Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
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Purification and Characterization of the Overproduced E. coli Endochitinase

과량 생산된 대장균 chitin 분해효소의 정제 및 특성 조사

  • 황희영 (중앙대학교 산업과학대학 생명공학과) ;
  • 김우연 (중앙대학교 산업과학대학 생명공학과)
  • Published : 2003.08.30
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Abstract

The putative endochitinase gene, yheB of Escherichia coli K-12 is not expressed under lab culture conditions. The endochitinase gene was amplified by PCR and subcloned into pET28c vector and pQE9 vector, respectively. The endochitinase produced in E. coli harboring pET28c containing yheB or pQE9 vector containing yheE was partly released into the growth medium. The overproduced endochitinase was partially purified by His affinity column chromatography and DE-52 column chromatography. The apparent molecular weight of the endochitinase determined by SDS-polyacrylamide gel electrophoresis was about 97,000. The purified E. coli endochitinase showed maximal chitinolytic activity at pH 6 and $40^{\circ}C$.

실험실 배양 조건에서는 발현되지 않는 대장균 K-12의 endochitinase 유전자(yheR)를 PCR로 증폭하여 pET28c와 pQE9벡터에 각각 클로닝 하였다. yheB유전자를 가진 pET28c와 pQE9 벡터를 함유한 대장균에서 생산된 endochitinase는 생장배지 내로 일부 분리되었다. 과량 생산된 endochitinase를 His-affinity 크로마토그래피와 DE-52 크로마토그래피로 부분 정제하였으며 SDS-PAGE에 의한 단백질의 분자량은 약 97,000 이었다. 정제된 효소의 최적 pH는 6이었으며 최적 온도는 $40^{\circ}C$이었다.

