- Email: [email protected]
Directed Evolution to Increase Pseudomonas sp. 42A2 LipC Thermostability
S404
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-I.167] Directed Evolution to Increase Pseudomonas sp. 42A2 LipC Thermostability Silvia Cesarini 1,∗ , Cristina Bofill 1 , Manfred Reetz 2 , Pilar Diaz 1 1
Universidad de Barcelona, Spain Max Planck Institut für Kohlenforschung, Germany Keywords: Directed Evolution; Lipases; Pseudomonas; Thermostability 2
Pseudomonas sp. 42A2 LipC is a foldase-dependent lipase showing an interesting optimal temperature at 4-20 ◦ C but with a moderate resistance to higher temperatures (Bofill et al., 2010). In order to increase the thermostability of LipC without losing its psychrophilic behaviour, we performed directed evolution based on a rational design mutation schedule. Since highly thermostable enzymes are known as rigid molecules, using the B-FITTER program (Reetz and Carballeira, 2007) on the LipC 3D-homology model, sites displaying the highest degree of flexibility were targeted to introduce appropriate mutations. Eight libraries were designed using different codon degeneracy systems, and the minimum number of clones to saturate each aminoacid position was calculated by means of CASTER program (Reetz and Carballeira, 2007). QuickChange PCR (Stratagene, 2003) was performed to mutate plasmid construction pBBlipClipH, bearing lipase and foldase genes in cis, and the resulting variants were electroporated into PABST7.1, a Pseudomonas lipase and foldase deficient strain (Jaeger et al., 1999). Clones showing activity on tributyrin agar plates were selected and used for highthroughput screening of increased thermostability. Supernatants of above 3000 clones of PABS-pBBlipClipH were analyzed for lipolytic activity on pNP-caprylate before and after exposure to 60 ◦ C for 15 min, and the residual activity was determined in comparison with that of the wild type enzyme. Only mutants showing more than 20% residual activity after heat-shock were considered as potential hits and further characterized in detail. Seven hits were selected and their activity analyzed individually after standardizing the protein concentration, and analysis of their nucleotide sequence was performed to check the mutations occurred. When all aminoacidic mutations have been analysed, new rounds of directed evolution through iterative saturation mutagenesis will be performed to get a mutant LipC with an even higher thermostability. References Bofill, et al., 2010. Biochimie 92, 307–316. Reetz, Carballeira, 2007. Nat.Prot (2), 891–903. Stratagene, 2003. La Jolla California. Jaeger, et al., 1999. Annu.Rev.Microbio (53), 315–351.
doi:10.1016/j.jbiotec.2010.09.533 [P-I.168] Optimization of hydrogen production from glycerol by Enterobacter aerogenes J. Song 1,2 , S. Kim 2 , D. Lee 1,∗ , J.K. Cho 1 , C. Park 3 , S. Kim 1 1
Korea Institute of Industrial Technology, Korea Republic of Korea University, Korea 3 Republic of Kwangwoon University, Republic of Korea 2
Massive amounts of waste glycerol are recently generated as a byproduct in the biodiesel industry and finding proper usage of them is a critical issue nowadays. Production of a future energy, hydrogen, from carbohydrates derived from renewable resources using biological processes has been one of the main research top-
ics in the relevant research communities and ceaseless efforts have been made to improve the process. However, hydrogen production from glycerol has gained relatively little attention despite the merit of glycerol as a low cost feedstock. As an attempt to transform the waste glycerol to valuable chemicals, hydrogen was produced biologically using a facultative microorganism, Enterobactero aerogenes ATCC 29007 from crude glycerol. In order to achieve the best yield of hydrogen production, production medium of the E. aerogenes was optimized statistically by Plackett-Burman and BoxBehnken methods. The result of Plackett-Burman design showed that KH2 PO4 , crude glycerol, and peptone concentrations have significant influences on the hydrogen production yield. And then, optimum concentrations of the main factors were determined to be 6.38 g/L KH2 PO4 , 184.4 mM crude glycerol, and 5.94 g/L peptone through Box-Behnken design and response surface method (RSM). The maximum yield of hydrogen production from crude glycerol was statistically predicted to be 2,045 mL/L under the optimum conditions. It is expected that the ongoing research would make this system attractive as a sustainable hydrogen production method, considering the fact that the growing biodiesel industry will provide low-cost glycerol for an extended period of time. doi:10.1016/j.jbiotec.2010.09.534 [P-I.169] Modifying Culture Media for Production of Plasmid DNA for Leishmaniasis Vaccine Fabiola Islas Lugo 1,∗ , Myriam Sánches Casco 1 , Erik Dumonteil 2 , Jaime Ortega López 1 , Maria del Carmen Montes Horcasitas 1 1
Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Mexico 2 Laboratorio de Parasitología, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mexico Keywords: Plasmid DNA; Vaccines; Production; Culture media Introduction: Plasmid DNA is an experimental vaccine, produced in E. coli for viral, bacterial and parasitic diseases. Therefore the productions yields are important for the vaccines development future, there are several factors that affec the plasmid production, some of them are: plasmid design, culture media and process. Culture media can drastically affect plasmid quality and yield, as well as temperature, O2 requirements and pH. Recently, the pVax-NH36 plasmid has been studied as a leishmaniasis vaccine. In this work, the plasmid DNA of E. coli DH5␣ as a host of pVax-NH36, was analyzed. In the corresponding studies we used two cultures media at 30 and 37 ◦ C. Under these conditions a plasmid production 155 mg pDNA/L and a specific plasmid yield of 5.45 mg pDNA / g cell·h were obtained. Materials and methods Bacterial strain and plasmid: E.coli DH5␣; Plasmid pVax-NH36 (leishmaniasis vaccine) Culture media Carbon source Na2 HPO4 KH2 PO4 NH4 Cl (NH4 )2 SO4 Na2 SO4 MgSO4 7H2 O NaCl Trace elements
M1 (g/L) 11 8,954 3,402 2,675 0,000 0,710 2,465 0,000
M2 (g/L) 50 6 3 0 5 0 0,7 0,5
M3 (g/L) 50 19 7 0 7 0 2.465 2 0.5 ml
The batch fermentation was carried out in a 0.5 L bioreactor. During the fermentation, pH was controlled at 6.8 - 7.0 by the auto-
Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576
[P-I.167] Directed Evolution to Increase Pseudomonas sp. 42A2 LipC Thermostability Silvia Cesarini 1,∗ , Cristina Bofill 1 , Manfred Reetz 2 , Pilar Diaz 1 1
Universidad de Barcelona, Spain Max Planck Institut für Kohlenforschung, Germany Keywords: Directed Evolution; Lipases; Pseudomonas; Thermostability 2
Pseudomonas sp. 42A2 LipC is a foldase-dependent lipase showing an interesting optimal temperature at 4-20 ◦ C but with a moderate resistance to higher temperatures (Bofill et al., 2010). In order to increase the thermostability of LipC without losing its psychrophilic behaviour, we performed directed evolution based on a rational design mutation schedule. Since highly thermostable enzymes are known as rigid molecules, using the B-FITTER program (Reetz and Carballeira, 2007) on the LipC 3D-homology model, sites displaying the highest degree of flexibility were targeted to introduce appropriate mutations. Eight libraries were designed using different codon degeneracy systems, and the minimum number of clones to saturate each aminoacid position was calculated by means of CASTER program (Reetz and Carballeira, 2007). QuickChange PCR (Stratagene, 2003) was performed to mutate plasmid construction pBBlipClipH, bearing lipase and foldase genes in cis, and the resulting variants were electroporated into PABST7.1, a Pseudomonas lipase and foldase deficient strain (Jaeger et al., 1999). Clones showing activity on tributyrin agar plates were selected and used for highthroughput screening of increased thermostability. Supernatants of above 3000 clones of PABS-pBBlipClipH were analyzed for lipolytic activity on pNP-caprylate before and after exposure to 60 ◦ C for 15 min, and the residual activity was determined in comparison with that of the wild type enzyme. Only mutants showing more than 20% residual activity after heat-shock were considered as potential hits and further characterized in detail. Seven hits were selected and their activity analyzed individually after standardizing the protein concentration, and analysis of their nucleotide sequence was performed to check the mutations occurred. When all aminoacidic mutations have been analysed, new rounds of directed evolution through iterative saturation mutagenesis will be performed to get a mutant LipC with an even higher thermostability. References Bofill, et al., 2010. Biochimie 92, 307–316. Reetz, Carballeira, 2007. Nat.Prot (2), 891–903. Stratagene, 2003. La Jolla California. Jaeger, et al., 1999. Annu.Rev.Microbio (53), 315–351.
