생명과학회지 (Journal of Life Science)

  • 제15권4호
  • /
  • Pages.633-640
  • /
  • 2005
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

Bacillus Pumilus TX703 유래 Xylanase의 활성에 관여하는 아미노산 잔기의 확인

Identification of Amino Acid Residues Involved in Xylanase Activity from Bacillus Pumilus TX703

  • 박영서 (경원대학교 생명공학부)
  • 발행 : 2005.08.01
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

Bacillus pumilus TX703으로부터 xylanase를 정제한 후 효소의 활성부위를 조사하기 위하여 여러 가지 화학수식제를 사용하여 효소활성의 저해도를 측정하였다. 여러 가지 화학수식제 중에서 carbodiimide와 N-bromosuccinimide가 효소활성을 완전히 저 해시 켜 glutamic acid 또는 aspartic acid 잔기와 tryptophan 잔기가 효소의 활성부위에 관여하리라 추측되었다. 각각의 경우에 효소 실활은 수식제의 첨가농도에 따라 pseudo first-order kinetics 양식을 보여주었으며, car-bodiimide와 N-bromosuccinimide는 각각 비경쟁적 저해와 경쟁적 저해방식을 나타내었다. 기질첨가에 의한 효소활성 보호실험을 통하여 tryptophan 잔기가 기질결합부위라 판단되었다. 효소실활속도의 분석에 의해 효소활성에는 2개의 glutamic acid 또는 aspartic acid 잔기와 1개의 tryptophan 잔기가 관여하는 것으로 나타났다.

The purified xylanase from Bacillus pumilus TX703 was modified with various chemical modifiers to determine the active sites of the enzyme. Treatment of the enzyme with group-specific reagents such as carbodiimide or N-bromosuccinimide resulted in complete loss of enzyme activity. These results assumed that these reagents reacted with glutamic acid or aspartic acid and tryptophan residues located at or near the active site. In each case, inactivation was performed by pseudo first-order kinetics. Inhibition of enzyme activity by carbodiimide and W-bromosuccinimide showed non-competitive and competitive inhibition type, respectively. Addition of xylan to the enzyme solution containing N-bromosuccinimide prevented the inactivation, indicating the presence of tryptophan at the substrate binding site. Analysis of kinetics for inactivation showed that the loss of enzyme activity was due to modification of two glutamic acid or aspartic acid residues and single tryptophan residue.

