한국미생물·생명공학회지 (Microbiology and Biotechnology Letters)

  • 제36권4호
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  • Pages.314-319
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  • 2008
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  • 1598-642X(pISSN)
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  • 2234-7305(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Penicillin G Amidase생산을 위한 재조합 대장균의 유가배양에 관한 연구

Fed-batch Culture of Recombinant E.coli for the Production of Penicillin G Amidase

  • 발행 : 2008.12.28
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초록

Penicillin G amidase(PGA, benzylpenicillinamidohydrolase, EC 3.5.1.11)는 penicillin G를 phenylacetic acid(PAA)와 6-aminopenicillanic acid(6-APA)로 분해하는 효소이다. Escherichia coli(E. coli) ATCC 11105의 PGA는 24 kDa의 small subunit과 65 kDa의 large subunit으로 구성되어 있고, precursor polypeptide에서 signal peptide와 spacer peptide가 절단되어 활성을 가진 heterodimer가 형성된다. 본 연구에서는 E. coli ATCC 11105에서 PCR(polymerase chain reaction)을 통해 증폭한 pga gene을 expression vector에 넣어 pET-pga plasmid를 제작하였고, 이것을 E. coli BL21 (DE3) 균주에 형질 전환하여 PGA를 발현하고 그 활성을 분석하였다. E. coli BL21(DE3)/pET-pga 균주의 고밀도 배양액을 SDS-PAGE로 분석 했을 때, PGA의 precursor, large subunit, 그리고 small subunit으로 보이는 protein band가 나타났으며, PGA가 soluble form의 precursor로 발현되어 processing을 거쳐서 large subunit과 small subunit으로 절단되기도 하고, 일부는 insoluble form의 precursor로 발현되기도 하는 것으로 생각된다. 유가배양시 온도변화 전략을 사용하여 고농도 배양에서 발현을 유도하였다. 온도변화 전략은 $37^{\circ}C$에서 $28^{\circ}C$를 거쳐 $22^{\circ}C$로 3단계로 변화시켰다. 이러한 전략으로 PGA활성은 19.6 U/mL이며 균체량은 600 nm에서 흡광도가 62까지 도달하였다.

Penicillin G amidase (PGA, benzylpenicillinaminohydrolase, EC 3.5.1.11) is industrially important enzyme which converts penicillin G to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA). The PGA in E. coli ATCC 11105 is secreted into the periplasm after removing signal sequences and becomes heterodimer which composed of two subunits, small subunit (24 kDa) and large subunit (65 kDa). In this study, the PGA gene was obtained from E. coli ATCC 11105 using PCR (polymerase chain reaction) technique. The active PGA was successfully secreated into periplasm in E. coli BL2 1(DE3) harboring pET-pga plasmid. The optimized fed-batch fermentation, consisting of a three-step shift of culture temperature from $37^{\circ}C$ to $22^{\circ}C$, gave a productivity of 19.6 U/mL with a cell growth of 62 O.D. at 600 nm.

