纳豆激酶及其生物工程研究进展
Research Progress on Nattokinase and Its Bioengineering
DOI: 10.12677/HJBM.2023.132027, PDF,   
作者: 林飞龙*, 李泰明#：中国药科大学生命科学与技术学院，江苏 南京
关键词: 纳豆激酶；溶栓；分子生物学；研究进展；Nattokinase； Thrombolysis； Molecular Biology； Research Progress
摘要: 纳豆激酶是从传统发酵食品纳豆中提取分离得到的一种由枯草杆菌分泌的碱性丝氨酸蛋白酶，因其具有强大的溶解血栓的能力，受到广大研究者的关注，有望成为新一代溶栓药物。本文首先从结构、理化特性、催化及溶栓机制方面对纳豆激酶做了介绍，其次简述了纳豆激酶全酶的分子生物学研究，包括信号肽、前导肽和成熟肽的改造，最后进行展望。本文期望为后续纳豆激酶结构和功能关系、药物开发及生物工程研究提供理论基础和新思路。
Abstract: Nattokinase is a kind of alkaline serine protease secreted by Bacillus subtilis, which is extracted and isolated from traditional fermented food natto. Due to its powerful thrombolysis ability, nattokinase has attracted the attention of most researchers and is expected to become a new generation of thrombolysis drugs. In this paper, the structure, physicochemical properties, catalysis and thrombolytic mechanism of nattokinase were firstly introduced. Secondly, the molecular biological research of nattokinase was briefly introduced, including the modification of signal peptide, propeptide and mature peptide. Finally, the prospect was made. The paper is expected to provide theoretical basis and new ideas for the relationship between structure and function of nattokinase, bioengineering research and drug development.
文章引用：林飞龙, 李泰明. 纳豆激酶及其生物工程研究进展[J]. 生物医学, 2023, 13(2): 240-245. https://doi.org/10.12677/HJBM.2023.132027

