Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterifica...Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications, was previously mostly expressed intracellularly as inclusion bodies in Escherichia coli. Denaturation and renaturation of inclusion bodies had a significant influence on the lipase activity. Thereupon, our present work described the secretion expression of gene encoding of this lipase in Pichia pastoris GS 115 and characterization of the recombinant enzyme. Firstly, the obtained lipA gene fragment was introduced into P. pastoris expression vector pPIC9K, the lipA gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOXI promoter, and the recombinant plasmid pPIC9K-lipA was transformed into P. pastoris strain GS115 by electroporation, and this recombinant P. pastoris were identified by PCR. Then lipase activity was detected on BMMY-tributyrin and olive oil agar plates containing Rhodamine B. Transformants with lipase activity by screening were induced 6 days by methanol, one band of 77 kDa protein could be observed by 10% SDS-PAGE. p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The pH and temperature optimum of lipase were pH 8.5 and 40℃ respectively. The stability and effects of metal ions and other reagents were also determined. However, the recombinant fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 4 U of lipase activity per milliliter of culture media supernatant.展开更多
Thales of Miletus （640？-546 BC） is famous for his prediction of the total solar eclipse in 585 BC. In this paper, the author demonstrate how Thales may have used the same principle for prediction of solar eclipses ...Thales of Miletus （640？-546 BC） is famous for his prediction of the total solar eclipse in 585 BC. In this paper, the author demonstrate how Thales may have used the same principle for prediction of solar eclipses as that used on the Antikythera Mechanism. At the SEAC conference in Alexandria in 2009, the author presented the paper ＂Ten solar eclipses show that the Antikythera Mechanism was constructed for use on Sicily.＂ The best defined series of exeligmos cycles started in 243 BC during the lifetime of Archimedes （287-212 BC） from Syracuse. The inscriptions on the Antikythera Mechanism were made in 100-150 BC and the last useful exeligmos started in 134 BC. The theory for the motion of the moon was from Hipparchus （ca 190-125 BC）. A more complete investigation of the solar eclipses on the Antikythera Mechanism reveals that the first month in the first saros cycle started with the first new moon after the winter solstice in 542 BC. Four solar eclipses 537-528 BC, from the first saros cycle, and three one exeligmos cycle later, 487-478 BC, are preserved and may have been recorded in Croton by Pythagoras （ca 575-495 BC） and his school.展开更多
文摘Serratia marcescens ECUI010, as an extracellular lipase and a significant catalyst, which had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications, was previously mostly expressed intracellularly as inclusion bodies in Escherichia coli. Denaturation and renaturation of inclusion bodies had a significant influence on the lipase activity. Thereupon, our present work described the secretion expression of gene encoding of this lipase in Pichia pastoris GS 115 and characterization of the recombinant enzyme. Firstly, the obtained lipA gene fragment was introduced into P. pastoris expression vector pPIC9K, the lipA gene without its signal sequence were cloned downstream to the alpha-mating factor signal and expressed in P. pastoris GS115 under the control of AOXI promoter, and the recombinant plasmid pPIC9K-lipA was transformed into P. pastoris strain GS115 by electroporation, and this recombinant P. pastoris were identified by PCR. Then lipase activity was detected on BMMY-tributyrin and olive oil agar plates containing Rhodamine B. Transformants with lipase activity by screening were induced 6 days by methanol, one band of 77 kDa protein could be observed by 10% SDS-PAGE. p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The pH and temperature optimum of lipase were pH 8.5 and 40℃ respectively. The stability and effects of metal ions and other reagents were also determined. However, the recombinant fusion intended for secretion was efficiently secreted in a percentage of close to 90% and remained stable even in rich medium at high cell density cultures, yielding values of over 4 U of lipase activity per milliliter of culture media supernatant.
文摘Thales of Miletus （640？-546 BC） is famous for his prediction of the total solar eclipse in 585 BC. In this paper, the author demonstrate how Thales may have used the same principle for prediction of solar eclipses as that used on the Antikythera Mechanism. At the SEAC conference in Alexandria in 2009, the author presented the paper ＂Ten solar eclipses show that the Antikythera Mechanism was constructed for use on Sicily.＂ The best defined series of exeligmos cycles started in 243 BC during the lifetime of Archimedes （287-212 BC） from Syracuse. The inscriptions on the Antikythera Mechanism were made in 100-150 BC and the last useful exeligmos started in 134 BC. The theory for the motion of the moon was from Hipparchus （ca 190-125 BC）. A more complete investigation of the solar eclipses on the Antikythera Mechanism reveals that the first month in the first saros cycle started with the first new moon after the winter solstice in 542 BC. Four solar eclipses 537-528 BC, from the first saros cycle, and three one exeligmos cycle later, 487-478 BC, are preserved and may have been recorded in Croton by Pythagoras （ca 575-495 BC） and his school.
