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Bacillus megaterium J-65에 의한 xylose로부터 poly-3-hydroxybutyrate 생산

Production of Poly-3-hydroxybutyrate from Xylose by Bacillus megaterium J-65

  • 전홍기 (부산대학교 자연과학대학 미생물학과) ;
  • 진영희 (부산대학교 자연과학대학 미생물학과) ;
  • 김해남 (마산대학 뷰티케어과) ;
  • 김윤태 (서울의과학연구소) ;
  • 김삼웅 (부산대학교 자연과학대학 미생물학과) ;
  • 백형석 (부산대학교 자연과학대학 미생물학과)
  • Jun, Hong-Ki (Department of Microbiology, College of Natural Science, Pusan National University) ;
  • Jin, Young-Hi (Department of Microbiology, College of Natural Science, Pusan National University) ;
  • Kim, Hae-Nam (Department of Beauty Care, Masan College) ;
  • Kim, Yun-Tae (Seoul Medical Science Institute and Seoul Clinical Laboratories) ;
  • Kim, Sam-Woong (Department of Microbiology, College of Natural Science, Pusan National University) ;
  • Baik, Hyung-Suk (Department of Microbiology, College of Natural Science, Pusan National University)
  • 발행 : 2008.12.30

초록

본 연구는 생분해성 플라스틱인 poly-${\beta}$-hydroxybutyrate (PHB)의 생산단가를 낮추기 위한 노력으로 저가의 기질로부터 PHB 대량생산을 위한 기초자료를 얻는데 그 목적을 두었다. Hemicellulose hydrolysate는 지구상에 풍부하게 존재하는 저가의 waste by-product로서 xylose가 많이 포함되어 있다. 본 연구에서는 xylose로부터 PHB를 생산할 수 있는 균주를 토양에서 분리하여, 분류학적 위치를 밝히고, 균체 생육 최적 조건, PHB 생산을 위한 최적 발효 배양 조건, PHB의 구조 확인 등을 검토 하였으며, 그 결과는 다음과 같다. 토양으로부터 분리한 균주 J-65는 형태학적, 배양적, 생화학적 및 partial 16S rRNA sequence에 근거하여 Bacillus megaterium로 동정하였다. B. megaterium J-65의 균체 생육 최적 조건은 온도 $37^{\circ}C$, 초발 pH 8.0이었으며 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, 0.1% $KH_2PO_4$였다. PHB 축적에 영향을 미치는 요인을 검토하기 위해 균체생육 최적배지에서 $37^{\circ}C$, 24시간 1차 배양한 후, 균체를 회수하여 각종 영양분이 결핍된 배지에 2차 배양을 실시한 결과 B. megaterium J-65는 균형생육조건(balanced-growth condition)에서 PHB를 합성하는 균주로 나타났다. PHB보다 물성이 향상된 PHB/HV 공중 합체를 생산하기 위하여 보조기질로 propionic acid를 첨가하였을 때, 0.1% propionic acid 농도에서 HV mol%가 14%인 PHB/HV 공중합체가 합성되었다. 5 l 용량의 발효조에 B. megaterium J-65를 회분배양하였을 때 배양 21시간에 건조균체량 10 g/l, PHB 3.5 g/l를 얻을 수 있었고, 유가배양을 실시한 결과 배양 48시간에 건조균체량 26.52g/l, PHB 9.28 g/l를 얻을 수 있었다. 생산된 PHB를 alkaline solution 처리와 chloroform을 이용한 유기용매 추출법을 이용하여 추출.정제한 후 Gas Chromatography로 정제를 확인하고 300MHz 1H-NMR을 실시한 결과 3-hydroxybutyrate의 homopolymer임을 확인하였다.

A microorganism capable of producing high level of poly-3-hydoxybutyrate (PHB) from xylose was isolated from soil. The isolated strain J-65 was identified as Bacillus megaterium based on the morphological, biochemical and molecular biological characteristics. The optimum temperature and pH for the growth of B. megaterium J-65 were $37^{\circ}C$ and 8.0, respectively. The optimum medium composition for the cell growth was 2% xylose, 0.25% $(NH_4)_2SO_4$, 0.3% $Na_2HPO_4{\cdot}12H_2O$, and 0.1% $KH_2PO_4$. The optimum condition for PHB accumulation was same to the optimum condition for cell growth. Copolymer of ${\beta}$-hydroxybutyric and ${\beta}$-hydroxyvaleric acid was produced when propionic acid was added to shake flasks containing 20 g/l of xylose. Fermenter culture was carried out to produce the high concentration of PHB. In batch culture, cell mass was 9.82 g/l and PHB content was 35% of dry cell weight. PHB produced by B. megaterium J-65 was identified as homopolymer of 3-hydoxybutyric acid by GC and NMR.

키워드

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