고도 호염성 세균의 분리 및 생리학적·효소학적 특성

Abstract

In this thesis, halophilic bacteria were isolated and their physiological characteristics and salt-dependent activation of enzyme were studied. Fourteen halophilic bacteria were isolated from salterns by cultivating them in Sehgal & Gibbons medium supplemented with 15% and 25% NaCl. Of them, the morphological and physiological characteristics of nine strains which grew at 20-25% NaCl were investigated. The result was that six strains EH8, EH10, EH17, EH18, EH19 and EH23 were tentatively identified as Halobacterium sp. and three strains EH11, EH12, and EH13 were tentatively identified as Halococcus sp. Among them, typical two strains Halobacterium sp. EH8 and EH10 which grew fast at 25-30% NaCl were selected and their physiological characteristics and salt-dependent activation of enzymes were studied. The optimal NaCl concentration for growth of Halobacterium sp. EH8, EH10 were 25% and growth were inhibited when the NaCl concentration fell down to 10%. The optimal temperature for growth of Halobacterium sp. EH8, EH10 were 45˚C and 5O˚C, resprectively. But these strains did'nt grow when NaCl was replaced with KCl. Some enzymes involved in metabolic pathway such as Lactate dehydrogenase, Glucokinase, Glucose-6-phosphate dehydrogenase, Alanine dehydrogenase and Isocitrate dehydrogenase were also tested. Consequently, Glucose-6-phosphate dehydrogenase was not detected. But the rest enzyme activities were observed at 0-5M NaCl concentration. Especially, Lactate dehydrogenase activity was highest at 5M NaCl and 4M KCl. Other enzyme involved in electron transport system such as NADH oxidase and NADH dehydrogenase were tested, and their enzyme activities increased to a maximum level at 2M NaCl and observed the highest level when Na^(+) was replaced with cations like K^(+), NH_(4)^(+) and anions like sodium carbonate, sodium acetate. Their optimal pH was 9. Km value for NADH oxidase was 0.052mM. NADH oxidase activity increased when SDS and Tween 80 were added.;정상적인 환경이 아닌 높은 NaCl농도에서도 충분히 생육할 수 있는 호염성 세균을 분리, 동정하고 그들의 생리적 특성과 효소의 염 의존성을 조사하였다. 염전의 토양을 10％ 식염수에 현탁하여 15％와 25％ NaCl의 Sehagal & Gibbons 배지에서 배양하여 14균주의 호염성 세균을 분리하였다. 이렇게 분리된 호염성 세균중 20-25％ NaCl 농도에서 증식하는 9균주의 고도 호염성 세균을 대상으로 형태적, 생리적 특성을 조사하였다. 그 결과 EH8, EH10, EH17, EH19, EH23은 Halobacterium. sp. 로 EH11, EH12, EH13은 Halococcus sp.로 잠정적으로 동정되었다. 이 균주들 중 25% NaCl에서 비교적 빠른 증식을 나타내는EH8,EH10을 선별하여 생리적 특성과 효소의 염 의존성을 조사하였다. EH8, EH10의 최적 증식 NaCl농도는 25％이며 10％이하에서는 증식이 억제 됐다. 최적 증식 온도는 EH8이 45℃, EH10이 50％였으며 NaCl을 KCl로 대치했을때 두 균주 모두 증식하지 못했다. 세포내 대사경로에 관여하는 효소들인 Lactate dehydrogenase, Glucokinase, Glucose-6-phosphate dehydrogenase, Alanine dehydrogenase, Isocitrate dehydrogenase의 활성을 조사한 결과 Glucose-6-phosphate dehydrogenase는 활성이 없었고 나머지 효소들은 모두 활성이 있는 것으로 나타났으며, 특히 Lactate dehydrogenase의 경우는 5M NaCl일때 높은 활성을 가졌으며, KCl은 4M일 때 활성이 높게 나타났다. 전자 전달계에 관여하는 효소인 NADH oxidase와 NADH dehydrogenase의 염 의존성을 Halobacterium sp.EH10에서 조사한 결과 이들 효소는 2M NaCl에서 최적 활성을 나타냈으며, KCI, NH_(4)Cl등의 양이온과 sodium acetate, sodium carbonate등의 음이온에 의해서도 활성화 되었으며 최적 pH는 9였다. NADH oxidase의 Km치는 0.0526mM이었고 SDS와 Tween80의 첨가에 의해 효소의 활성이 증가되었다.