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알칼리성 Bacillus species AL-8의 amylase 생성과 gene cloning

Title
알칼리성 Bacillus species AL-8의 amylase 생성과 gene cloning
Other Titles
STUDIES ON THE CLONING AND PRODUCTION OF ALKALINE AMYLASE BY ALKALINE BACILLUS SPECIES AL-8
Authors
황재원
Issue Date
1986
Department/Major
대학원 생물학과
Keywords
알칼리성Bacillus species AL-8Aamylasegene cloning
Publisher
이화여자대학교 대학원
Degree
Master
Abstract
알칼리성 조건하에서도 생육할 수 있는 알칼리성 세균을 분리하고 그 균주에 의해 생성된 알칼리성 amylase의 성질을 조사한 후 해당 gene을 E. coli에 cloning하였다. 알칼리성 세균을 분리하기 위하여 여러곳의 토양을 채취해 소량을 멸균생리식염수에 현탁하고 80℃에서 10분간 열처리한 후 알칼리성 배지(pH10.3)에 접종하였다. 접종 후 30℃에서 48시간 배양시키고 0.1N 요오드용액을 뿌려 투명대가 형성되는 균주들을 1차선정한 다음 이 균주들을 다시 알칼리성 액체배지에(pH10.3) 3일간 30℃ 에서 배양한 다음 amylase 활성을 조사하여 알칼리성 amylase 활성이 높은 균주를 최종적으로 선별하였다. 이 균주의 형태학적, 생리적, 생화학적 특징을 조사한 결과 Bacillus 속에 속하며 Bacillus megaterium과 유사하였으며 Bacillus Species AL-8이라 명명하였다. 이 균주의 생육을 위한 pH범위는 pH 6.5-11이였고 최적생육 pH는 9.0이였다. 생육온도 범위는 10℃에서 45℃이고 최적생육온도는 35℃ 였다. 알칼리성 Bacillus species AL-8의 알칼리성 amylase 생성을 위한 조건은 다음과 같다. 알칼리성 배지에서(pH10.3) 35℃, 36시간 진탕배양한 경우 알칼리성 amylase가 가장 많이 생성되었다. 배지 조성 중 탄소원으로는 soluble starch를 첨가하였을 때 알칼리성 amylase가 가장 많이 생성되었으며 maltose, glucose는 저해역할을 하였다. 유기질소원으로는 yeast extract가 가장 효과적이였으며 탄산염으로는 Na_(2)CO_(3) 1.0%를 첨가하여 pH를 조절했을 때 가장 많이 생성되었다. 금속염의 효과는 배지에 Na_(2)WO_(4)·2H_(2)O 와 MgSO_(4)·7H_(2)O를 첨가하였을 때 가장 많이 생성되었다. 알칼리성 amylase의 gene cloning을 위해, Bacillus species AL-8로부터 chromosomal DNA를 추출하여 알칼리성 amylase gene source로 하였고 vector plasmid로는 E. coli RD103에서 추출한 pBR322를 사용하여, 얻은 hybrid plasmid DNA를 E. coli HB101에 형질전환시켰다. 형질전환 빈도는 1.1×10^(-5) 이였고 형질전환주 중 amylase 활성을 가진 균주를 1차, 2차선별을 통해 최종 3균주를 선발하여 그 hybrid DNA를 추출하였다. 추출한 hybrid DNA는 전기 영동으로 확인되었다.;Bacillus species AL-8 isolated from soil produced alkaline amylase in alkaline media (pH 10.3) ,Amylase gene of B.species AL-8 was cloned in E. coli HB 101 using pBR322 as a vector. To isolate Bacillus species which produce alkaline amylase, various samples of soil were taken, small amounts of samples were suspended in sterile saline solution and heated at 80 ˚C for ten minutes, and then spreaded on petri-dishes containing alkaline media. 0.1N KI-I, solution was sprayed on the colonies grown on the petri-dishes after 2 days incubation at 3O ˚C. First, colonies with a larger clear zone around them were selected. The strains were incubated in alkaline liquid media. Thereafter Amylase activity was tested and a strain with the highest amylase activity in alkaline condition was selected. The morphological, physiological and biochemical characteristics of this strain were investigated. It was identified to be Bacillus species and resembled with Bacillus megaterium. It was named Bacillus species AL- 8.The optimum growth pH of this strain was 9.0 and it grew at pH 6.5-11.0. The optimum growth temperature of this strain was 35˚C. The minimum growth temperature was 10˚C and the maximum was 45˚C. Optimum cultural conditions for the production of alkaline amylase by Bacillus species AL-8 were following; when this strain was incubated in an alkaline media (pH 10.3) with shaking at 35˚C for 36 hours, the highest amylase productivity was showed. The alkaline amylase was most active at pH 10.0 and at 50 ˚C. Amylase was produced the highest level when soluble starch was added as carbon source, yeast extract as organic nitrogen source, Na_(2)CO_(3)(1.0%) as reagent for pH control of the media and Na_(2)SO_(4)·7H_(2)0, MgSO_(4)·7H_(2)O as metal salts. Bacillus species AL-8 chromosomal DNA was used as alkaline amylase gene source and pBR322 used as a vector was extracted from E. coli RD 103. Chromosmal DNA of Bacillus species AL-8 was partially digested with EcoRI and pBR322 was cleaved with EcoRI. The hybrid plasmid DNA was constructed by shot gun method and transformed to E. coli. HB 101. The amount of amylase produced by transformants was about 25-30% of that produced by Bacillus species AL-8. As the result of agarose gel electrophoresis, hybrid plasmid DNA was found to be various in its size.
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