Applied Biological Chemistry

The Korean Society for Applied Biological Chemistry (한국응용생명화학회)
권호 표지

Purification and Characterization of Overproduced E. coli Laccase

과량 생산된 대장균 laccase의 정제 및 특성

  • 홍준혁 (중앙대학교 산업과학대학 생명공학과) ;
  • 김현정 (중앙대학교 산업과학대학 생명공학과) ;
  • 김우연 (중앙대학교 산업과학대학 생명공학과)
  • Published : 2007.06.30
Download PDF
⟨ Previous Next ⟩

Abstract

The putative laccase gene, yacK of Escherichia coli, K-12 is not expressed in lab culture conditions. The laccase gene was amplified by PCR and subcloned into pET28a vector. The laccase overproduced in E. coli harboring pET28a was purified by His-affinity column chromatography. The purified laccase, which has the apparent molecular weight of 55,000 on the SDS-polyacrylamide gel showed enzyme activity on the guaiacol solution and agar plate. Optimum temperature and pH were around 65$^{\circ}C$ and 5.0, respectively.

일반적 대장균 배지 조건에서는 발현되지 않는 대장균 K-12의 laccase gene(yacK)을 PCR로 증폭한 후 pET28c에 클로닝하여 과량 발현시켰다. 과량 생산된 laccase를 His-affinity 칼럼 크로마토그래피로 정제하였다. SDS-PAGE 방법으로 확인한 과량 발현된 단백질의 분자량은 약 55,000이었으며, guaiacol 용액과 agar 배지에서 역가를 보여주었고 최적 온도는 65$^{\circ}C$, 최적 pH는 5이었다.

Keywords

References

  1. Solomon, E. I., Sundaram, U. M. and Machonkin, T. E. (1996) Multicopper oxidases and oxygenases. Chem. Rev. 96, 2563- 2605 https://doi.org/10.1021/cr950046o
  2. Alexandre, G. and Zhulin, L. B. (2000) Laccases are widespread in bacteria. Trends Biotechnol. 18, 41-42 https://doi.org/10.1016/S0167-7799(99)01406-7
  3. Mayer, A. M. and Harel, E. (1979) Polyphenoloxidases in plant. Phytochemistry 18, 193-215 https://doi.org/10.1016/0031-9422(79)80057-6
  4. Mayer, A. M. (1987) Polyphenoloxidase in plants-recent progress. Phytochemistry 26, 11-20 https://doi.org/10.1016/S0031-9422(00)81472-7
  5. Raghukumar, C., Raghukumar, S., Chinnaraj, A., Chandramohan, D., Dsouza, T. M. and Reddy, C. A. (1994) Laccase and other lignocillulose modifying enzymes of marine fungi isolated from the coast of India. Bot. Marina 37, 515-523 https://doi.org/10.1515/botm.1994.37.6.515
  6. Pelaez, F., Martinez, M. J. and Martinez, A. T. (1995) Screening of 68 species of basidiomycetes for enzymes involved in lignin degradation. Mycol. Res. 99, 37-42 https://doi.org/10.1016/S0953-7562(09)80313-4
  7. Andersen, S. O. (1985) Sclerotization and tanning of the cuticle, In: G. P. Kerkut and L. I. Gilbert (eds). Comparative Insect Physiology, Biochemistry, and Pharmacology, Vol. 3. Pergamon Press, New York, pp 59-74
  8. Andersen, S. O., Peter, M. G. and Roepstorff, P. (1996) Cuticular sclerotization in insects. Comp. Biochem. Physol. 113B, 689-705
  9. Dean, J. F. D. and Eriksson, K. E. L. (1994) Laccase and the deposition of lignin. Holzforschung 48, 21-33 https://doi.org/10.1515/hfsg.1994.48.s1.21
  10. Peter, M. G. and Wollenberg, U. W. (1997) Phenol-oxidizing enzymes: Mechanisms and applications in biosensors. Frontiers in Biosensorics I, pp 63-82
  11. Kim, C. W., Lorenz, W., Hoopes, T. and Dean, J.F.D. (2001) Oxidation of phenolate siderophores by the Multicopper oxidase encoded by the Escherichia coli yacK gene. J. Bacteriol. 183, 4866-4875 https://doi.org/10.1128/JB.183.16.4866-4875.2001
  12. Link, A., Robinson, J., K. and Church, G. M. (1997) Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 18, 1259-1313 https://doi.org/10.1002/elps.1150180807
  13. 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
  14. Doerge, D. R., Divi, R. L. and Churchwell, M. I. (1997) Identification of the Colored Guaiacol Oxidation Product Produced by Peroxidases. Anal. Biochem. 250, 10-17 https://doi.org/10.1006/abio.1997.2191
  15. Liu, W., Chao, Y., Liu, S., Bao, H. and Qian, S. (2004) Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris. Appl. Microbiol. Biotechnol. 63, 174-181