Keywords

References

  1. Muzzarelli, R. A. A. (1977) Chitin. Pergamon Press, Inc., New York
  2. Connell, T. D., Metzger, D. J., Lynch, J. and Folster, J. P. (1998) Endochitinase is transported to the extmcellular milieu by the esp-encoded general secretory pathway of Vibrio choIerae. J. BacterioI. 180, 5591-5600v
  3. Cohen-Kupiec, R. and Chet, I. (1998) The molecular biology of chitin digestion. Curr. Opin. Biotechnol. 9, 270-277 https://doi.org/10.1016/S0958-1669(98)80058-X
  4. Miyashita, K., Fujii, T., Watanabe, A. and Ueno, U. (1997) Nucleotide sequence and expression of a gene (chiB) for a chitinase from Streptomyces lividans. J. Ferment. Bioeng. 83, 26-31 https://doi.org/10.1016/S0922-338X(97)87322-9
  5. Watanabe, T., Oyanagi, W., Suzuki, K. and Tanaka, H. (1990) Chitinase system of BacilIus cifcuIans WL-12 and importance of chitinase A1 in chitin degradation. J. BacterioI. 172, 4017-4022 https://doi.org/10.1128/jb.172.7.4017-4022.1990
  6. Jones, J. D. G., Grady, K. L., Suslow, T. V. and Bedbrook, J. R. (1986) Isolation and characterization of genes encoding two chitinase enzymes from Serratia macescens. EMBO J. 5, 467-473
  7. Lin, C. S., Chen, H. C. and Lin, F. P. (1997) Expression and characterization of the recombinant gene encoding chitinase from Aeromonas caviae. Enz. Microb. Technol. 21, 472-478 https://doi.org/10.1016/S0141-0229(96)00249-9
  8. Wortman, A. T., Somerville, C. C. and Clowell, R. R. (1986) Chitinase determinants of Vtbrio vulnificus: Gene cloning and applications of a chitinase probe. Appl. Environ. Microbiol. 52, 142-145
  9. Kirsch, C., Hahlbrock, K. and Kombrink, E. (1993) Purification and characterizadon of extracellular, acidic chitinase isoenzymes from elicitor-stimulated parsley cells. Eur. J. Biochem. 213, 419-425
  10. Whitechurch, C. B. and Mattick, J. S. (1994) Escherichia coli contains a set of genes homologous to those involved in protein secretion, DNA uptake and the assembly of type-4 fimbnae in other bacteria. Gene 150, 9-15 https://doi.org/10.1016/0378-1119(94)90851-6
  11. Stojiljkovic, I., Schonherr, R. and Kusters, J. G. (1995) Identification of the hopG gene, a component of Escherichia coIi K-12 type II export system and its conservation among different pathogenic Escherichia coli and Shigella isolates. J. Bacteriol. 177, 1892-1895 https://doi.org/10.1128/jb.177.7.1892-1895.1995
  12. Prancetic, O. and Pugsley, A. P. (1996) The cryptic general secretory pathway (gsP) operon of Escherichia coli K-12 encodes functional proteins. J. Bacteriol. 178, 3544-3549 https://doi.org/10.1128/jb.178.12.3544-3549.1996
  13. Francetic, O., Badaut, C., Rimsky, S. and Pugsly, A. P. (2000) The ChiA (YheB) Protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS. Mol. Microbiol. 35, 1506-1517
  14. Francetic, O., Belin, D., Badaut, C. and Pugsly, A. P. (2000) Expression of the endochitinase type II secretion pathway in Escherichia coli lead to chitinase secretion. EMBO J. 19, 6697-6703 https://doi.org/10.1093/emboj/19.24.6697
  15. Ueda, M., Kawaguchi, T. and Arai, M. (1994) Molecular cloning and nucleotide sequence of the gene encoding chitinase II from Aeromonas sp. No. 10S-24. J. Ferment. Bioene. 78, 205-211 https://doi.org/10.1016/0922-338X(94)90291-7
  16. Laemmli, U.K. (1970) Cleavage of structural proteins dunng the assembly of the head of bacteriophage T4. Nature 227, 680-685 https://doi.org/10.1038/227680a0
  17. Tronsmo, A. and Harman, G. E. (1993) Detection and quantification of N-acetyl-B-D-glucosaminidase, chitobiosidase, and endochitinase in solutions and on gels. Anal. Biochem. 208, 74-79 https://doi.org/10.1006/abio.1993.1010
  18. Ahn, Y. K. (1994) Enzyme Purification Method, Yangseogak, Seoul
  19. Wang, S. L. and Chang, W. T. (1997) Purification and characterization of two bifunctional chitinase/lysozyme extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. AppI. Environ. Micmbiol. 63, 380-386
  20. Lee, H.-S., Han, D.-S., Choi, S.-J., Choi, S.-W., Kim, D.-S., Bai, D.-H. and Yu, J.-H. (2000) Purification, characterization and primary structure of a chitinase from Pseudomonas sp. YHS-A42. Appl. Microbiol. Biotechnol. 54, 397-405 https://doi.org/10.1007/s002530000408
  21. Kenji, M., Shuichi, K., Tetsuya, M., Kazuo, S. and Kunio, O. (2001) Characterization of Ctostridium paraputrificum chitinase A from a recombinant Escherichia coli. J. Biosci. Bioeng. 5, 466-468
  22. Tujibo, H., Hantano, N., Endo, H., Miyamoto, K. and Inamori, Y. (2000) Purification and characterization of a thermostable chitinase from Streptomyces thermoviolaceus OPC-520 and cloning of the encoding gene. Biosci. Biotechnol. Biochem. 64, 96-102
  23. Wiwat, C., Swayaprapom, P. and Bhumiratana, A. (2000) Purification and characterization of chitinase from Bacillus circulans No. 4.1. Curr. Microbiol. 39, 134-140 https://doi.org/10.1007/s002849900434
  24. Aji, M. U., Hiroshi I., Takashi, K., Yoshihisa, N., Motoo, A. and Kazutaka, M. (2001) Expression of a gene encoding chitinase (pCA 8 ORF) from Aeromonas sp. No. 10S-24 in Escherichia coli and enzyme characterization. J. Biosci. Bioene. 6, 599-602
  25. Kenji., S., Akira, Y., Hajime., K., Mamoru., W. and Mitsuaki, M. (1998) Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-l, isolated from chitin-containing compost. Appl. Environ. Micmbiol. 9, 3397-3402