Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

The Signal Sequence of Sporulation-Specific Glucoamylase Directs the Secretion of Bacterial Endo-1,4-β-D-Glucanase in Yeast

효모에서 포자형성 특이 글루코아밀라제의 분비서열에 의한 세균 endo-1,4-β-D-glucanase의 분비

  • Ahn, Soon-Cheol (Department of Microbiology and Immunology, School of Medicine, Pusan National University) ;
  • Kim, Eun-Ju (Chemistry Education Major, Division of Science Education, College of Education, Daegu University) ;
  • Chun, Sung-Sik (Department of Food Science, International University of Korea) ;
  • Cho, Yong-Kweon (Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University) ;
  • Moon, Ja-Young (Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University) ;
  • Kang, Dae-Ook (Department of Biochemistry and Health Science, College of Natural Sciences, Changwon National University)
  • 안순철 (부산대학교 의과대학 미생물학교실) ;
  • 김은주 (대구대학교 사범대학 과학교육학부 화학교육) ;
  • 전성식 (한국국제대학교 식품의약학과) ;
  • 조용권 (창원대학교 자연과학대학 보건의과학과) ;
  • 문자영 (창원대학교 자연과학대학 보건의과학과) ;
  • 강대욱 (창원대학교 자연과학대학 보건의과학과)
  • Received : 2012.01.10
  • Accepted : 2012.02.20
  • Published : 2012.02.28
Download PDF
⟨ Previous Next ⟩

Abstract

The sporulation-specific glucoamylase (SGA) of Saccharomyces diastaticus is known to be produced in the cytoplasm during sporulation. For the purpose of proving that SGA has secretory potential, we constructed a hybrid plasmid, pYESC25, containing the promoter and the putative signal sequence of the SGA fused in frame to the endo-1,4-${\beta}$-D-glucanase (CMCase) gene of Bacillus subtilis without its own signal sequence. The recipient yeast strain of S. diastaticus YIY345 was transformed with the hybrid plasmid. CMCase secretion from S. diastaticus harboring pYESC25 into culture medium was confirmed by the formation of yellowish halos around transformants after staining with Congo red on a CMC agar plate. The transformant culture was fractionated to the extracellular, periplasmic, and intracellular fraction, followed by the measurement of CMCase activity. About 63% and 13% enzyme activity were detected in the culture supernatant (extracellular fraction) and periplasmic fraction, respectively. Furthermore, ConA-Sepharose chromatography, native gel electrophoresis, and activity staining revealed that CMCase produced in yeast was glycosylated and its molecular weight was larger than that of the unglycosylated form from B. subtilis. Taking these findings together, SGA has the potential of secretion to culture medium, and the putative signal sequence of SGA can efficiently direct bacterial CMCase to the yeast secretion pathway.

효모 Saccharomyces diastaticus가 포자형성기에 세포질에서 생산된다고 알려진 포자형성 특이 glucoamylase (SGA)가 세포 외로 분비되는 단백질임을 증명하고자 S. dastaticus의 SGA promoter와 예상되는 분비신호서열 다음에 reporter gene으로 사용한 고초균의 CMCase 구조유전자를 융합한 재조합 플라스미드 pYSC25를 제작하고 수주세포인 S. diastaticus YIY345에 형질전환 하였다. 형질전환체를 1% CMC를 포함하는 최소한천배지에서 배양한 후 Congo red 염료로 염색하여 생성된 투명환으로부터 SGA의 분비서열에 의해 세균의 CMCase가 효모세포외로 분비되는 것을 확인하였다. 효모세포부위 별 CMCase의 활성분포를 측정하여 SGA 분비서열의 분비효율을 추정하기 위해 효모세포 배양액을 배양상등액, periplasmic 및 세포질 분획으로 나눈 다음 효소활성을 측정한 결과 CMCase 활성의 76%가 배양상등액과 periplasmic 부위에 존재하였으며 N-연결형 당쇄가 일어났으므로 SGA 분비서열은 효과적으로 작용함을 알 수 있었다. 대조균인 고초균에서 생산된 CMCase에서는 당쇄가 일어나지 않은 것을 확인하였다. 이상의 결과로부터 SGA는 아미노 말단에 존재하는, 24개의 아미노산으로 구성된 분비서열을 보유한 분비성 단백질임을 확인하였다.