키워드

참고문헌

  1. Bandivadekar, K. R. and V. V. Deshpande. 1996. Structurefunction relationship of xylanase: fluorimetric analysis of the tryptophan environment. Biochem. J. 315, 583-587
  2. Biely, P. 1985. Microbial xylanolytic systems. Trends Biotechnol. 3, 286-290 https://doi.org/10.1016/0167-7799(85)90004-6
  3. Blanke, S. R. and L. P. Hager. 1990. Chemical modification of chloroperoxidase with diethylpyrocarbonate. Evidence for the presence of an essential histidine residue. J. Bioi. Chem. 265, 12454-12461
  4. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation 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
  5. Church, F. C., R. L. Lundblad and C. M. Noyes. 1985. Modification of histidines on human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin. J. biol. Chem. 260, 4936-4940
  6. Coughlan, M. P. and G. P. Hazelwood. 1993. Beta-1,4-Dxylan degrading enzyme systems: Biochemistry, molecular biology and applications. Biotechnol. Appl. Biochem. 17, 259-289
  7. Das, N. N., S. C. Das, A K. Sarkar and A K. Mukherjee. 1984. Lignin-xylan ester linkage in mesta fiber (Hibiscus cannabinus). Carbohydr. Res. 129, 197-207 https://doi.org/10.1016/0008-6215(84)85312-4
  8. Davoodi, J., W. W. Wakarchuk, R. L. Campbell, P. R. Carey and W. K. Surewicz. 1995. Abnormally high pKa of an activesite glutamic acid residue in Bacillus circulans xylanase. The role of electrostatic interactions. Eur. J. Biochem. 232, 839-843
  9. Khasin, A., I. Alchanati and Y. Shoham. 1993. Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6. Appl. Environ. Microbiol. 59, 1725-1730
  10. Kulkarni, N., A. Shendye and M. Rao. 1999. Molecular and biotechnological aspects of xylanases. FEMS Microbiol. Rev. 23, 411-456 https://doi.org/10.1111/j.1574-6976.1999.tb00407.x
  11. 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
  12. McCarthy, A A, D. D. Morris, P. L. Bergquist and E. N. Baker. 2000. Structure of XynB, a highly thermostable beta-1,4-xylanase from Dictyoglomus thermophilum Rt46B.1, at 1.8 A resolution. Acta Crystallogr. D. Biol. Crystallogr. 11, 1367-1375
  13. Nakamura, S., K. Wakabayashi, R. Nakai, R. Aono and K Horikoshi. 1993. Purification and some properties of an alkaline xylanase from alkaliphilic Bacillus sp. strain 41M-1. Appl. Environ. Microbiol. 59, 2311-2316
  14. Nath, D. and M. Rao. 1998. Structural and functional role of tryptophan in xylanase from an extremophilic Bacillus: assessment of the active site. Biochem. Biophys. Res. Commun. 249, 207-212 https://doi.org/10.1006/bbrc.1998.9107
  15. Park, Y. S. 2002. Molecular cloning and analysis of nucleotide sequence of xylanase gene (xynK) from Bacillus pumilus TX703. Korean J. Life Sci. 12, 188-199 https://doi.org/10.5352/JLS.2002.12.2.188
  16. Park, Y. S., M Y. Kang, H. G. Chang, G. G. Park, J. B. Kang, J. K. Lee and T. K. Oh. 1999. Isolation of xylanase-producing thermo-tolerant Bacillus sp. and its enzyme production. Kor. J. Appl. Microbiol. Biotechnol. 27, 370-377
  17. Scalbert, A., B. Monities, J. Y. Lallemand, E. Guittet and C. Rolando. 1985. Ether linkage between phenolic acids and lignin franctions from wheat straw. Phytochemistry 24, 1359-1362 https://doi.org/10.1016/S0031-9422(00)81133-4
  18. Somogyi, M. 1952. Notes on sugar determination. J. Biol. Chem. 195, 19-23
  19. Takeuchi, M, A. Asano, Y. Kameda and K. Matsui. 1986. Chemical modification by diethylpyrocarbonate of an essential histidien residue in 3-ketovalidoxylamine A C-N lyase. J. Biochem. 99, 1571-1577
  20. Velikodvorskaya, T. V., I. Y. Volkov, V. T. Vasilevko, V. V. Zverlov and E. S. Piruzian. 1997. Purification and some properties of Thermotoga neapolitana thermostable xylanase B expressed in E. coli cells. Biochemistry 62, 66-70
  21. Wakarchuk, W. W., R. L. Campbell, W. L. Sung, J. Davoodi and M Yaguchi. 1994. Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase. Protein Sci. 3, 467-475 https://doi.org/10.1002/pro.5560030312
  22. Wang, K. K. Y., L. U. L. Tan and J. N. Saddler. 1988. Multiplicity of beta-1,4-xylanase in microorganisms: Function and applications. Microbiol. Rev. 52, 305-317
  23. Wong, K. K. Y. and J. N. Saddler. 1992. Applications of hemicellulases in the food, feed, and pulp and paper industries, pp. 127-143, In Coughlen, P. P. and G. P. Hazlewood (eds), Hemicelolose and Hemicellulases, Portland Press, London
  24. Wong, K. K. Y., L. U. L. Tan and J. N. Saddler. 1988. Multiplicity of $\beta$-1,4-xylanase in microorganism: Functions and applicaitons. Microbiol. Rev. 52, 305-317
  25. Xie, H., H. J. Gilbert, S. J. Charnock, G. J. Davies, M. P. Williamson, P. J. Simpson, S. Raghothama, C. M. Fontes, F. M. Dias, L. M. Ferreira and D. N. Bolam. 2001. Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding. Biochemistry 40, 9167-9176 https://doi.org/10.1021/bi0106742
  26. Zhu, H., F. W. Paradis, P. J. Krell, J. P. Phillips and C. W. Forsberg. 1994. Enzymatic specificities and modes of action of the two catalytic domains of the XynC xylanase from Fibrobacter succinogenes 585. J Bacteriol. 176, 3885-3894