알칼리성 Bacillus sp. 9-18의 고온 알칼리성 protease의 분리 정제와 그 특성에 관한 연구

Abstract

알칼리의 조건하에서 잘 자라는 알칼리성 세균을 분리, 동정하고 그 균주에 의해 생성되는 알칼리성 단백분해효소의 성질을 조사한 후, 정제하고 분자량을 알아보았다. 알칼리성 세균을 분리하기 위해 여러 곳의 퇴비를채취해서 소량을 멸균 생리 식염수에 현탁하고 알칼리성 배지(pH10.3)에 접종하여 55℃에서 48시간 배양하였다. 생성된 균주들은 알칼리성 액체배지(pH10.3)에 접종하여 50℃에서 3일간 배양한 후 protease 활성을 조사하여 알칼리 조건하에서 protease의 활성이 높은 균주를 선별하였다. 이 균주의 형태적, 배양학적, 생리적 특징을 조사한 결과 Bacillus alkaophilus subsp. halodurans와 유사한 것으로 추정되었다. 이 균주의 생육을 위한 pH 범위는 7.5-12이었고 최적 생육 pH는 10이었다. 생육 온도 범위는 15-58℃이고 최적 생육 온도는 50℃였다. 알칼리성 Bacillus sp. 9-18의 알칼리성 protease의 생성과 반응을 위한 조건은 다음과 같다. 알칼리성 배지에서 45℃, 3일간 진탕 배양한 경우 알칼리성 protease가 가장 많이 생성되었다. 탄소원으로는 glucose를 첨가하였을 때, 유기 질소원으로는 beef extract가 가장 효과적이었으며, 탄산염으로는 Na_(2)Co_(3)를 첨가하여 pH를 조절하였을 때 가장 많이 생성되었다. 금속염의 효과는 MgSO_(4) 7H_(2)O를 첨가하였을 때 가장 좋았다. 조효소로서 protease의 특성은 최적 pH는 12였고, 넓은 pH 범위에서 안정하였으며, 최적 온도는 70℃였고, 60℃에서 1시간 동안 안정하였다. 그리고, 알칼리성 Bacillus sp. 9-18의 protease를 아세톤 침전, Sephadex G-100, Sephadex G-75 gel filtration을 통해 정제하였고, 회수율은 8.6％였으며 ㎞치는 1.0㎎/㎖였다. Sephadex G-100 gel filtration을 통한 결과 분자량은 31,000인 것으로 나타났다. 이 효소는 Mg, Ca, Mn 등에 의해서는 효소 활성이 촉매되었고, Cu, Hg에 의해서는 현저히 저하되었다. 또, 다양한 범위의 detergent에 대해 안정하였는데, Sodium dodecyl benzen sulfonate에 의해서는 저해되었다.;An alkaline protease was prepared from microorganism isolated from composts. The characteristic point of this microorganism is especially good growth in alkaline and thermal condition. To isolate these strains, various samples of composts were collected. A few samples were suspended in sterile saline solution, spreaded on petridishes containing alkaline media and incubated at 55˚C. After 2 days of incubation, colonies grown were isolated. The strains were incubated in alkaline liquid medium at 50˚C for 3 days. Thereafter protease activity was tested and a strain with the highest protease activity in alkaline condition was selected. The selected strain was resembled with Bacillus alkalophilus subsp. halodurans from the point of morphological, physiological, and biochemical characteristics. The optimum growth pH of the strain was 10, and it grew at the range of pH 7.5-12. The optimum growth temperature of the strain was 50˚C. The minimum growth temperature was 15˚ C, and the maximum growth temperature was 58˚C. Optimum cultural conditions for the formation of protease by the strain were as follows : When the strain was incubated in an alkaline media with shaking at 45˚C for 3days, the highest protease productivity was shown. Protease was produced at the highest level when glucose was added as carbon source, beef extract and casein as organic nitrogen source, Na_(2)C0_(3) as reagent for pH control of media and MgS0_(4)7H_(2)0 as metal salts. Protease was purified from crude enzyme by acetone precipitation, Sephadex G-100 and Sephadex 6-75 gel filtration Through the simple Sephadex gel chromatographies, the enzyme was purified to homogeneity with specific activity of 67.4 unit/ug protein fold higher than that of the culture broth. Characteristics of the partially purified enzyme were as follows. Km value for the enzyme was 1mg/ml. The enzyme sample showed a maximal activity at 70˚C, pH 12. The enzyme was stable in various pH at room temperature, and stable at 60˚C for 1 hr. The enzyme was stable in various detergents and metal ions, but was inactivated by HgCl_(2), and DBS. The molecular weight o f the enzyme was estimated by Sephadex G-100 gel filtration to be 31,000.