Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Cloning of Isoamylase Gene of Pectobacterium carotovorum subsp. carotovorum LY34 and Identification of Essential Residues of Enzyme

Pectobacterium carotovorum subsp. carotovorum LY34에서 Lsoamylase 유전자 클로닝 및 효소 활성의 필수 잔기 확인

  • Cho, Kye-Man (Division of Applied Life Science, Gyeongsang National University) ;
  • Kim, Eun-Ju (Division of Applied Life Science, Gyeongsang National University) ;
  • Math, Renukaradhya K. (Division of Applied Life Science, Gyeongsang National University) ;
  • Asraful Islam, Shah Md. (Division of Applied Life Science, Gyeongsang National University) ;
  • Hong, Sun-Joo (Division of Applied Life Science, Gyeongsang National University) ;
  • Kim, Jong-Ok (Division of Applied Life Science, Gyeongsang National University) ;
  • Shin, Ki-Jae (Division of Applied Life Science, Gyeongsang National University) ;
  • Lee, Young-Han (Division of Plant Environmental Research, Gyeongsangnam-do Agricultural Research and Extension Service) ;
  • Kim, Hoon (Department of Bio-environmental chemistry, Sunchon National University) ;
  • Yun, Han-Dae (Research Institute of Agriculture & Life Science, Gyeongsang, National University)
  • Published : 2007.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

The gene encoding for isoamylase of the Pectobacterium carotovorum subsp. carotovorum (Pcc) LY34 was cloned and expressed into Escherichia coli $DH5{\alpha}$. Isoamylase catalyzes the hydrolysis of ${\alpha}-1,6-glycosidic$ linkages specifically in amylopectin, glycogen, and derived oligosaccharides, while the enzyme did not hydrolyze ${\alpha}-1,4-glycosidic$ linkages of amylose. The isoamylase gene (glgX) had an open reading frame of 1,977 bp encoding 658 amino acid residues with a calculated molecular weight of 74,188 Da. The molecular weight of the enzyme was also estimated to be 74 kDa by activity staining of a SDS-PA gel. The mature GlgX had a calculated pI of 4.91. Isoamylase from Pcc LY34 had 70% amino acid identity with isoamylase from Pectobacterium chrysanthemi and contained the four regions conserved among all amylolytic enzymes. The isoamylase was optimally active at pH 7.0 and $40^{\circ}C$. GlgX was $Ca^{2+}-dependent$. The changes of Asp-335, Glu-370, and Asp-442 into Ala, respectively, using site-directed mutagenesis techniques showed that three residues are essential to isolamyalse (GlgX) activity. The sequences around those residues were highly conserved in isoamylase of different origins and GlgX of the glg operon in glycongen biosynthesis.

연부균인 Pectobacterium carotovorum subsp. carotovorum LY34로부터 이소아밀라제 유전자 (glgX)를 클로닝한 후 대장균 숙주에서 발현시켰다. 이 효소는 ${\alpha}-1$,6-글루코시드 결합을 가수분해하였으나 ${\alpha}-1$,4-글루코시드 결합은 가수분해 하지 못하였다. 유전자는 658개의 아미노산을 암호화하는 1,977개의 DNA 염기서열로 이루어져 있었고 이 유전자에 의해 암호화되는 아미노산 서열을 다른 아밀라제 효소들과 비교한 결과 이소아밀라제 유전자와 유사하였으며 4개의 보존 지역을 확인하였다. SDS-PAGE에 의해 확인된 단백질의 크기는 약 74 kDa 이었다. 효소 활성은 pH 7.0, $40^{\circ}C$에서 가장 높은 활성을 나타났으며 $Ca^{2+}$ 첨가로 활성이 증가되었다. 이 효소의 보존되어 있는 아미노산 중에 글루탐산 370번, 아스파르트산 335번 및 442번 잔기를 알라닌으로 치환시킨 결과 활성이 약해졌다. 이 결과로부터 이들 잔기들이 효소활성에 중요한 역할을 하는 것으로 추정된다.

