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Enhancement of the Thermostability of a Fibrinolytic Enzyme from Bacillus amyloliquefaciens CH51

Bacillus amyloliquefaciens CH51이 생산하는 혈전용해효소의 열안정성 개선

  • Kim, Jieun (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Choi, Kyoung-Hwa (Department of Microbiology, College of Natural Sciences, Pusan National University) ;
  • Kim, Jeong Hwan (Institute of Agriculture & Life Science, Gyeongsang National University) ;
  • Song, Young-Sun (School of Food and Life Science, Inje University) ;
  • Cha, Jaeho (Department of Microbiology, College of Natural Sciences, Pusan National University)
  • Received : 2012.11.20
  • Accepted : 2012.12.07
  • Published : 2013.01.30

Abstract

AprE51 from Bacillus amyloliquefaciens CH51 is a 27 kDa subtilisin-like protease with fibrinolytic activity. AprE51-6 showing increased catalytic activity was produced previously. To enhance the thermostability of AprE51-6, 2 residues, Gly-166 and Asn-218 based on B. subtilis subtilisin E were mutated by site-directed mutagenesis. The results of the mutational analysis showed that substitution of arginine for Gly-166 (AprE51-7) increased the fibrinolytic activity 1.8-fold. An N218S mutant (AprE51-8) also increased the fibrinolytic activity up to 4.5-fold in a fibrin plate assay. Purified AprE51-7 and AprE51-8 mutants had a 1.9- and a 2.5-fold higher $k_{cat}$, respectively, and a 2.1-1.9-fold lower $K_m$, respectively. This resulted in a 3.8- and a 4.7-fold increase in catalytic efficiency ($k_{cat}/K_m$), respectively, relative to that of wild-type AprE51. AprE51-8 had a broader pH range than AprE51-6 and nattokinase, especially at an alkaline pH value. In addition, AprE51-8 showed higher thermostability than AprE51-6 at $60^{\circ}C$. The half-lives of AprE51-7 and AprE51-8 at $50^{\circ}C$ were 21.5 and 27.3 min, respectively, which are 2.0 and 2.6 times longer, respectively, than that of the wild-type AprE51.

Bacillus amyloliquefaciens CH51은 분자량 27 kDa 크기의 subtilisin 타입의 혈전용해능을 지니는 단백질분해효소인 AprE51을 생산하였다. 이전연구에서 더 우수한 혈전용해 활성을 갖는 AprE51-6이 세포외 돌연변이법으로 생산되었으며, 본 연구에서는 이 개선된 효소인 AprE51-6의 열안정성을 증진시킬 목적으로 B. subtilis subtilisin E의 아미노산과의 상동성 분석을 통하여 두 아미노산인 Gly-166과 Asn-218이 치환되었다. 그 결과 G166R과 N218S 돌연변이체는 혈전용해능을 보이는 용해능 배지에서 원 효소보다 각각 1.8배와 4.5배 높은 혈전용해능을 보였다. 정제된 두 돌연변이효소인 AprE51-7과 AprE51-8는 원효소인 AprE51-6에 비하여 1.9 그리고 2.5배 높은 $k_{cat}$값을 나타내었고, 2.1과 1.9배 낮은 기질친화력을 나타내는 $K_m$값을 보여주었다. 특히 AprE51-8는 나토키나아제에 비하여 알칼리 pH 영역에서 높은활성을 유지하였고, $60^{\circ}C$에서 더 우수한 열안정성을 보여주었다. 열안정성의 정도를 나타내는 척도인 반감기 값에서도 AprE51-7과 AprE51-8는 $50^{\circ}C$에서 21.5분과 27.3분으로 기존의 AprE51보다 2배 그리고 2.6배 더 긴 반감기를 보였다.