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참고문헌

  1. Buttrick, P. 2006. The regulation of heat shock protein expression: how, when and where. J. Mol. Cell. Cardiol. 41: 785-786 https://doi.org/10.1016/j.yjmcc.2006.07.020
  2. Carty, S. M., K. R. Sreekumart, and C. R. H. Raetz. 1999. Effect of cold shock on lipid A biosynthesis in Escherichia coli. J. Biol. Chem. 274: 9677-9685 https://doi.org/10.1074/jbc.274.14.9677
  3. Cheng, S. W., Q. X. Song, D. Z. Wei, and B. X. Gao. 2007. High-level production penicillin G acylase from Alcaligenes faecalis in recombinant Escherichia coli with optimization of carbon sources. Enzyme. Microb. Technol. 41: 326-330 https://doi.org/10.1016/j.enzmictec.2007.02.011
  4. Choi, K. S., J. A. Kim, and H. S. Kang. 1992. Effects of sitedirected mutations on processing and activities of penicillin G acylase from Escherichia coli ATCC 11105. J. Bacteriol. 174: 6270-6276 https://doi.org/10.1128/jb.174.19.6270-6276.1992
  5. Deak, P. M., S. Lutz-Wahl, H. Bothe, and L. Fisher. 2003. Bioreactor cultivation of Escherichia coli for production of recombinant penicillin G amidase from Alcaligenes faecalis. Biotech. Lett. 25: 397-400 https://doi.org/10.1023/A:1022498314354
  6. French, C., E. K. Moore, and J. M. Ward. 1996. Development of a simple method for the recovery of recombinant proteins from the Escherichia coli periplasm. Enzyme Microb. Technol. 19: 332-338 https://doi.org/10.1016/S0141-0229(96)00003-8
  7. Fukushima, H., C. E. Martin, H. Iida, Y. Kitajima, and G .A. Nozawa. 1976. Changes in membrane lipid composition during temperature adaptation by a thermotolerant strain of Tetrahymena pyriformis. Biochim. Biophys. Acta. 43: 165-179
  8. Kheirolomoom, A., M. Ardjmand, H. Fazelinia, and A. Zakeri. 2001. Clarification of penicillin G acylase reaction mechanism. Process Biochem. 36: 1095-1101 https://doi.org/10.1016/S0032-9592(01)00145-5
  9. Kim, J. H., H. J. Kang, E. S. Kim, J. H. Kim, and Y. M. Kim. 2004. One-step purification of poly-his tagged penicillin G acylase expressed in E.coli. J. Microbial. Biotechnol. 14: 231-236
  10. Lamotte, D., M. Ouzzine, S. Fournel-Gigleux, J. Magdalou, and J. Boudrant. 1996. A temperature profile in batch culture to increase the production of the recombinant UDPglucuronosyltransferase 2B4 in Escherichia coli. Process Biochem. 31: 235-241 https://doi.org/10.1016/0032-9592(95)00055-0
  11. Lan, J. C., T. C. Ling, G. Hamilton, and A. Lyddiatt. 2006. A fermentation strategy for anti-MUC1 C595 diabody expression in recombinant Escherichia coli. Biotechnol. Bioprocess Eng. 11: 425-431 https://doi.org/10.1007/BF02932310
  12. Oh, S. J., Y. C. Kim, Y. W. Park, S. Y. Min, I. S. Kim, and H. S. Kang. 1987. Complete nucleotide sequence of the penicillin G acylase gene and the flanking regions, and its expression in Escherichia coli. Gene 56: 87-97 https://doi.org/10.1016/0378-1119(87)90161-2
  13. Panoff, J. M., B. Thammavongs, M. Gueuen, and P. Boutibonnes. 1998. Cold stress responces in mesophilic bacteria. Cryobiology 36: 75-83 https://doi.org/10.1006/cryo.1997.2069
  14. Park, Y. C., S. J. Kim, J. H. Choi, W. H. Lee, K. M. Park, M. Kawamukai, Y. W. Ryu, and J. H. Seo. 2005. Batch and fedbatch production of coenzyme Q(10) in recombinant Escherichia coli containing the decaprenyl diphosphate synthase gene from Gluconobacter suboxydans. Appl. Microbiol. Biotechnol. 67: 192-196 https://doi.org/10.1007/s00253-004-1743-y
  15. Pierce, J. J., C. Turner, E. K. Moore, and P. Dunnil. 1997. Factors determining more efficient large-scale release of a periplasmic enzyme from E. coli using lysozyme. J. Biotech. 58: 1-11 https://doi.org/10.1016/S0168-1656(97)00116-8
  16. Rinas, U., H. A. Krackehelm, and K. Schugerl. 1989. Glucose as a substrate in recombinant strain fermentation technology by-product formation, degradation and intracellular accumulation of recombinant protein. Appl. Microbiol. Biotechnol. 31: 163-167 https://doi.org/10.1007/BF00262456
  17. Sambrook, J., E. F. Fritch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A
  18. Sobotkova, L., V. Stepanek, K. Plhackova, and P. Kyslík. 1996. Development of a high-expression system for penicillin G acylase based on the recombinant Escherichia coli strain RE3(pKA18). Enzyme and Microbial Technology. 19: 389-397 https://doi.org/10.1016/S0141-0229(96)00052-X
  19. Strandberg, L,. and S. O. Enfors. 1991. Batch and fed batch cultivations for the temperature induced production of a recombinant protein in Escherichia coli. Biotechnol. Lett. 13: 609-614 https://doi.org/10.1007/BF01033419
  20. Vaara, M. and M. Nuaminen. 1999. Outer membrane permeability barrier in Escherichia coli mutant that are defective in the late acyltransferase of lipid A biosynthsis. Antimicrob. Agents Chemother. 43: 1459-1462
  21. Vorachek-Warren, M. K., S. M. Carty, S. Lin, R. J. Cotter, and C. R. H. Raetz. 2002. An Escherichia coli mutant lacking the cold shock-induced palmitoleoyltransferase of lipid A biosynthesis. J. Biol. Chem. 277: 14186-14193 https://doi.org/10.1074/jbc.M200408200
  22. Wouters, J. A., H. H. Kamphuis, J. Hugenholtz, O. P. Kuipers, W. M. De-Vos, and T. Abee. 2000. Changes in glycolytic activity of Lactococcus lactis induced by low temperature. Appl. Environ. Microbiol. 66: 3686-3691 https://doi.org/10.1128/AEM.66.9.3686-3691.2000
  23. Yang, Y., R. Biedendieck, W. Wang, M. Gamer, M. Malten, D. Jahn, and W. D. Deckwer. 2006. High yield recombinant penicillin G amidase production and export into the growth medium using Bacillus megaterium. Microbial Cell Factories 5: 36-50 https://doi.org/10.1186/1475-2859-5-36
  24. Yim, S. C., K. J. Jeong, H. N. Chang, and S. Y. Lee. 2001. High-level secretory production of granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli. Bioprocess and Biosystems Engineering 24: 249-254 https://doi.org/10.1007/s004490100267