내알카리성 Bacillus species BC No-1의 α-amylase 생성과 특성에 관한 연구

Abstract

정상적 환경이 아닌 알칼리성 환경하에서도 충분히 생육할 수 있는 세균에 의해 생성된 α-amylase의 성질을 규명하기 위하여 토양(pH 5.9)에서 포자를 형성하는 알칼리성 세균을 분리·동정하여 Bacillus속임을 밝히고 알칼리성 세균이 생성하는 α-amylase 의 생성과 특성을 조사하였다. 알칼리성 amylase를 생성하는 Bacillus속을 분리하기 위하여 여러 토양의 시료를 채취하여 소량을 멸균생리식염수에 현탁시키고 80℃에서 10분간 가열한 후 알칼리성 배지(pH 10.3)를 넣은 제트리 접시들에 접종했다. 그 페트리 접시들을 35℃에서 하룻밤 배양한 후 0.1N KI-Ⅰ_(2)용액을 뿌려 보아 투명대가 크게 형성된 균주들을 1차 선정하였다. 이 균주들을 다시 알칼리성 액체 배지(pH 10.3)에 배양한 다음 균주들의 α-amylase활성을 조사하여 알칼리 조건하에서 α-amylase활성이 가장 높은 균주를 선택하였다. 이 균주의 형태적, 생리적, 생화학적 특징을 조사한 결과 Bacillus circulance와 유사하였으며 Bacillus species BC No.-1 이라 명명하였다. 이 균주의 최적 생육 pH는 10.5였다. 그리고 pH6-11까지 모두 성장하였다. 최적 생육 온도는 40℃였으며 최소 생육 온도는 10℃이고 최대 생육 온도는 50℃였다. 내알칼리성 Bacillus species BC №-1의 amylase 생성을 위한 최적배양조건을 보면 다음과 같다. 알칼리성 배지(pH 10.5)에서 40℃, 3일간 진탕배양한 경우 α-amylase가 가장 많이 생성되었다. 탄소원의 경우는 α-amyase는 soluble starch(2％)를 첨가하였을 때 가장 많이 생성되었으며 β-amylase는 lactose(2%)를 첨가하였을 때 가장 많이 생성되었다. 유기 질소원의 경우는 α-amylase나 β-amylase가 모두 polypeptone(2%)를 첨가하였을 때 가장 많이 생성되었고 배지의 pH를 조절하기 위한 시료로써는 α-amyase의 경우 Na_(2)CO_(3) 1.0%를 첨가하였을 때 가장 많이 생성되었고 β-amylase의 경우 Na_(2)CO_(3) 1.0%를 첨가하였을 때 가장 많이 생성되었다. 금속염의 효과를 보면 배지에 CaCl_(2)를 첨가하였을 때 α-amylase가 가장 많이 생성되었다. 조효소로써 α-amylase의 특성을 보면 다음과 같다. α-amylase의 ㎞치는 5.88 ㎎/㎖였고 최적 pH는 9였으며 pH8.5에서 가장 안정하였다. 최적 온도는 55℃ 였으며 50℃까지 안정하였고 3mM Cacl_(2)를 첨가할 경우에 55℃까지 안정성을 보임으로써 온도에 대한 안정성에 Ca_(++)이온이 큰 역할을 함을 알 수 있었다. 그리고 내알칼리성 Bacillus의 α-amylase를 유안침전, 투석, DEAE-cellulose column chromatography등의 과정을 통해서 정제하여 23.7배 농축되었으며 회수율은 4.92%였다.;A alkalophilic bacteria isolated from soil(pH 5.9) were identified to be Bacillus species. The formation of amylase by these bacteria and its properties were investigated. To isolate Bacillus species which produce alkaline amylase from soil, various samples of soil were collected. Small amounts of samples were suspended in sterile saline solution and heated at 80℃ 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 an overnight incubation at 35℃. First, colonies with a large clear zone around them were selected. The strains were incubated in alkaline liquid media. Thereafter α-amylase activity was tested and a strain with the highest α-amylase activity in alkaline condition was selected. The morphological, physiological and biochemical characteristics of this strain were investigated. The conclusion was that it resembled with Bacillus circulance, and it was named "Bacillus species BC No-1". The optimum growth pH of this strain was 10.5, and it grew at the range of pH 6 - pH 11. The optimum growth temperature of this strain was 40℃. The minimum growth temperature was 10℃ and the maximum growth temperature was 50℃. Optimum cultural conditions for the formation of amylase by alkaline-tolerant Bacillus species BC No-1 were following ; When this strain was incubated in an alkaline midia(pH 10.5) with shaking at 40℃ for three days, the highest α-amylase productivity was showed. α-Amylase was produced the highest level when soluble starch(Z%) was added as one of carbon sources and B-amylase was produced the highest level when lactose was added as one of carbon sources. Either α-amylase or β-amylase was produced the highest level when polypeptone(2%) was added as one of organic nitrogen sources. α-Amylase was produced the highest level when Na_(2) CO_(3) (1.5%) was added as one of reagents for pH control of the media, and β-amylase was produced the highest level when Na_(2) CO_(3)(l% was added, (0.05%) was added as one of minerals, α-Amylase was produced the highest level when CaCl_(2) (0.05%) was added as one of minerals. Characteristics of α-amylase as crude enzyme were as followes Km value for the enzyme was 5.88mg/ml. Its optimum pH was 9 at 55℃ and it had the highest stability at 8.5. Its optimum tem- perature was 55℃ was stable up to 50℃. It was verified that Ca^(+) played an important role in the temperature stability of α- amylase, since α-amylase was stable up to 55℃ when 3mM CaCl_(2) was added to buffer solution. α-Amylase produced by alkaline-tolerant Bacillus species BC No-1 was concentrated up to 23.7 times and its recovery rate was 4.92% through all the processes of ammonium sulfate salting out, dialysis and DEAE-cellulose column chromatography.