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Verification of GMO analytical methods: DNA isolation and quantitative detection methods for Roundup Ready soy flour
New Biotechnology · Volume 29S · September 2012
effect of aeration rate on the lipase activity was investigated in the range of 1–4 vvm. The maximum lipase activities in the media containing glucose and sucrose as main C sources were found to be 61.07 mol/L min and 102.83 mol/L min, respectively at the stationary phase of growth (96th hour of fermentation) at 3 vvm and 2 vvm aeration rates, respectively. The inclusion of 10% PFR in the fermentation medium resulted in higher lipase production. The maximum lipase activities in the media containing glucose and sucrose were determined as102.67 mol/L min and 171.20 mol/L min, respectively at 2 vvm aeration rate. http://dx.doi.org/10.1016/j.nbt.2012.08.549 Poster 5.0.110 Modification of explant’s metabolic activity to increase regeneration capacity of flax (Linum usitatissimum L.) hypocotyl segments Cansu Telci Kahramanogullari1 , Behrouz Alizadeh2 , Ramazan Beyaz3 , Mustafa Yildiz1,∗ 1
University of Ankara, Faculty of Agriculture, Department of Field Crops 06110 Dıs¸kapı, Ankara, Turkey 2 University of Ankara, Graduate School of Natural and Applied Sciences, Department of Field Crops, Gölbas¸ı, Ankara, Turkey 3 University of Ankara, Biotechnology Institute, Gölbas¸ı, Ankara, Turkey This study was conducted to investigate the effects of removing one of the cotyledon leaves of in vitro-grown flax (Linum usitatissimum L.) seedling on regeneration capacity of hypocotyls. Seeds from two flax cvs. ‘Madaras’ and ‘1886 Sel.’ were surface sterilized with 40% commercial bleach containing 5% sodium hypochlorite at 10◦ C for 12 min with continuous stirring and then were washed three times with sterile distilled water at the same temperature. Sterilized seeds were germinated on a basal medium containing the mineral salts and vitamins of Murashige and Skoog (MS), 3% (w/v) sucrose and 0.7% (w/v) agar in Magenta vessels. One of the cotyledon leaves of some seedling was removed 5 days after culture initiation while other seedlings kept as they were. 14 days after seed sown for germination, seedling and root lengths, seedling fresh and dry weights, seedling water and dry matter contents, chlorophyll a, chlorophyll b and total chlorophyll contents in cotyledon leaves of seedlings were recorded. Hypocotyl segments were cultured for regeneration for 4 weeks. Sixteen explants were cultured at 1.0 cm × 1.0 cm distance with a 4 × 4 matrix in a Petri dish. At the end of the culture, shoot regeneration percentage, shoot number per explant, regenerated shoot length and total shoot number per Petri dish were recorded in hypocotyls excised from complete- and one cotyledon-absent-seedlings. According to the results, it could be concluded that removing one cotyledon leaf forced seedlings to increase metabolic activity to sustain their viability as seedlings with two cotyledons. Keywords: Flax; Metabolic activity; Regeneration capacity http://dx.doi.org/10.1016/j.nbt.2012.08.550
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Poster 5.0.111 Recent advances in microbial degradation of sulfur, nitrogen and oxygen heterocycles Ping Xu1,2 1
State Key Laboratory of Microbial Metabolism & School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 2 State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Shandong Province, People’s Republic of China Sulfur (S), nitrogen (N) and oxygen (O) heterocycles are among the most potent environmental pollutants. Microbial degradation of these pollutants is attracting more and more attention because such bioprocesses are environmentally friendly. The biotechnological potential of these processes is being investigated, for example, to achieve better sulfur removal by immobilized biocatalysts with magnetite nanoparticles or by solvent-tolerant bacteria, and to obtain valuable intermediates from these heterocycles. Our recent advances have demonstrated the mechanism of the important nitrogen heterocycle-nicotine degradation by Pseudomonas. However, these technologies are not yet available for large-scale applications so future research must investigate proper modifications for industrial applications of these processes. This presentation focuses on recent progress in understanding how microbes degrade S, N and O heterocycles. Supporting publications: Tang HZ et al., 2012, Sci. Rep. 2:377; Li A et al., Biotechnol. Bioeng. 2012, 109:609–613; Tang HZ et al., J. Biol. Chem. 2011, 286:39179; Tang HZ et al., J. Bacteriol. 2011, 193:6789–6790; Yu H et al., J. Bacteriol. 2011, 193:5541–5542; Gai ZH et al., PLoS ONE 2010, 5:e10018; Li QG et al., Environ. Sci. Technol. 