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The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Isolation of Mutant Strains from Keratinase Producing Bacillus subtilis SMMJ-2 and Comparision of Their Enzymatic Properties

Keratinase 생산균 Bacillus subtilis SMMJ-2의 변이주 분리와 효소학적 특성 비교

  • Ko, Hee-Sun (Department of Microbiology, College of Natural Science, Keimyung University) ;
  • Kim, Hyun-Soo (Department of Microbiology, College of Natural Science, Keimyung University)
  • 고희선 (계명대학교 자연과학대학 미생물학과) ;
  • 김현수 (계명대학교 자연과학대학 미생물학과)
  • Received : 2010.05.27
  • Accepted : 2010.10.21
  • Published : 2010.10.31
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Abstract

Keratinase is widely used in certain industrial applications. The present study sought to improve the culture conditions of Bacillus subtilis SMMJ-2 to facilitate mass production of keratinase. Strain SMMJ-2 was irradiated by ultraviolet light and the resulting isolates were tested for keratinase activity. Isolates displaying elevated keratinase activity were selected and used to determine the optimum temperature (24, 30, 37, 45, $55^{\circ}C$) for bacterial keratinase production during a 4 day incubation period. The highest enzyme activity (55 units/mL/min), from a Bacillus subtilis SMMJ-2 mutant (mutant No. 2) was demonstrated following incubation at $30^{\circ}C$. The effects of carbon and nitrogen sources on keratinase production were confirmed by measuring the enzyme activity from the culture broth of the mutant strain cultured in various media containing different carbon source and nitrogen sources during a 4 day period. The optimal medium composition for producing keratinase consisted of 1% glucose, 0.7% $K_2HPO_4$, 0.2% $K_2HPO_4$, and 1.2% soybean meal. Optimal initial pH and temperature for producing keratinase were 7.0 and $30^{\circ}C$, respectively. Keratinases produced by B. subtilis SMMJ-2 and the mutant No. 2 were purified from the culture broth which used soybean meal as a nitrogen source. Membrane ultrafiltration, DEAE-sephacel ion exchange and Sephadex G-100 gel chromatography were used to purify the enzymes. The purified keratinases from both B. subtilis SMMJ-2 and the mutant No. 2 showed single bands and their molecular weights were estimated as 28 kDa and 42 kDa, respectively on SDS-polyacrylamide gel electrophoresis.

본 연구는 선행연구에서 얻은 keratinase 효소의 활성이 높은 균주 Bacillus subtilis SMMJ-2를 UV 조사에 의해 개량하여 mutant No. 2를 얻었으며, keratinase의 생산성 향상을 위한 최적 탄소원, 최적 질소원의 조건 하에서 본 효소를 대량생산하여 정제하고, 야생주와 변이주 간의 효소활성의 변화 및 정제된 효소 간의 효소화학적인 성질을 비교하였다. Mutant No. 2의 keratinase 생산을 위한 최적 탄소원과 질소원은 각각 glucose와 soybean meal로 나타나, 야생주의 경우와는 최적 질소원을 달리했으며, 효소활성에 있어서는 야생주보다 40% 정도 상승하였다. 변이주의 효소가 야생주의 효소보다 높은 배양온도에 대하여 더 안정적인 활성으로 생산되며, 효소 생산을 위한 최적 pH는 7.0으로, 비교적 효소생산이 가능한 pH 영역대은 6~9로 나타났다. Bacillus subtilis SMMJ-2와 mutant No. 2에서 생산된 keratinase는 DEAEsephacel 크로마토그래피법와 겔여과 크로마토그래피법으로 최종 정제되었다. 정제과정 중 DEAE-sephacel 크로마토그래피 상에서 나타나는 2개의 효소피크는 Bacillus subtilis SMMJ-2의 메인 효소피크의 위치와 mutant No. 2에서의 메인 효소피크의 위치가 전환되어 나타났다. SDS-PAGE 상에서의 각각의 효소 분자량은 B. subtilis SMMJ-2의 경우에 28 kDa, mutant No. 2의 경우에 42 kDa 로 추산되었다

Keywords

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