Keywords

References

  1. Abe, J. L, C. Ushijima and S. Hizukuri, 1999. Expression of the isoamylase of Flavobacterium ordoraium KU in Escherichia coli and identification of essential residues of the by site-directed mutagenesis. Appl. Environ. Microbiol. 65, 4163-4170
  2. Beguin, P. 1990. Molecular biology of cellulose degradation, Annu. Rev. Microbiol. 44, 219-248 https://doi.org/10.1146/annurev.mi.44.100190.001251
  3. Bernfeld, P. 1955. Amylases a and $\beta$. Methods Enzymol. 1, 149-155 https://doi.org/10.1016/0076-6879(55)01021-5
  4. Bonafonte, M. A., C. Solano, B. Sesma, M. Alvarez, L. Montuenga, D. Garcia-Ros and C. Gamazo. 2000. The relationship between glycogen synthesis, biofilm formation and virulence in Salmonella enteritidis. FEMS Microbiol. Lett. 191, 31-36 https://doi.org/10.1111/j.1574-6968.2000.tb09315.x
  5. Bradford, M. M. 1976. A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  6. Brooker, J. D. and J. M. McCarthy. 1997. Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis. Curr. Microbiol. 35, 133-138 https://doi.org/10.1007/s002849900226
  7. Cho, S. J. and H. D. Yun. 2005. Cloning of $\alpha$-amylase gene from unculturable bacterium using cow rumen metagenome. J. Life Sci. 15, 1013-1021 https://doi.org/10.5352/JLS.2005.15.6.1013
  8. Harada, T., K. Yokobayashi and A. Misaki. 1968. Formation of isoamylase by Pseudomonas. Appl. Microbiol. 16, 1439-1444
  9. Hong, S. Y., K. M. Cho, Y. H. Kim, S. J. Hong, S. J. Cho, Y. U. Cho, H. Kim and H. D. Yun. 2006. Cloning and identification of essential residues for thermostable $\beta$-glucosidase gene from Thermotoga maritima MSB8. J. Life Sci. 16, 1148-1157 https://doi.org/10.5352/JLS.2006.16.7.1148
  10. Jeanningros, R., N. Creuzet-Sigal, C. Frixon and J. Cattaneo. 1976. Purification and properties of a debranching enzyme from Escherichia coli. Biochim. Biophys. Acta 438, 186-199 https://doi.org/10.1016/0005-2744(76)90235-7
  11. Katsuya, Y., Y. Metaki, M. Kubota and Y. Matsusuura. 1998. Three-dimensional structure of Pseudomonas isoamylase at 2.2 ${\AA}$ resolution. J. Mol. Biol. 281, 885-897 https://doi.org/10.1006/jmbi.1998.1992
  12. Keen, N. T., C. Boyd and B. Henrissat. 1996. Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi. Mol. Plant Microbe Interact 9, 651-657 https://doi.org/10.1094/MPMI-9-0651
  13. Krohn, B. M., G. F. Barry and G. M. Kishore. 1997. An isoamylase with neutral pH optimum from a Flavobacterium species: Cloning, characterization and expression of the iam gene. Mol. Gen. Genet. 254, 469-478 https://doi.org/10.1007/s004380050441
  14. Lim, W. J., S. R. Park, S. J. Cho, M. K. Kim, S. K. Ryu, S. Y. Hong, W. T. Seo, H. Kim and H. D. Yun. 2001. Cloning and characterization of an intracellular isoamylase gene from Pectooacierium chrysanthemi PY35. Biochem. Biophys. Res. Commun. 287, 348-54 https://doi.org/10.1006/bbrc.2001.5594
  15. Lim, W. J., S. R. Park, C. L. An, J. Y. Lee, S. Y. Hong, E. C. Shin, E. J. Kim, J. O. Kim, H. Kim and H. D. Yun. 2003. Cloning and characterization of a thermostable intracellular $\alpha$-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8. Res. Microbiol. 154, 681-687 https://doi.org/10.1016/j.resmic.2003.09.005
  16. 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 Pectobacierium carotovorum subsp. carotovorum LY34 and its comparison to those of Pectooacierium sp. J. Microbiol. Biotechnol. 15, 302-309
  17. May, B. J., Q. Zhang, L. L. Li, M. L. Paustian, T. S. Whittam and V. Kapur. 2001. Complete genomic sequence of Pasteurella multocida, Pm70. Proc. Natl. Acad. Sci. 98, 3460-3465 https://doi.org/10.1073/pnas.051634598