Keywords

References

  1. Astrup, T. and Mullertz, S. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 6, 346-351.
  2. Baruah, D. B., Dash, R. N., Chaudhari, M. R. and Kadam, S. S. 2006. Plasminogen activators: A comparison. Vasc Pharmacol 44, 1-9. https://doi.org/10.1016/j.vph.2005.09.003
  3. Cai, Y., Bao, W., Jiang, S., Weng, M., Jia, Y., Yin, Y., Zheng, Z. and Zou, G. 2011. Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto. FEMS Microbiol Lett 325, 155-161. https://doi.org/10.1111/j.1574-6968.2011.02423.x
  4. Choi, N. S. and Kim, S. H. 2001. The effect of sodium chloride on the serine-type fibrinolytic enzymes and the thermostability of extracellular protease from Bacillus amyloliquefaciens DJ-4. J Biochem Mol Biol 34, 134-138.
  5. Collen, D. and Lijnen, H. R. 2005. Thrombolytic agents. Thromb Haemost 93, 627-630.
  6. Desantis, G., Shang, X. and Jones, J. B. 1999. Toward tailoring the specificity of the S1 pocket of subtilisin B. lentus: chemical modification of mutant enzymes as a strategy for removing specificity. Biochemistry 38, 13391-13397. https://doi.org/10.1021/bi990861o
  7. Ho, S. N., Hunt, H. D., Morton, R. M., Pullen, J. K. and Pease, L. R. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59. https://doi.org/10.1016/0378-1119(89)90358-2
  8. Hsieh, C., Lu, W., Hsieh, W., Huang, Y., Lai, C. and Ko, W. 2009. Improvement of the stability of nattokinase using γ-polyglutamic acid as a coating material for microencapsulation. LWT-Food Sci Technol 42, 144-149. https://doi.org/10.1016/j.lwt.2008.05.025
  9. Hwang, K. J., Choi, K. H., Kim, M. J., Park, C. S. and Cha, J. 2007. Purification and characterization of a new fibrinolytic enzyme of Bacillus licheniformis KJ-31, isolated from Korean traditional Jeot-gal. J Microbiol Biotechnol 17, 1469-1476.
  10. Ito, M. and Nagane, M. 2001. Improvement of the electo- transformation efficiency of facultatively alkaliphilic Bacillus pseudofirmus OF4 by high osmolarity and glycine treatment. Biosci Biotechnol Biochem 65, 2773-2775. https://doi.org/10.1271/bbb.65.2773
  11. Kannel, W. B. 2005. Overview of hemostatic factors involved in atherosclerotic cardiovascular disease. Lipids 40, 1215- 1220. https://doi.org/10.1007/s11745-005-1488-8
  12. Kim, G. M., Lee, A. R., Lee, K. W., Park, J., Lee, M., Chun, J., Cha, J., Song, Y. and Kim, J. H. 2009. Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from Cheonggukjang. J Microbiol Biotechnol 19, 997-1004. https://doi.org/10.4014/jmb.0811.600
  13. Kim, J., Kim, J. H., Choi, K. H., Kim, J. H., Song, Y. S. and Cha, J. 2011. Enhancement of the catalytic activity of a 27 kDa subtilisin-like enzyme from Bacillus amyloliquefaciens CH51 by in vitro mutagenesis. J Agric Food Chem 59, 8675-8682. https://doi.org/10.1021/jf201947m
  14. Kim, W., Choi, K., Kim, Y., Park, H., Choi, J., Lee, Y., Oh, H., Kwon, I. and Lee, S. 1996. Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang. Appl Environ Microbiol 62, 2482-2488.
  15. Law, D. and Zhang, Z. 2007. Stabilization and target delivery of nattokinase using compression coating. Drug Dev Ind Pharm 33, 495-503. https://doi.org/10.1080/03639040601050247
  16. Omura, K., Hitosugi, M., Zhu, X., Ikeda, M., Maeda, H. and Tokudome, S. 2005. A newly derived protein from Bacillus subtilis natto with both antithrombotic and fibrinolytic effects. J Pharmacol 99, 247-251.
  17. Peng, Y., Huang, Q., Zhang, R. H. and Zhang, Y. Z. 2003. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chineses soybean food. Comp Biochem Physiol 134, 45-52.
  18. Price, N. C. and Stevens, L. 2000. Fundamentals of Enzymology; The cell and molecular biology of catalytic proteins. 3rd edition. Oxford University Press.
  19. Sumi, H., Hamada, H., Nakanishi, K. and Hiratani, H. 1990. Enhancement of the fibrinolytic activity in plasma by oral administration of NK. Acta Haematol 84, 139-143. https://doi.org/10.1159/000205051
  20. Sumi, H., Hamada, H., Tsushima, H. and Mihara, H. 1987. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 43, 1110-1111. https://doi.org/10.1007/BF01956052
  21. Wang, C., Du, M., Zheng, D., Kong, F., Zu, G. and Feng, Y. 2009. Purification and characterization of nattokinase from Bacillus subtilis Natto B-12. J Agric Food Chem 57, 9722-9729. https://doi.org/10.1021/jf901861v
  22. Wells, J. A., Cunningham, B. C., Graycar, T. P. and Estell, D. A. 1987. Recruitment of substrate-specificity properties from one enzyme into a related one by protein engineering. Proc Natl Acad Sci 84, 5167-5171. https://doi.org/10.1073/pnas.84.15.5167
  23. Weng, M., Zheng, Z., Bao, W., Cai, Y., Yin, Y. and Zou, G. 2009. Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli. Biochim Biophys Acta 1794, 1566-1572. https://doi.org/10.1016/j.bbapap.2009.07.007
  24. Wu, B., Wu, L., Ruan, L., Ge, M. and Chen, D. 2009. Screening of endophytic fungi with antithrombic activity and identification of a bioactive metabolite from the endophytic fungal strain CPCC 480097. Curr Microbiol 58, 522-527. https://doi.org/10.1007/s00284-009-9361-7
  25. Xue, G., Johnson, J. S. and Dalrymple, B. P. 1999. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J Microbiol Methods 34, 183-191. https://doi.org/10.1016/S0167-7012(98)00087-6
  26. Yang, Y., Jiang, L., Yang, S., Zhu, L., Wu, Y. and Li, Z. 2000. A mutant subtilisin E with enhanced thermostability. World J Microbiol Biotechnol 16, 249-251. https://doi.org/10.1023/A:1008959825832
  27. Zhao, H. and Arnold, F. H. 1999. Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng 12, 47-53. https://doi.org/10.1093/protein/12.1.47