2009, 43:8635–8642; Tang HZ et al., Appl. Environ. Microbiol. 2009, 75:772–778; Tang HZ et al., Appl. Environ. Microbiol. 2008, 74:1567–1574; Xu P. et al., Trends Microbiol. 2006, 14:398; Wang SN et al., Environ. Sci. Technol. 2005, 39:6877–6880. http://dx.doi.org/10.1016/j.nbt.2012.08.551 Poster 5.0.112 Verification of GMO analytical methods: DNA isolation and quantitative detection methods for Roundup Ready soy flour Remziye Yılmaz∗ , Ceren Bayrac¸, Pelin Mutlu, Erdem Boy, Meral Yücel Middle East Technical University, Molecular Biology and Biotechnology R&D Center, Genome Analysis Laboratory, 06800 Ankara, Turkey Quality assurance is a prerequisite for accurate and reliable results in food and feed testing, ISO/IEC 17025 being recognized worldwide as the base standard. Accreditation is a suitable system for harmonising procedures in each testing laboratory. Precisely defined procedures for the verification of methods are the key points for accreditation. Testing for genetically modified organisms (GMOs) is a challenging exercise, especially with more GMOs entering the world market. We describe here the organization and performance of verifying DNA isolation and quantitative detection methods for GMO testing of Roundup Ready soy flour in the con-
New Biotechnology · Volume 29S · September 2012
text of the European Union legislation. Operational procedures for method verification organized by the Middle East Technical University, Molecular Biology and Biotechnology R&D Center, Genome Analysis Laboratory, are described. The system setup can be an example for other similar fields of analytical work. Keywords: Verification; Genetically modified organisms; Accreditation; Testing http://dx.doi.org/10.1016/j.nbt.2012.08.552 Poster 5.0.113 Biochemical and molecular characterization of copperresistant bacterial isolates Turalı Gamze, Icgen Bulent, Tan Sema∗
enzyme was stable at pH 1.0–4.5. It was also heat stable with 76% residual activity after heating at 70◦ C for 80 min. For comparison, the appA gene of E. coli K12 was transformed and expressed in E. coli BL21(DE3). Some of the properties of the purified recombinant phytase were similar to those of the GM potato phytase. However, the enzyme was not glycosylated and the N-terminal sequence was determined to be QSEP. It was not as heat stable as the phytase expressed in line 2-1. Only 50% activity remained after the same heat treatment. These differences should be taken into account when evaluating the safety of the transgenic phytase. Acknowledgements: We thank Dr. Su-May Yu for providing plasmids and Taiwan Agricultural Research Institute for providing potatoes. This work was supported by grant FDA 99-FS031 from the Taiwan Food and Drug Administration.
Kırıkkale University, Department of Biology, 71450 Kırıkkale, Turkey Mining activities of modern societies, extensive industrial use of copper and its widespread use as a pesticide in crop production are major sources of copper pollution of soils and water. The objective of present study is to isolate and identify copperresistant bacteria from the river Kızılırmak along the city Kırıkkale, Turkey. Copper-resistant isolate with a minimal inhibitory concentration of 450 mg L−1 was isolated and identified as Pseudomonas putida by using biochemical tests and 16S rDNA sequencing. Pseudomonas putida was shown to be resistant to other heavy metals like aluminum, lithium, silver, nickel, zinc and resistance to the antibiotics like aztreonam, gentamicin bacitracin, erythromycin, cephalothin, pefloxacin, ticarsillin, and vancomycin. The copper resistance genes of Pseudomonas putida were found out to be located on the chromosomal DNA. Total and outer membrane protein profiles revealed that membrane porins were functional in the copper resistance of Pseudomonas putida. Keywords: Copper-resistant bacteria; Molecular characterization; 16S DNA sequencing; Heavy metal resistance http://dx.doi.org/10.1016/j.nbt.2012.08.553 Poster 5.0.114 Assessment of substantial equivalence of phytase from genetically modified potato and recombinant Escherichia coli Mei-Li Chao, Jen-Tao Chen, Bo-Chou Chen, Wen-Shen Chu∗ Food Industry Research and Development Institute, Hsinchu 30062, Taiwan, ROC To solve the problem of essentially unavailable phosphorus in phytate to monogastric animals, Dr. Su-May Yu’s team in Taiwan developed a genetically modified (GM) potato line 2-1 by transforming the appA gene of E. coli K12 encoding an enzyme with phytase activity. For safety assessment, the phytase of line 2-1 was purified 53.7fold with a specific activity of 218 U/mg. The molecular weight of the enzyme was 48.5 kDa with a pI of 6.5. It was glycosylated and the N-terminal sequence was SEPEL. The optimal pH and temperature for the enzyme activity were 4.5 and 60◦ C, respectively. The