doi:10.1016/j.jbiotec.2010.09.533 [P-I.168] Optimization of hydrogen production from glycerol by Enterobacter aerogenes J. Song 1,2 , S. Kim 2 , D. Lee 1,∗ , J.K. Cho 1 , C. Park 3 , S. Kim 1 1
Korea Institute of Industrial Technology, Korea Republic of Korea University, Korea 3 Republic of Kwangwoon University, Republic of Korea 2
Massive amounts of waste glycerol are recently generated as a byproduct in the biodiesel industry and finding proper usage of them is a critical issue nowadays. Production of a future energy, hydrogen, from carbohydrates derived from renewable resources using biological processes has been one of the main research top-
ics in the relevant research communities and ceaseless efforts have been made to improve the process. However, hydrogen production from glycerol has gained relatively little attention despite the merit of glycerol as a low cost feedstock. As an attempt to transform the waste glycerol to valuable chemicals, hydrogen was produced biologically using a facultative microorganism, Enterobactero aerogenes ATCC 29007 from crude glycerol. In order to achieve the best yield of hydrogen production, production medium of the E. aerogenes was optimized statistically by Plackett-Burman and BoxBehnken methods. The result of Plackett-Burman design showed that KH2 PO4 , crude glycerol, and peptone concentrations have significant influences on the hydrogen production yield. And then, optimum concentrations of the main factors were determined to be 6.38 g/L KH2 PO4 , 184.4 mM crude glycerol, and 5.94 g/L peptone through Box-Behnken design and response surface method (RSM). The maximum yield of hydrogen production from crude glycerol was statistically predicted to be 2,045 mL/L under the optimum conditions. It is expected that the ongoing research would make this system attractive as a sustainable hydrogen production method, considering the fact that the growing biodiesel industry will provide low-cost glycerol for an extended period of time. doi:10.1016/j.jbiotec.2010.09.534 [P-I.169] Modifying Culture Media for Production of Plasmid DNA for Leishmaniasis Vaccine Fabiola Islas Lugo 1,∗ , Myriam Sánches Casco 1 , Erik Dumonteil 2 , Jaime Ortega López 1 , Maria del Carmen Montes Horcasitas 1 1
Departamento de Biotecnología y Bioingeniería, CINVESTAV-IPN, Mexico 2 Laboratorio de Parasitología, Centro de Investigaciones Regionales “Dr. Hideyo Noguchi”, Universidad Autónoma de Yucatán, Mexico Keywords: Plasmid DNA; Vaccines; Production; Culture media Introduction: Plasmid DNA is an experimental vaccine, produced in E. coli for viral, bacterial and parasitic diseases. Therefore the productions yields are important for the vaccines development future, there are several factors that affec the plasmid production, some of them are: plasmid design, culture media and process. Culture media can drastically affect plasmid quality and yield, as well as temperature, O2 requirements and pH. Recently, the pVax-NH36 plasmid has been studied as a leishmaniasis vaccine. In this work, the plasmid DNA of E. coli DH5␣ as a host of pVax-NH36, was analyzed. In the corresponding studies we used two cultures media at 30 and 37 ◦ C. Under these conditions a plasmid production 155 mg pDNA/L and a specific plasmid yield of 5.45 mg pDNA / g cell·h were obtained. Materials and methods Bacterial strain and plasmid: E.coli DH5␣; Plasmid pVax-NH36 (leishmaniasis vaccine) Culture media Carbon source Na2 HPO4 KH2 PO4 NH4 Cl (NH4 )2 SO4 Na2 SO4 MgSO4 7H2 O NaCl Trace elements
M1 (g/L) 11 8,954 3,402 2,675 0,000 0,710 2,465 0,000
M2 (g/L) 50 6 3 0 5 0 0,7 0,5
M3 (g/L) 50 19 7 0 7 0 2.465 2 0.5 ml
The batch fermentation was carried out in a 0.5 L bioreactor. During the fermentation, pH was controlled at 6.8 - 7.0 by the auto-