Enhancing thermostability of nattokinase by site-directed mutagenesis

被引：0
|
作者：
Liu, Zhong-Mei [1 ]
He, Xiao-Tian [1 ]
Cui, Wen-Jing [1 ]
Zhou, Zhe-Min [1 ]
机构：
[1] The Key Laboratory of Industrial Biotechnology, School of Biotechnology, Jiangnan University, Wuxi
来源：
Modern Food Science and Technology | 2015年 / 31卷 / 02期
关键词：
Bacillus subtilis; Nattokinase; Site-directed mutagenesis; Thermostability;
D O I：
10.13982/j.mfst.1673-9078.2015.2.007
中图分类号：
学科分类号：
摘要：
Cardiovascular and cerebrovascular diseases caused by thrombi are often life-threatening, contributing to the highest mortality rate. This has fuelled rapid development of thrombolytic drugs and functional foods with thrombolytic activity in the past few years. Due to its strong thrombolytic effect, nattokinase (NK) has become the focus of international interest. The aim of this study was to enhance the thermostability of NK. Based on its sequence alignments and crystal structure, NK mutants were prepared by site-directed mutagenesis and the amino acids that might affect NK thermostability were studied. Among the five mutants generated in this study, P14L and N76D showed significantly increased thermostability and their half-life at 65 ℃ improved from 20 min (wild type) to 30 and 50 min, respectively. Homology modeling and molecular dynamics analysis showed that two amino acid sites influenced thermostability and catalytic activity of NK through different mechanisms. This study provides a theoretical and technical basis for the genetic reconstruction, production, and application of NK. ©, 2015, South China University of Technology. All right reserved.
引用
收藏
页码：37 / 41
页数：4
相关论文
共 12 条
  • [1] Sumi H., Hamada H., Tsushima H., Et al., A noval fibrinolytic enzyme in the vegetable cheese natto: a typical land popular soybean food in the Japanese diet, Experientia, 43, 20, pp. 1110-1117, (1987)
  • [2] Nakamura T., Yamagata Y., Ichishima E., Nucleotide sequence of the Subtilisin NAT gene, aprN, of Bacillus subtilis (natto), Biosci Biotech Biochem, 56, 11, pp. 1869-1871, (1992)
  • [3] Sumi H., Banba T., Kishitei A., Pro-urokinase activation enzyme and thrombolysis ability in natto beans, Journal of Japanese Food Science, 43, 10, pp. 46-49, (1996)
  • [4] Zheng Z.L., Ye M.Q., Zuo Z.Y., Et al., Probing the importance of hydrogen bonds in the active site of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation, Biochem. J., 395, 3, pp. 509-515, (2006)
  • [5] Yongjun C., Wei B., Shujun J., Et al., Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto, FEMS Microbiol. Lett., 325, 2, pp. 155-161, (2011)
  • [6] Yanagisawa Y., Chatake T., Chiba-Kamoshida K., Et al., Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto, Acta Crystallographica Section F Structural Biology and Crystallization Communications, 66, 12, pp. 1670-1673, (2010)
  • [7] Nguyen T.T., Quyen T.D., Le H.T., Cloning and enhancing production of a detergent- and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01 by using an eight-protease-gene-deficient Bacillus subtilis WB800, Microb. Cell Fact., 12, (2013)
  • [8] He X.-T., Liu Z.-M., Cui W.-J., Et al., Comparison of signal peptides for nattokinase secretory expression, Modern Food Science and Technology, 30, 5, pp. 62-68, (2014)
  • [9] Zhao H., Arnold F.H., Directed evolution converts subtilisin E into a functional equivalent of thermitase, Protein Eng., 12, 1, pp. 47-53, (1999)
  • [10] Weng M., Zheng Z., Bao W., Et al., Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli, Biochim. Biophys. Acta, 1794, 11, pp. 1566-1572, (2009)
12