http://dx.doi.org/10.1016/j.nbt.2012.08.554 Poster 5.0.115 Anthocyanins from Mexican cultivars of colored maize and pomegranate: extraction, characterization, comparison and effect of geographic source A.E. Ortega-Regules1,∗ , A.B. Bautista-Ortín2 , J.D. Lozada-Ramírez3 , E. Gómez-Plaza2 1
Universidad Politécnica de Tlaxcala, Tlaxcala, Mexico Universidad de Murcia, Murcia, Spain 3 Universidad de las Américas Puebla, Puebla, Mexico 2
Anthocyanins are natural antioxidants of biotechnological interest widely distributed in plants with a number of possible applications in medicine, pharmacy, cosmetology, the food industry, as alternative sources of energy, and nanotechnology. Blue and red maize (Zea mays) and pomegranate (Punica granatum) are important sources of anthocyanins and count with a strong cultural legacy. The objective of this work was to evaluate phenolic and anthocyanic content, and determine antioxidant properties of anthocyanic extracts from colored maize and pomegranate, using methanol, ethanol, water and ethanol(80):water(20) for extraction, besides the chromatographic identification of anthocyanins. Maize was cultivated in Tlaxcala, Mexico (19◦ 25 44 N, 98◦ 9 39 W; 4,420 m.a.s.l.) and pomegranates were collected in two different localities, Hidalgo (20◦ 7 12 N, 98◦ 44 9 W; 2,400 m.a.s.l.) and Tlaxcala, Mexico. Anthocyanic and total phenolic content of red maize showed a significant difference (5- and 2.5-fold, respectively) to blue maize, consistent with 3-fold higher antioxidant activity of red maize extracts. Anthocyanic and phenolic content in pomegranate was lower than those extracted from red maize, but higher than those extracted from blue maize. Slight differences related to the geographic source between pomegranates were found. Antioxidant capacity between pomegranates is very similar, slightly lower than that from red maize, but higher than that from blue maize. Chromatographic identification at 520 and 280 nm evidenced the existence of the same kind of principal anthocyanins but in different ratios. In pomegranate, chromatographic analysis confirms the same composition with different ratios, associated to geographic source. The above confirms the potential use
www.elsevier.com/locate/nbt S197
effect of aeration rate on the lipase activity was investigated in the range of 1–4 vvm. The maximum lipase activities in the media containing glucose and sucrose as main C sources were found to be 61.07 mol/L min and 102.83 mol/L min, respectively at the stationary phase of growth (96th hour of fermentation) at 3 vvm and 2 vvm aeration rates, respectively. The inclusion of 10% PFR in the fermentation medium resulted in higher lipase production. The maximum lipase activities in the media containing glucose and sucrose were determined as102.67 mol/L min and 171.20 mol/L min, respectively at 2 vvm aeration rate. http://dx.doi.org/10.1016/j.nbt.2012.08.549 Poster 5.0.110 Modification of explant’s metabolic activity to increase regeneration capacity of flax (Linum usitatissimum L.) hypocotyl segments Cansu Telci Kahramanogullari1 , Behrouz Alizadeh2 , Ramazan Beyaz3 , Mustafa Yildiz1,∗ 1
University of Ankara, Faculty of Agriculture, Department of Field Crops 06110 Dıs¸kapı, Ankara, Turkey 2 University of Ankara, Graduate School of Natural and Applied Sciences, Department of Field Crops, Gölbas¸ı, Ankara, Turkey 3 University of Ankara, Biotechnology Institute, Gölbas¸ı, Ankara, Turkey This study was conducted to investigate the effects of removing one of the cotyledon leaves of in vitro-grown flax (Linum usitatissimum L.) seedling on regeneration capacity of hypocotyls. Seeds from two flax cvs. ‘Madaras’ and ‘1886 Sel.’ were surface sterilized with 40% commercial bleach containing 5% sodium hypochlorite at 10◦ C for 12 min with continuous stirring and then were washed three times with sterile distilled water at the same temperature. Sterilized seeds were germinated on a basal medium containing the mineral salts and vitamins of Murashige and Skoog (MS), 3% (w/v) sucrose and 0.7% (w/v) agar in Magenta vessels. One of the cotyledon leaves of some seedling was removed 5 days after culture initiation while other seedlings kept as they were. 14 days after seed sown for germination, seedling and root lengths, seedling fresh and dry weights, seedling water and dry matter contents, chlorophyll a, chlorophyll b and total chlorophyll contents in cotyledon leaves of seedlings were recorded. Hypocotyl segments were cultured for regeneration for 4 weeks. Sixteen explants were cultured at 1.0 cm × 1.0 cm distance with a 4 × 4 matrix in a Petri dish. At the end of the culture, shoot regeneration percentage, shoot number per explant, regenerated shoot length and total shoot number per Petri dish were recorded in hypocotyls excised from complete- and one cotyledon-absent-seedlings. According to the results, it could be concluded that removing one cotyledon leaf forced seedlings to increase metabolic activity to sustain their viability as seedlings with two cotyledons. Keywords: Flax; Metabolic activity; Regeneration capacity http://dx.doi.org/10.1016/j.nbt.2012.08.550
