Journal of Microbiology and Biotechnology

  • 제16권8호
  • /
  • Pages.1325-1329
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  • 2006
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  • 1017-7825(pISSN)
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  • 1738-8872(eISSN)
한국미생물·생명공학회 (The Korean Society for Microbiology and Biotechnology)
권호 표지

Substitutions for Cys-472 and His-509 at the Active Site of $\beta$-Galactosidase from Lactococcus lactis ssp. lactis 7962 Cause Large Decreases in Enzyme Activity

  • Chung Hye-Young (Department of Food and Nutrition, Chosun University) ;
  • Yang Eun-Ju (Department of Food and Nutrition, Chosun University) ;
  • Chang Hae-Choon (Department of Food and Nutrition, Chosun University)
  • 발행 : 2006.08.01
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초록

Structural modeling of $\beta$-galactosidase from L. lactis ssp. lactis 7962 has shown that the residues Cys-472 and His-509 are located in the wall of the active-site cavity. To examine the functions of Cys-472 and His-509, we generated five site-specific mutants: Cys-472-Ser, Cys-472-Thr, Cys-472-Met, His-509-Asn, and His-509-Phe. $\beta$-Galactosidase substituted at Cys-472 with Met or His-509 with Phe had <3% of the activity of the native enzyme when assayed using ONPG as substrate. The other mutants Cys-472-Ser, Cys-472-Thr, and His-509-Asn had ca. 10-15% of the native enzyme activity. The V$_max$ values of the five mutated enzymes were lower (60-7,000-fold) than that of native enzyme. These results show that the catalytic ability of $\beta$-galactosidase is significantly affected by mutations at Cys-472 or His-509.

키워드

참고문헌

  1. Chang, H. C, Y. D. Choi, and H. J. Lee. 1996. Molecular cloning of a $\beta$-$_D$-galactosidase gene from Lactococcus lac tis subsp. lactis ATCC7962. J. Microbial. Biotechnol. 6: 386-390
  2. De Vos, W. M. and E. E. Vaughan. 1994. Genetics oflactose utilization in lactic acid bacteria. FEMS Microbiol. Rev. 15: 217-237 https://doi.org/10.1111/j.1574-6976.1994.tb00136.x
  3. Huber, R. E., M. N. Gupta, and S. K. Khare. 1994. The active site and mechanism of the beta-galactosidase from Escherichia coli. lnt. J. Biochem. 26: 309-318 https://doi.org/10.1016/0020-711X(94)90051-5
  4. Jacobson, R. H., X. J. Zhang, R. F. DuBose, and B. W. Mattews. 1994. Three-dimensional structure of $\beta$-galactosidase from E. coli. Nature 369: 761-766 https://doi.org/10.1038/369761a0
  5. Kim, J. H., J. Park, S. Jeong, J. Chun, J. H. Lee, D.K. Chung, and J. H. Kim. 2005. Characterization of the a-galactosidase gene from Leuconostoc mesenteroides SY1. J. Microbiol. Biotechnol. 15: 800-808
  6. Lee, J. H., J. Y. Choi, J. M. Lee, J. H. Kim, H. C. Chang, D. K. Chung, and H. J. Lee. 2000. Expression of the galactosidase mutarose gene from Lactococcus lactis ssp. lactis ATCC7962 in Escherichia coli. J. Microbiol. Biotechnol. 10: 840-843
  7. Lee, J. M., D. K. Chung, J. H. Park, W. K. Lee, H. C. Chang, J. H. Kim, and H. J. Lee. 1997. Cloning and nucleotide sequence of the p-galactosidase gene from Lactococcus lactis ssp. lactis ATCC7962. Biotechnol. Lett. 19: 179-183 https://doi.org/10.1023/A:1018376717545
  8. Lim, W. J., S. K. Ryu, S. R. Park, M. K. Kim, C. L. An, S. Y. Hong, E. C. Shin, J. Y. Lee, Y. P. Lim, and H. D. Yun. 2005. Cloning of celC, third cellulase gene, from Pectobacterium carotovorum subsp. carotovorum LY34 and its comparison to those of Pectobacterium sp. J. Microbiol. Biotechnol. 15: 302-309
  9. Miller, J. H. 1972. Experiments in Molecular Genetics. pp. 352-355. CSH NY
  10. Moon, K., S. Yoast, A. L. Palombella, S. E. Mainzer, and B. F. Schmidt. 1994. Two histidines are essential for the activity of the beta-galactosidase from Lactobacillus delbruckii subsp. Bulgaricus. Biochem. Biophys. Res. Commun. 201: 1167-1174 https://doi.org/10.1006/bbrc.1994.1828
  11. Poch, O., H. L'Hote, V. Dallery, F. Debeaux, R. Fleer, and R. Sodoyer. 1992. Sequence of the Kluyveromyces lactis beta-galactosidase: Comparison with prokaryotic enzymes and secondary structure analysis. Gene 118: 55-63 https://doi.org/10.1016/0378-1119(92)90248-N
  12. Richmond, M. L. and C. M. Stine. 1981. Beta-galactosidase: Review of recent research related to technological application, nutritional concerns and immobilization. J. Dairy Sci. 64: 1759-1771 https://doi.org/10.3168/jds.S0022-0302(81)82764-6
  13. Ryoo, H. J., E. J. Yang, H. Y. Chung, H. J. Lee, J. H. Kim, D. K. Chung, J. H. Lee, and H. C. Chang. 2002. $Mg^{2+}$ ligands (Glu-384, Glu-429) of $\beta$-galactosidase from Lactococcus lactis ssp. lactis 7962. Biotechnol. Lett. 24: 691-696 https://doi.org/10.1023/A:1015277932318
  14. Ryu, H.-J., D. Kim, E.-S. Seo, H.-K. Kang, J.-H. Lee, S.-H. Yoon, J.-Y. Cho, J. F. Robyt, D.-W. Kim, S.-S. Chang, S.-H. Kim, and A. Kimura. 2004. Identification of amino-acids residues for key role in dextransucrase activity of Leuconostoc mesenteroides B-742CB. J. Microbiol. Biotechnol. 14: 1075-1080
  15. Sanger, F, S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain termination inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5467
  16. Schroeder, C. J., C. Robert, G Lenzen, L. L. McKay, and A. Mercenier. 1991. Analysis of the lacZ sequences from two Streptococcus thermophilus strains: Comparison with the Escherichia coli and Lactobacillus bulgaricus betagalactosidase sequences. J. Gen. Microbiol. 137: 369-380 https://doi.org/10.1099/00221287-137-2-369
  17. Shukla, T. 1975. Beta-galactosidase technology: Solution to the lactose problem. CRC Crit. Rev. Food Technol. 1: 325-356
  18. Yang, E. J., J. M. Lee, H. J. Lee, J. H. Kim, D. K. Chung, J. H. Lee, and H. C. Chang. 2003. The importance of Tyr-475 and Glu-506 in the $\beta$-galactosidase from Lactococcus lactis ssp. lactis ATCC7962. J. Microbiol. Biotechnol. 13: 134-138