  18. Nakajima, R., T. Imanaka and S. Aiba. 1986. Comparison of amino acid sequences of eleven different $\alpha$-amylases. Appl. Microbiol. Biotechnol. 23, 355-360
  19. Park, S. R., S. J. Cho and H. D. Yun. 2000. Cloning and sequencing of pel gene responsible for CMCase activity from Erwinia chrysanthemi PY35. Biosci. Biotechnol. Biochem. 64, 820-830
  20. Park, Y. W., S. T. Lim and H. D. Yun. 1998. Cloning and characterization of a CMCase gene, celB, of Erwinia carotovra subsp. carotovora LY34 and its comparison to celA, Mol. Cells 8, 280-285
  21. Park, Y. W., S. T. Lim, S. J. Cho and H. D. Yun, 1997. Characterization of Erwinia caroiovora subsp. carotovora LY34 endo-1,4-$\beta$-glucanase genes and rapid identification of their gene products. Biochem. Biophysic. Res. Commun. 241, 636-641 https://doi.org/10.1006/bbrc.1997.7747
  22. Raha, M., I. Kawagishi, V. Muller, M. Kihara and R. M. Macnab. 1992. Escherichia coli produces a cytoplasmic alpha-amylase, AmyA. J. Bacteriol. 174, 6644-6652 https://doi.org/10.1128/jb.174.20.6644-6652.1992
  23. Robert, Y. I. and C. R. Meyer. 2000. Cloning and sequencing of glycogen metabolism genes from Rhodobacier sphaeroides 2.4.1. expression and characterization of recomibinant ADP-glucose pyrophosphorylase. Arch. Biochem. Biophys. 376, 47-58 https://doi.org/10.1006/abbi.1999.1689
  24. Romeo, T., A. Kumar and J. Preiss. 1988. Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes. Gene 70, 363-376 https://doi.org/10.1016/0378-1119(88)90208-9
  25. Rouanet, C., K. Nemura, S. Tsuyumu and W. Nasser. 1999. Regulation of pelD and pelE, encoding major alkaline pectate lyases in Erwinia chrysanthemi: Involvement of the main transcriptional factors. J. Bacteriol. 181, 5948-5957
  26. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor
  27. Satoh, E., T. Uchimura, T. Kudo and K. Komagata. 1997. Purification, characterization, and nucleotide sequence of an intracellular maltotriose-producing alpha-amylase from Streptococcus bovis 148. Appl. Environ. Microbiol. 63, 4941-4944
  28. Simpsen, H. D. and F. Barras. 1999. Functional analysis of the carbohydrate-binding domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli putative chitinase. J. Bacteriol. 181, 4011-4616
  29. Svenssion, B. 1994. Protein engineering in the $\alpha$-amylase family: catalytic mechanism, substrate specificity, and stability. Plant. Mol. Biol. 25, 141-157 https://doi.org/10.1007/BF00023233
  30. Takase, K. 1992. Interaction of catalytic-site mutants of Bacillus subtilis $\alpha$-amylase with substrates and acarbose. Biocheim. Biophys. Acta 1122, 278-282 https://doi.org/10.1016/0167-4838(92)90405-3
  31. Takata, H., T. Kuriki, S. Okada, Y. Takesada, M. Lizuka, N. Minamiura and T. Imanaka. 1992. Action of neopullulanase: neopullulanase catalyzes both hydrolysis and transglycosylation at $\alpha$-(1$\rightarrow$4)-and $\alpha$-(1$\rightarrow$6)-glucosidic linkages. J. Biol. Chem. 267, 18447-18452
  32. Urlaub, Hand G. Woeber. 1975. Identification of isoamylase, a glycogen-debranching enzyme, from Bacillus amyloliquefaciens. FEBS Lett. 57, 1-4 https://doi.org/10.1016/0014-5793(75)80138-4
  33. Vihinen, M. and P. Mantsala. 1989. Microbial amylolytic enzymes. Crit. Rev. Biochem. Mol. Biol. 24, 329-418 https://doi.org/10.3109/10409238909082556
  34. Yang, H, M. Y. Liu and T. Romeo. 1996. Coordinate genetic regulation of glycogen catabolism and biosynthesis in Escherichia coli via the CsrA gene product. J. Bacteriol. 178, 1012-1017 https://doi.org/10.1128/jb.178.4.1012-1017.1996
  35. Zhou, S., F. C. Davis and L. O. Ingram. 2001. Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2. Appl. Environ. Miorobiol. 67, 6-14 https://doi.org/10.1128/AEM.67.1.6-14.2001