S196
www.elsevier.com/locate/nbt
Poster 5.0.111 Recent advances in microbial degradation of sulfur, nitrogen and oxygen heterocycles Ping Xu1,2 1
State Key Laboratory of Microbial Metabolism & School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, People’s Republic of China 2 State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, Shandong Province, People’s Republic of China Sulfur (S), nitrogen (N) and oxygen (O) heterocycles are among the most potent environmental pollutants. Microbial degradation of these pollutants is attracting more and more attention because such bioprocesses are environmentally friendly. The biotechnological potential of these processes is being investigated, for example, to achieve better sulfur removal by immobilized biocatalysts with magnetite nanoparticles or by solvent-tolerant bacteria, and to obtain valuable intermediates from these heterocycles. Our recent advances have demonstrated the mechanism of the important nitrogen heterocycle-nicotine degradation by Pseudomonas. However, these technologies are not yet available for large-scale applications so future research must investigate proper modifications for industrial applications of these processes. This presentation focuses on recent progress in understanding how microbes degrade S, N and O heterocycles. Supporting publications: Tang HZ et al., 2012, Sci. Rep. 2:377; Li A et al., Biotechnol. Bioeng. 2012, 109:609–613; Tang HZ et al., J. Biol. Chem. 2011, 286:39179; Tang HZ et al., J. Bacteriol. 2011, 193:6789–6790; Yu H et al., J. Bacteriol. 2011, 193:5541–5542; Gai ZH et al., PLoS ONE 2010, 5:e10018; Li QG et al., Environ. Sci. Technol. 2009, 43:8635–8642; Tang HZ et al., Appl. Environ. Microbiol. 2009, 75:772–778; Tang HZ et al., Appl. Environ. Microbiol. 2008, 74:1567–1574; Xu P. et al., Trends Microbiol. 2006, 14:398; Wang SN et al., Environ. Sci. Technol. 2005, 39:6877–6880. http://dx.doi.org/10.1016/j.nbt.2012.08.551 Poster 5.0.112 Verification of GMO analytical methods: DNA isolation and quantitative detection methods for Roundup Ready soy flour Remziye Yılmaz∗ , Ceren Bayrac¸, Pelin Mutlu, Erdem Boy, Meral Yücel Middle East Technical University, Molecular Biology and Biotechnology R&D Center, Genome Analysis Laboratory, 06800 Ankara, Turkey Quality assurance is a prerequisite for accurate and reliable results in food and feed testing, ISO/IEC 17025 being recognized worldwide as the base standard. Accreditation is a suitable system for harmonising procedures in each testing laboratory. Precisely defined procedures for the verification of methods are the key points for accreditation. Testing for genetically modified organisms (GMOs) is a challenging exercise, especially with more GMOs entering the world market. We describe here the organization and performance of verifying DNA isolation and quantitative detection methods for GMO testing of Roundup Ready soy flour in the con-
New Biotechnology · Volume 29S · September 2012
text of the European Union legislation. Operational procedures for method verification organized by the Middle East Technical University, Molecular Biology and Biotechnology R&D Center, Genome Analysis Laboratory, are described. The system setup can be an example for other similar fields of analytical work. Keywords: Verification; Genetically modified organisms; Accreditation; Testing http://dx.doi.org/10.1016/j.nbt.2012.08.552 Poster 5.0.113 Biochemical and molecular characterization of copperresistant bacterial isolates Turalı Gamze, Icgen Bulent, Tan Sema∗
enzyme was stable at pH 1.0–4.5. It was also heat stable with 76% residual activity after heating at 70◦ C for 80 min. For comparison, the appA gene of E. coli K12 was transformed and expressed in E. coli BL21(DE3). Some of the properties of the purified recombinant phytase were similar to those of the GM potato phytase. However, the enzyme was not glycosylated and the N-terminal sequence was determined to be QSEP. It was not as heat stable as the phytase expressed in line 2-1. Only 50% activity remained after the same heat treatment. These differences should be taken into account when evaluating the safety of the transgenic phytase. Acknowledgements: We thank Dr. Su-May Yu for providing plasmids and Taiwan Agricultural Research Institute for providing potatoes. This work was supported by grant FDA 99-FS031 from the Taiwan Food and Drug Administration.