Keywords

References

  1. Colonna, W. J. and P. T. Magee. 1978. Glycogenolytic enzymes in sporulating yeast. J. Bacteriol. 134, 844-853.
  2. Curry, C., N. Gilkes, G. O'Neill, JR. R. C. Miller, and N. Skipper. 1988. Expression and secretion of a Cellulomonas fimi exoglucanase in Saccharomyces cerevisiae. Appl. Environ. Microbiol. 54, 476-484.
  3. Dohmen, R. J., A. W. M. Strasser, C. B. Honer, and C. P. Hollenberg. 1991. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast 7, 691-692. https://doi.org/10.1002/yea.320070704
  4. Kang, D. O., I. K. Hwang, W. K. Oh, H. S. Lee, S. C. Ahn, B. Y. Kim, T. I. Mheen, and J. S. Ahn. 1999. Molecular cloning and analysis of sporulation specific glucoamylase (SGA) gene of Saccharomyces diastaticus. J. Microbiol. 37, 35-40.
  5. Kim, E. J., J. S. Ahn, and D. O. Kang. 2010. Characterization of sporulation-specific glucoamylase of Saccharomyces diastaticus. J. Life Sci. 20, 683-690. https://doi.org/10.5352/JLS.2010.20.5.683
  6. Kleinman, M. J., A. E. Wilkinson, I. P. Wright, and I. H. Evands. 1988. Purification and properties of an extracellular glucoamylase from a diastatic strain of Saccharomyces cerevisiae. Biochem. J. 249, 163-170
  7. 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
  8. Lambrechts, M. G., I. S. Pretorius, J. Marmur, and P. Sollitti. 1995. The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae. Yeast 11, 783-787. https://doi.org/10.1002/yea.320110810
  9. Miller, G. L. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 55, 952-959.
  10. Nielsen, H., J. Engelbrecht, S. Brunak, and G. von Heijne. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Engineer. 10, 1-6. https://doi.org/10.1093/protein/10.1.1
  11. Park, S. H., H. K. Kim, and M. Y. Pack. 1991. Characterization and structure of the cellulase gene of Bacillus subtilis BSE 616. Agric. Biol. Chem. 55, 441-448. https://doi.org/10.1271/bbb1961.55.441
  12. Pretorius, L. S., M. G. Lambrechts, and J. Marmur. 1991. The glucoamylase multigene family in Saccharomyces cerevisiae var. diastaticus: an overview. Crit. Rev.Biochem. Mol. Biol. 26, 53-76. https://doi.org/10.3109/10409239109081720
  13. Pugh T. A., J. C. Shah, P. T. Magee, and M. J. Clancy. 1989. Characterization and localization of the sporulation glucoamylase of Saccharomyces cerevisiae. Biochim. Biophys. Acta 994, 200-209. https://doi.org/10.1016/0167-4838(89)90294-X
  14. Schwarz W. H., K. Bronnenmeier, F. Grabnitz, and W. L. Staudenbauer. 1987. Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans. Anal. Biochem. 164, 72-77. https://doi.org/10.1016/0003-2697(87)90369-1
  15. Seidman, C. E. 1988. Escherichia coli, plasmid, and bacteriophages, In: Current protocols in molecular biology. Eds. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. John Wiley & Sons Inc. New York, unit 1.8.1-1.8.3.
  16. Theater, R. M. and P. J. Wood. 1982. Use of Congo red polysaccharide interactions in enumeration and characterization of cellulolytic bacteria from the bovine rumen. Appl. Environ. Microbiol. 43, 777-780.
  17. Verner, K. and G. Schatz. 1988. Protein translocation across membranes. Science 241, 1307-1313. https://doi.org/10.1126/science.2842866
  18. von Heinje, G. 1983 Patterns of amino acids near signal-sequence cleavage sites. Eur. J. Biochem. 133, 17-21. https://doi.org/10.1111/j.1432-1033.1983.tb07424.x