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http://dx.doi.org/10.5352/JLS.2013.23.1.15

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

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)
Publication Information
Journal of Life Science / v.23, no.1, 2013 , pp. 15-23 More about this Journal
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.
Keywords
Bacillus amyloliquefaciens; Cheonggukjang; fibrinolytic enzyme; in vitro mutagenesis; thermostability;
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1 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.   DOI   ScienceOn
2 Kannel, W. B. 2005. Overview of hemostatic factors involved in atherosclerotic cardiovascular disease. Lipids 40, 1215- 1220.   DOI   ScienceOn
3 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.   과학기술학회마을   DOI
4 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.   DOI   ScienceOn
5 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.
6 Law, D. and Zhang, Z. 2007. Stabilization and target delivery of nattokinase using compression coating. Drug Dev Ind Pharm 33, 495-503.   DOI   ScienceOn
7 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.
8 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.
9 Price, N. C. and Stevens, L. 2000. Fundamentals of Enzymology; The cell and molecular biology of catalytic proteins. 3rd edition. Oxford University Press.
10 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.   DOI   ScienceOn
11 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.   DOI   ScienceOn
12 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.   DOI   ScienceOn
13 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.   DOI
14 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.   DOI   ScienceOn
15 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.   DOI   ScienceOn
16 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.   DOI   ScienceOn
17 Baruah, D. B., Dash, R. N., Chaudhari, M. R. and Kadam, S. S. 2006. Plasminogen activators: A comparison. Vasc Pharmacol 44, 1-9.   DOI   ScienceOn
18 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.   DOI   ScienceOn
19 Zhao, H. and Arnold, F. H. 1999. Directed evolution converts subtilisin E into a functional equivalent of thermitase. Protein Eng 12, 47-53.   DOI
20 Astrup, T. and Mullertz, S. 1952. The fibrin plate method for estimating fibrinolytic activity. Arch Biochem Biophys 6, 346-351.
21 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.   DOI   ScienceOn
22 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.
23 Collen, D. and Lijnen, H. R. 2005. Thrombolytic agents. Thromb Haemost 93, 627-630.
24 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.   DOI   ScienceOn
25 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.   DOI   ScienceOn
26 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.   DOI   ScienceOn
27 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.   과학기술학회마을