参考文献

[1] Chen, H., McGowan, E.M., Ren, N., et al. (2018) Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases. Biomark, 13. [Google Scholar] [CrossRef] [PubMed]
[2] Sumi, H., Hamada, H., Tsushima, H., et al. (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. [Google Scholar] [CrossRef] 
[3] Jensen, G.S., Lenninger, M., Ero, M.P., et al. (2016) Consumption of Nattokinase Is Associated with Reduced Blood Pressure and von Willebrand Factor, a Cardiovascular Risk Marker: Results from a Randomized, Double-Blind, Placebo-Controlled, Multicenter North American Clinical Trial. Integrative and Comparative Biology, 9, 95-104. [Google Scholar] [CrossRef] 
[4] 孟繁宇, 薛菲, 施慧. 纳豆激酶对动脉粥样硬化模型大鼠血脂及血液流变学影响[J]. 中国实验诊断学, 2013, 17(9): 1567-1569.
[5] Ji, H., Yu, L., Liu, K., et al. (2014) Mechanisms of Nattokinase in Protection of Cerebral Ischemia. European Journal of Pharmacology, 745, 144-151. [Google Scholar] [CrossRef] [PubMed]
[6] 占慧琴, 袁志兰, 杨翎, 等. 纳豆激酶对视网膜中央静脉阻塞动物模型中闪光视网膜电图的影响[J]. 南京医科大学学报(自然科学版), 2010, 30(7): 974-976+1016.
[7] Takabayashi, T., Imoto, Y., Sakashita, M., et al. (2017) Nattokinase, Profibrinolytic Enzyme, Effectively Shrinks the Nasal Polyp Tissue and Decreases Viscosity of Mucus. Allergology International, 66, 594-602. [Google Scholar] [CrossRef] [PubMed]
[8] (2012) Optimisation of Preparation Conditions and Properties of Phytosterol Liposome-Encapsulating Nattokinase. Natural Product Research, 26, 548-556. [Google Scholar] [CrossRef] [PubMed]
[9] 陈景鑫. 纳豆激酶微胶囊的制备及其稳定性研究[D]: [硕士学位论文]. 大庆: 黑龙江八一农垦大学, 2010.
[10] 谢建飞. 一种含有纳豆激酶和益生菌的微胶囊及其制备方法和应用[P]. 中国, CN201410618436.7. 2015-02-25.
[11] Zhang, X., Lyu, X., Tong, Y., et al. (2020) Chitosan/Casein Based Microparticles with a Bilayer Shell-Core Structure for Oral Delivery of Nattokinase. Food & Function, 11, 10799-10816. [Google Scholar] [CrossRef] 
[12] Deepak, V., Kalishwaralal, K., Ramkumarpandian, S., et al. (2008) Optimization of Media Composition for Nattokinase Production by Bacillus subtilis Using Response Surface Methodology. Bioresource Technology, 99, 8170-8174. [Google Scholar] [CrossRef] [PubMed]
[13] 李佳增. 纳豆激酶毕赤酵母X33高效表达双启动子系统构建及发酵条件优化[D]: [硕士学位论文]. 沈阳: 辽宁大学, 2021.
[14] Nakamura, T., Yamagata, Y. and Ichishima, E. (1992) Nucleotide Sequence of the Subtilisin NAT Gene, aprN, of Bacillus subtilis (Natto). Bioscience, Biotechnology, and Biochemistry, 56, 1869-1871. [Google Scholar] [CrossRef] [PubMed]
[15] Yanagisawa, Y., Chatake, T., Chiba-Kamoshida, K., et al. (2010) Purification, Crystallization and Preliminary X-Ray Diffraction Experiment of Nattokinase from Bacillus subtilis Natto. Acta Crystallographica Section F, 66, 1670-1673. [Google Scholar] [CrossRef] 
[16] Carter, P. and Wells, J.A. (1988) Dissecting the Catalytic Triad of a Serine Protease. Nature, 332, 564-568. [Google Scholar] [CrossRef] [PubMed]
[17] Weng, Y., Yao, J., Sparks, S., et al. (2017) Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease. International Journal of Molecular Sciences, 18, 523. [Google Scholar] [CrossRef] [PubMed]
[18] 李睿, 阮文辉, 熊晓辉. 纳豆激酶的酶学性质研究[J]. 扬州大学学报(农业与生命科学版), 2015, 36(2): 106-110.
[19] Li, Y., et al. (2010) Bioproperties of Potent Nattokinase from Bacillus subtilis YJ1. Journal of Agricultural and Food Chemistry, 58, 5737-5742. [Google Scholar] [CrossRef] [PubMed]
[20] Li, Y., et al. (2022) Biotechnology, Bioengineering and Applications of Bacillus nattokinase. Biomolecules, 12, 980. [Google Scholar] [CrossRef] [PubMed]
[21] Liang, X., Jia, S., Sun, Y., et al. (2007) Secretory Expression of Nattokinase from Bacillus subtilis YF38 in Escherichia coli. Molecular Biotechnology, 37, 187-194. [Google Scholar] [CrossRef] [PubMed]
[22] 何孝天, 刘中美, 崔文璟, 等. 介导纳豆激酶分泌表达的信号肽比较[J]. 现代食品科技, 2014, 30(5): 62-68.
[23] Cai, D., Wei, X., Qiu, Y., et al. (2016) High-Level Expression of Nattokinase in Bacillus licheniformis by Manipulating Signal Peptide and Signal Peptidase. Journal of Applied Microbiology, 121, 704-712. [Google Scholar] [CrossRef] [PubMed]
[24] Jia, Y., Liu, H., Bao, W., et al. (2010) Functional Analysis of Propeptide as an Intramolecular Chaperone for in Vivo Folding of Subtilisin Nattokinase. FEBS Letters, 584, 4789-4796. [Google Scholar] [CrossRef] [PubMed]
[25] Yang, M., Wu, J., Huang, Q. and Jia, Y. (2021) Probing the Role of Catalytic Triad on the Cleavage between Intramolecular Chaperone and NK Mature Peptide. Journal of Agricultural and Food Chemistry, 69, 2348-2353. [Google Scholar] [CrossRef] [PubMed]
[26] Ikemura, H., Takagi, H. and Inouye, M. (1987) Requirement of Pro-Sequence for the Production of Active Subtilisin E in Escherichia coli. Journal of Biological Chemistry, 262, 7859-7864. [Google Scholar] [CrossRef] 
[27] Jain, S.C., Shinde, U., Li, Y., et al. (1998) The Crystal Structure of an Autoprocessed Ser221 Cys-Subtilisin E-Propeptide Complex at 2.0 A Resolution. Journal of Molecular Biology, 284, 137-144. [Google Scholar] [CrossRef] [PubMed]
[28] 田莉. 构建高产纳豆激酶基因工程菌株及酶活研究[D]: [硕士学位论文]. 武汉: 武汉工程大学, 2019.
[29] Weng, M., Zheng, Z., Bao, W., et al. (2009) Enhancement of Oxidative Stability of the Subtilisin Nattokinase by Site-Directed Mutagenesis Expressed in Escherichia coli. Biochimica et Biophysica Acta, 1794, 1566-1572. [Google Scholar] [CrossRef] [PubMed]
[30] Jia, Y., Cao, X., Deng, Y., et al. (2014) Four Residues of Propeptide Are Essential for Precursor Folding of Nattokinase. Acta Biochimica et Biophysica Sinica (Shanghai), 46, 957-964. [Google Scholar] [CrossRef] [PubMed]
[31] Cai, Y.J., et al. (2011) Directed Evolution Improves the Fibrinolytic Activity of Nattokinase from Bacillus natto. FEMS Microbiology Letters, 325, 155-161. [Google Scholar] [CrossRef] [PubMed]
[32] Wu, S., Feng, C., Zhong, J. and Huan, L. (2007) Roles of s3 Site Residues of Nattokinase on Its Activity and Substrate Specificity. The Journal of Biochemistry, 142, 357-364. [Google Scholar] [CrossRef] [PubMed]
[33] 赵菡, 周丽, 周哲敏. 通过定点突变提高纳豆激酶的酶活及热稳定性[J]. 食品与发酵工业, 2018, 44(9): 36-40+47.
[34] Weng, M., Deng, X., Bao, W., et al. (2015) Improving the Activity of the Subtilisin Nattokinase by Site-Directed Mutagenesis and Molecular Dynamics Simulation. Biochemical and Biophysical Research Communications, 465, 580-586. [Google Scholar] [CrossRef] [PubMed]
[35] 刘中美, 何孝天, 崔文璟, 周哲敏. 通过定点突变增强纳豆激酶的热稳定性[J]. 现代食品科技, 2015, 31(2): 37-41.
[36] Zhao, H. and Arnold, F.H. (1999) Directed Evolution Converts Subtilisin E into a Functional Equivalent of Thermitase. Protein Engineering, 12, 47-53. [Google Scholar] [CrossRef] [PubMed]
[37] 刘朔, 姜梅, 陈晓红, 等. 纳豆激酶第36位氨基酸突变对其活性及热稳定性的影响[J]. 南京农业大学学报, 2008(3): 130-136.

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