Kırıkkale University, Department of Biology, 71450 Kırıkkale, Turkey Mining activities of modern societies, extensive industrial use of copper and its widespread use as a pesticide in crop production are major sources of copper pollution of soils and water. The objective of present study is to isolate and identify copperresistant bacteria from the river Kızılırmak along the city Kırıkkale, Turkey. Copper-resistant isolate with a minimal inhibitory concentration of 450 mg L−1 was isolated and identified as Pseudomonas putida by using biochemical tests and 16S rDNA sequencing. Pseudomonas putida was shown to be resistant to other heavy metals like aluminum, lithium, silver, nickel, zinc and resistance to the antibiotics like aztreonam, gentamicin bacitracin, erythromycin, cephalothin, pefloxacin, ticarsillin, and vancomycin. The copper resistance genes of Pseudomonas putida were found out to be located on the chromosomal DNA. Total and outer membrane protein profiles revealed that membrane porins were functional in the copper resistance of Pseudomonas putida. Keywords: Copper-resistant bacteria; Molecular characterization; 16S DNA sequencing; Heavy metal resistance http://dx.doi.org/10.1016/j.nbt.2012.08.553 Poster 5.0.114 Assessment of substantial equivalence of phytase from genetically modified potato and recombinant Escherichia coli Mei-Li Chao, Jen-Tao Chen, Bo-Chou Chen, Wen-Shen Chu∗ Food Industry Research and Development Institute, Hsinchu 30062, Taiwan, ROC To solve the problem of essentially unavailable phosphorus in phytate to monogastric animals, Dr. Su-May Yu’s team in Taiwan developed a genetically modified (GM) potato line 2-1 by transforming the appA gene of E. coli K12 encoding an enzyme with phytase activity. For safety assessment, the phytase of line 2-1 was purified 53.7fold with a specific activity of 218 U/mg. The molecular weight of the enzyme was 48.5 kDa with a pI of 6.5. It was glycosylated and the N-terminal sequence was SEPEL. The optimal pH and temperature for the enzyme activity were 4.5 and 60◦ C, respectively. The
http://dx.doi.org/10.1016/j.nbt.2012.08.554 Poster 5.0.115 Anthocyanins from Mexican cultivars of colored maize and pomegranate: extraction, characterization, comparison and effect of geographic source A.E. Ortega-Regules1,∗ , A.B. Bautista-Ortín2 , J.D. Lozada-Ramírez3 , E. Gómez-Plaza2 1
Universidad Politécnica de Tlaxcala, Tlaxcala, Mexico Universidad de Murcia, Murcia, Spain 3 Universidad de las Américas Puebla, Puebla, Mexico 2
Anthocyanins are natural antioxidants of biotechnological interest widely distributed in plants with a number of possible applications in medicine, pharmacy, cosmetology, the food industry, as alternative sources of energy, and nanotechnology. Blue and red maize (Zea mays) and pomegranate (Punica granatum) are important sources of anthocyanins and count with a strong cultural legacy. The objective of this work was to evaluate phenolic and anthocyanic content, and determine antioxidant properties of anthocyanic extracts from colored maize and pomegranate, using methanol, ethanol, water and ethanol(80):water(20) for extraction, besides the chromatographic identification of anthocyanins. Maize was cultivated in Tlaxcala, Mexico (19◦ 25 44 N, 98◦ 9 39 W; 4,420 m.a.s.l.) and pomegranates were collected in two different localities, Hidalgo (20◦ 7 12 N, 98◦ 44 9 W; 2,400 m.a.s.l.) and Tlaxcala, Mexico. Anthocyanic and total phenolic content of red maize showed a significant difference (5- and 2.5-fold, respectively) to blue maize, consistent with 3-fold higher antioxidant activity of red maize extracts. Anthocyanic and phenolic content in pomegranate was lower than those extracted from red maize, but higher than those extracted from blue maize. Slight differences related to the geographic source between pomegranates were found. Antioxidant capacity between pomegranates is very similar, slightly lower than that from red maize, but higher than that from blue maize. Chromatographic identification at 520 and 280 nm evidenced the existence of the same kind of principal anthocyanins but in different ratios. In pomegranate, chromatographic analysis confirms the same composition with different ratios, associated to geographic source. The above confirms the potential use
www.elsevier.com/locate/nbt S197