DOI QR코드

DOI QR Code

Constitutive Expression of Bacillus stearothermophilus CGTase in Bacillus subtilis.

Bacillus subtilis에서 Bacillus stearothermophilus CGTase의 구성적 발현

  • Published : 2004.06.01

Abstract

To overproduce the cyclodextrin glucanotransferase(CGTase) of Bacillus stearothermophilus NO2 in B. subtilis, the pJH-CGTl plasmid (8.14 kb) was constructed, in which the ORF of CGTase gene could be transcribed by strong constitutive promoter, P$\_$JH/. To overproduce CGTase from a recombinant B. subtilis, the effect of media on the cell growth and expression level of CGTase were investigated with various media (LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG) in the flask culture. Among them, [5% molasses+2% CSL] medium revealed the maximum expression level of CGTase with 1.8 unit/$m\ell$ at 9 hr culture. In the batch culture on [10% molasses+5% corn steep liquor] medium the expression level of CGTase, the secretion efficiency, and plasmid stability were about 4.2 unit/$m\ell$, 90% and 90%, respectively, at 30 hr culture. The cell growth and expression level in the fermenter culture with the industrial molasses medium were increased by 2-folds over the flask culture.

B. stearothermophilus NO2의 CGTase 유전자 (cgtS)를 구성적 $P_{JH}$ promoter 하류에 subcloning 하여 재조합 plasmid pIH-CGT1 (8.14 kb)을 구축하고 B. subtilis DB431에 형질 전환하였다. B. subtilis DB431/pJH-CGT1를 5가지 배지(LB, 2${\times}$LB, 5% molasses+2% CSL, CS, LBG)로 flask 배양하여 균체증식과 CGTase발현량 및 분비국재성을 조사하여 최적 배지를 결정하였다. 그 중 〔5% molasses+2% CSL〕 배지에서 9시간에 1.8 unit/$m\ell$의 CGTase가 발현$.$생산되었다. 이 결과를 토대로 3. subtilis DB431/pJH-CGT1를 〔10% molasses + 5% corn steep liquor〕 배지에서 발효조 회분 배양한 결과, 30시간 배양시 CGTase의 최대 발현량은 4.2 unit/$m\ell$, 90%의 분비 효율, 90% 이상의 plasmid 안정성을 나타내었다. 저렴한 산업용 molasses 배지로 발효조 회분배양시 플라스크 배양보다 균체증식과 CGTase 발현량이 2배 이상의 증가된 값을 얻었다.

Keywords

References

  1. Fujiwara, S., H. Kakihara, B. W. Kim, A. Leujeune, M. Kanemoto, K. Sakaguchi and T. Imanaka. 1992. Cyclization chracteristics of cyclodextrin glucanotransferase are conferred by the NH2-terminal region of the enzyme. Appl. Environ. Microbiol. 58, 4016-4025
  2. Hara, K. and H. Hashimoto. 1986. Application of cyclodextrin. J. Jpn. Soc. Starch Sci. 3, 152-161
  3. Jeon, S. J., S. W. Nam, J. W. Yun, S. K. Song and B. W. Kim. 1998. Effect of C- or D-Domain deletion on enzymatic properties of cyclodextrin glucanotransferase from Bacillus stearothermophilus NO2. J. Microbiol. Biotechnol. 8, 152-157
  4. Kazuhido, T. and K. Ouchi. 1995. Effects of yeast invertase on ethanol production in molasses. J. Fermen. Bioen. 79, 513-515 https://doi.org/10.1016/0922-338X(95)91274-9
  5. Kim, C. S., N. S. Han, D. H, D. H. Kweon and J. H. Seo. 1999. Expression of Bacillus macerans cyclodextrin glucanotransferase in Bacillus subtilis. J. Microbiol. Biotechnol. 9, 230-233
  6. Kim, J. H., J. H. Kim., S. C. Kim and S. W. Nam. 2000. Constitutive overexpression of the endoxylanase gene in Bacillus subtilis. J. Microbiol. Biotechnol. 10, 551-553
  7. Miller, G. L. 1959. Use of dinitrosalicylic acid regent for determination of reducing sugar. Anal. Chem. 55, 952-959
  8. Misaki, M. 1984. Utilization of cyclodextrin for citrus fruit products. J. Jpn. Soc. Starch Sci. 31, 98-106 https://doi.org/10.5458/jag1972.31.98
  9. Nam, S. W., D. K. Chung and B. H. Chung. 1997. Consti-tutive expression of Clostridium thermocellum endoglucanase gene in Saccharomyces cerevisiae. Kor. J. Biotechnol. Bioeng. 12, 430-437
  10. Neu, H. C. and L. A. Heppel. 1995. The release of enzymes from E. coli by osmotic shock and during the formation of spheroplast. J. Biol. Chem. 240, 3685-3692
  11. Park, H. Y., S. W. Nam and B. W. Kim. 2001. Biochemical properties of recombinant cyclodextrin glucacanotransferase expressed in Saccharomyces cerevisiae. Kor. J. Life Sci. 11, 230-234.
  12. Suzuki, M and A. Satoh. 1983. Nutritional consequences of $\alpha$-cyclodextrin. J. Jpn. Soc. Starch Sci. 30, 240-246. https://doi.org/10.5458/jag1972.30.240
  13. Szejtli, J. 1990. The Cyclodextrins and their applications in biotechnology. Carbohydrate Polymers. 12, 375. https://doi.org/10.1016/0144-8617(90)90088-A
  14. Szente, L. and J. Szejitli. 1986. Molecular encapsulation of natural and synthetic coffee flavor with $\beta$-cyclodextrin. J. Food Sci. 51, 1024-1027. https://doi.org/10.1111/j.1365-2621.1986.tb11224.x
  15. Vokk, R., A. Mener and E. K. Saar. 1991. Biotechnology of $\beta$-cyclodextrin. BFE. 8, 510-516.
  16. Ye, R., J. H. Kim., B. G. Kim., S. Szarka., E. Sihota and S. L. Wong. 1998. High-level secretory production of intact, biologically active staphylokinase from Bacillus subtilis. Biotechnol. Bioeng. 62, 87-96.
  17. You, D. J., H. Y. Park, S. J. Jeon, H. J. Kwon, S. W. Nam and B. W. Kim. 2002. Expression of the Bacillus stearothermophilus NO2 CGTase gene in Saccharomyces cerevisiae. Kor. J. Microbiol. Biotechonol. 30, 206-209.
  18. Yu, E. K. C. 1988. Novel decaffeination process using cyclodextrin. Appl. Microbiol. Biotechnol. 28, 546-552.