摘要
Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation （HC, 〈10 000 g）, ultracentrifugation （UC, 70000 g）, and ultrafiltration （UF） with 100 kit molecular weight cutoff, were compared using two diatom species （Asterionellopsis glacialis and Thalassiosira weissflogii）, and two dinoflagellate species （Alexandriura catenella and Prorocentrum donghaiense） as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity （GSA） in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods （HC and UF） overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.
Glutamine synthetase （GS） is an important enzyme involved in nitrogen assimilation and metabolism in marine phytoplankton. However, little work has been done in situ due to the limitation of crude enzyme preparation methods. In this study, three enzyme preparation methods, high-speed centrifugation （HC, 〈10 000 g）, ultracentrifugation （UC, 70000 g）, and ultrafiltration （UF） with 100 kit molecular weight cutoff, were compared using two diatom species （Asterionellopsis glacialis and Thalassiosira weissflogii）, and two dinoflagellate species （Alexandriura catenella and Prorocentrum donghaiense） as experimental materials together with field samples collected from Xiamen Harbor, China. The results showed that HC is the best method to prepare crude enzymes for glutamine synthetase activity （GSA） in diatom species and diatom-dominant samples, while UF is the best method to extract GS from dinoflagellate species and dinoflagellate-dominant samples. For the HC method, the optimal centrifugal speed and time were 10 000 g and 35 min, respectively, and under these conditions, the highest GSA was obtained in all samples. This study indicates that both methods （HC and UF） overcome the limitation of centrifugal speed and could be applied to in situ GSA analysis, especially at sea.
基金
The National Natural Science Foundation of China under contract No 40331004
the Ministry of Science and Technology of China under contract No 2005DFA20430
