DOI QR코드

DOI QR Code

Enterococcus faecalsis 유래의 신규 L-aspartate β-decarboxylase의 cloning, 정제 및 활성 규명

Cloning, Purification and Characterization of Novel L-Aspartate β-decarboxylase from Enterococcus

  • 이동근 (신라대학교 공과대학 생명공학과) ;
  • 송태윤 (신라대학교 공과대학 생명공학과) ;
  • 김남영 (신라대학교 공과대학 생명공학과) ;
  • 이어진 (신라대학교 공과대학 생명공학과) ;
  • 하상안 (신라대학교 공과대학 환경공학과) ;
  • 이재화 (신라대학교 공과대학 생명공학과) ;
  • 하종명 (신라대학교 공과대학 생명공학과) ;
  • 하배진 (신라대학교 공과대학 생명공학과) ;
  • 이상현 (신라대학교 공과대학 생명공학과)
  • Lee Dong-Geun (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Song Tae-Yoon (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Kim Nam Young (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Lee Eo-Jin (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Ha Sang-An (Department of Environmental Engineering, College of Engineering, Silla University) ;
  • Lee Jae-Hwa (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Ha Jong-Myuong (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Ha Bae Jin (Department of Bioscience and Biotechnology, College of Engineering, Silla University) ;
  • Lee Sang-Hyeon (Department of Bioscience and Biotechnology, College of Engineering, Silla University)
  • 발행 : 2006.02.01

초록

L-alanine의 산업적 생산을 위한 신규의 L-aspartate $\beta-carboxylase$ 유전자를 Enterococcus faecalis에서 검색하고 이를 대장균에 형질전환시켰다. E. faecalis 유래의 ADC유전자 는 1611 bp의 염기서열로 구성되어 있으며 형질전환된 대장균에서 59 KDa의 효소를 생산하며 L-aspartate $\beta-carboxylase$의 촉매활성을 나타내는 것을 확인하였다. 본 연구결과로 기능은 모르고 유전체 서열만 아는 균체에서 신규 효소를 개발하는 방법을 확립하였으며 저가의 aspartate를 이용한 고부가가치 L-alanine을 생산할 수 있는 신규효소를 개발할 수 있었다.

The gene for a L-aspartate $\beta-decarboxylase$ (ADC) from Enterococcus faecalis was cloned and sequenced. The gene comprised an open reading frame of 1,611 base pairs, which encodes a protein of 58,960 Da consisting of 536 amino acid residues. The gene was subcloned into an expression plasmid for overexpression of the ADC. The recombinant ADC was produced using E. coli as the host and purified to homogeneity. Our result showed that the ADC may be obtained from bacteria known nucleotide sequence. Thus, we suggest that high value L-alanine might be produced by low value aspartate.

키워드

참고문헌

  1. Abe, K., F. Ohnishi, K. Yagi, T. Nakajima, T. Higuchi, M. Sano, M. Machida, R. I. Sarker and P. C. Maloney. 2002. Plasmid-Encoded asp operon confers a proton motive metabolic cycle catalyzed by an aspartate-alanine exchange reaction. J. Bacteriol. 184, 2906-2913 https://doi.org/10.1128/JB.184.11.2906-2913.2002
  2. Bodalo, A., J. Bastida, J. L. Gomez, E. Gomez, I. Alcaraz and M. L. Asanza. 1997. Stabilization studies of L-aminoacylase-producing Pseudomonas sp. BA2 immobilized in calcium alginate gel. Enzyme Microb. Tech. 21, 64-69 https://doi.org/10.1016/S0141-0229(96)00227-X
  3. Calik, G., H. Vural and T. H. Ozdamar. 1997. Bioprocess parameters and oxygen transfer effects in the growth of Pseudomonas dacunhae for L-alanine production. Chem. Eng. J. 65, 109-116 https://doi.org/10.1016/S1385-8947(96)03159-2
  4. Fusee, M. C. and J. E. Weber. 1984. Immobilization by polyurethane of Pseudomonas dacunhae cells containing l-aspartate ${\beta}-decarboxylase$ activity and application to l-alanine production. Appl. Environ. Microbiol. 48, 694-698
  5. Fusee, M. C., W. E. Swann and G. J. Calton. 1981. Immobilization of Escherichia coli cells containing aspartase activity with polyurethane and its application for L-aspartic acid production. Appl. Environ. Microbiol. 42, 672-676
  6. Hols, P., M. Kleerebezem, A. N. Schanck, T. Ferain, J. Hugenholtz, J. Delcour and W. M. de Vos. 1999. Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering. Nat. Biotechnol. 17, 588-592 https://doi.org/10.1038/9902
  7. http://www.ncbi.nlm.nih.gov/genomes/static/eub_g.html
  8. Kengen, S. W. and A. J. M. Stams. 1994. Formation of L-alanine as a reduced end product in carbohydrate fermentation by the hyperthermophilic archaeon Pyrococcus furiosus. Arch. Microbiol. 161, 168-175 https://doi.org/10.1007/BF00276479
  9. Kobayashi, T., S. Higuchi, K. Kimura, T. Kudo and K. Horikoshi. 1995. Properties of glutamate dehydrogenase and its involvement in alanine production in alanine production in a hyperthermophilic archaeon, Thermococcus Profundus. J. Biochem. 120(3), 531-539
  10. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  11. Orlygsson, J., R. Anderson, and B. H. Svensson. 1995. Alanine as an end product during fermentation of monosaccharides by Clostridium strain P2. Anton. Leeuw. 68, 273-280 https://doi.org/10.1007/BF00874136
  12. Ravot, G., B. Ollivier, M.-L. Fardeau, B. K. Patel, K. T. Andrews, M. Magot and J.-L. Garcia. 1996. L-Alanine production from glucose fermentation by hyperthermophilic members of the domains Bacteria and Archaea: a remnant of an ancestral metabolism? Appl. Environ. Microbiol. 62, 2657-2659
  13. Sambrook, J. and D. W. Russell. 1989. Molecular Cloning: A Laboratory Manual. 2nd eds., Cold Spring Harbor Laboratory Press, New York, New York
  14. Schaefer, T., K. B. Xavier, H. Santos and P. Schoenheit. 1994. Glucose fermentation to acetate and alanine in resting cell suspensions of Pyrococcus furiosus: proposal of a novel glycolytic pathway based on $^{13}C$ labelling data and enzyme activities. FEMS Microbiol. Lett. 21, 107-114
  15. Tate, S. S. and A. Meister. 1971. L-aspartate ${\beta}-decarboxylase;$ structure, catalytic activities, and allosteric regulation. Adv. Enzymol. Relat. Areas Mol. Biol. 35, 503-543 https://doi.org/10.1002/9780470122808.ch9
  16. Ward, D. E., S. W. Kengen, J. van der Oost and W. M. de Vos. 2000. Purification and characterization of the alanine aminotransferase from the hyperthermophilic archaeon Pyrococcus furiosus and its role in alanine production. J. Bacteriol. 182, 2559-2566 https://doi.org/10.1128/JB.182.9.2559-2566.2000
  17. 임번삼. 2003. 아미노산 발효공업. KISTI 기술동향분석보고서
  18. 임번삼. 2003. 아미노산 생산균주의 개량. KISTI 기술동향분석보고서
  19. 임번삼. 2004. 발효산업의 현황과 발전방안. KISTI 기술동향보고서