Keywords: Chaperonin; Aeropyrum pernix; Thermal Protection; Nucleotide Dependency In the present study, nucleotide dependency in thermal protection activities of Aeropyrum pernix chaperonins (ApCpnA and ApCpnB) was estimated using citrate synthase (CS) from porcine heart, alcohol dehydrogenase (ADH) from Saccharomyces cerevisiae, and malate dehydrogenase (MDH) from Thermus flavus as foreign proteins. The molecular chaperonins, ApCpnA and ApCpnB, were overexpressed in E. coli and purified by heat treatment at 80◦ C for 20 min and HiTrap Q anion-exchange chromatography. The addi-