纳豆激酶分子改造及其在心血管疾病防治中的研究进展

摘要/Abstract

摘要： 心血管疾病（CVDs）是全球首要死因,血栓形成是其关键的病理基础。纳豆激酶（NK）是一种鉴定自发酵食品纳豆、具有高效溶栓活性的丝氨酸蛋白酶,其通过直接降解纤维蛋白、激活内源性纤溶系统、改善血流特性及抗炎等多重机制发挥防治CVDs的作用,并具备口服有效性和良好的安全性优势。然而,野生型NK存在热稳定性差、耐酸性弱等缺陷,限制了其应用。本文系统综述了NK分子改造及其在心血管疾病防治中的研究进展。通过定点突变、表面残基工程、分子动力学模拟指导设计及协同进化分析等策略,显著提升了NK的催化活性、热稳定性、耐酸性和抗氧化性。利用枯草芽孢杆菌等安全宿主,通过启动子工程、信号肽优化及发酵工艺改进,实现了NK的高水平异源分泌表达,为规模化生产奠定了基础。功能研究表明NK能有效溶解血栓、降低血压、调节血脂并能减缓动脉粥样硬化进程;同时具有较高的使用安全性。新型递送系统的开发有效提高了NK的生物利用度和靶向性。

关键词: 纳豆激酶, 心血管疾病, 血栓, 溶栓, 分子改造, 靶向递送

殷英, 郭军玲, 周梁云. 纳豆激酶分子改造及其在心血管疾病防治中的研究进展[J]. 心血管病防治知识, 2026, 16(1): 132-138.

参考文献

[1] Kuipers E N, Dam A D V, Held N M, et al. Quercetin Lowers Plasma Triglycerides Accompanied by White Adipose Tissue Browning in Diet-Induced Obese Mice[J]. Int J Mol Sci, 2018, 19(6):1786.
[2] Shi H, Guo J, Xu K, et al.Study on the value of small dense low-density lipoprotein in predicting cardiovascular and cerebrovascular events in the high-risk stroke population[J]. J Clin Lab Anal, 2022, 36(4): e24278.
[3] Wu H, Lei Z, Gao S, et al.YiqiHuoxue Decoction and Its Ethanol Precipitation Show Anti-Platelet and Antithrombotic Effects by Suppressing Thromboxane B2 Formation[J]. Acta Cardiol Sin, 2019, 35(5): 524-533.
[4] 莫合塔伯尔·莫敏, 李建平, 张岩, 等. 急性冠状动脉综合征患者血浆同型半胱氨酸水平与左心室射血分数及心功能严重程度的相关性研究[J]. 中国介入心脏病学杂志, 2015, (5): 8.
[5] Shankar K N, Zhang Y, Sinno T, et al.A three-dimensional multiscale model for the prediction of thrombus growth under flow with single-platelet resolution[J]. PLoS Comput Biol, 2022, 18(1): e1009850.
[6] 陈佳仑, 李树仁, 吕晓, 等. 不同血栓负荷的ST段抬高型心肌梗死患者急诊经皮冠状动脉介入治疗中血栓抽吸效果的临床观察[J]. 中国心血管杂志, 2022, 27(4): 351-356.
[7] 张艳玲, 吕铁伟, 王娟莉. 儿童心肌病心腔内血栓研究进展[J]. 临床医学进展, 2023, 13(12): 18559-18565.
[8] Ayadurai S, Hattingh H L, Tee L B, et al.A Narrative Review of Diabetes Intervention Studies to Explore Diabetes Care Opportunities for Pharmacists[J]. J Diabetes Res, 2016: 5897452.
[9] Chen J, Du Z, Song B, et al.A natural heparinoid from mollusc Meretrix lusoria: Purification, structural characterization, and antithrombotic evaluation[J]. Curr Res Food Sci, 2022, 5: 1897-1905.
[10] Tian G, Ci H, Song W, et al.Study on the Correlation between the Prevalence of Venous Thromboembolism in Kazak Pregnant and Lying-In Women in Xinjiang[J]. Contrast Media Mol Imaging, 2022: 7001743.
[11] Sumi H, Hamada H, Tsushima H, et al.A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet[J]. Experientia, 1987, 43(10): 1110-1111.
[12] Fujita M, Nomura K, Hong K, et al.Purification and characterization of a strong fibrinolytic enzyme (nattokinase) in the vegetable cheese natto, a popular soybean fermented food in Japan[J]. Biochem Biophys Res Commun, 1993, 197(3): 1340-1347.
[13] Nagata C, Wada K, Tamura T, et al.Dietary soy and natto intake and cardiovascular disease mortality in Japanese adults: the Takayama study[J]. Am J Clin Nutr, 2017, 105(2): 426-431.
[14] Nakamura T, Yamagata Y, Ichishima E.Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto)[J]. Biosci Biotechnol Biochem, 1992, 56(11): 1869-1871.
[15] Rautengarten C, Steinhauser D, BüSsis D, et al. Inferring hypotheses on functional relationships of genes: Analysis of the Arabidopsis thaliana subtilase gene family[J]. PLoS Comput Biol, 2005, 1(4): e40.
[16] Yanagisawa Y, Chatake T, Chiba-Kamoshida K, et al.Purification, crystallization and preliminary X-ray diffraction experiment of nattokinase from Bacillus subtilis natto[J]. Acta Crystallogr Sect F Struct Biol Cryst Commun, 2010, 66(Pt 12): 1670-1673.
[17] Thu N T A, Khue N T M, Huy N D, et al. Characterizations and Fibrinolytic Activity of Serine Protease from Bacillus subtilis C10[J]. Curr Pharm Biotechnol, 2020, 21(2): 110-116.
[18] Luo J, Song C, Cui W, et al.Precise redesign for improving enzyme robustness based on coevolutionary analysis and multidimensional virtual screening[J]. Chem Sci, 2024, 15(38): 15698-15712.
[19] Yatagai C, Maruyama M, Kawahara T, et al.Nattokinase-promoted tissue plasminogen activator release from human cells[J]. Pathophysiol Haemost Thromb, 2008, 36(5): 227-232.
[20] Chen H, Mcgowan E M, Ren N, et al.Nattokinase: A Promising Alternative in Prevention and Treatment of Cardiovascular Diseases[J]. Biomark Insights, 2018, 13: 1177271918785130.
[21] Tai M W, Sweet B V.Nattokinase for prevention of thrombosis[J]. Am J Health Syst Pharm, 2006, 63(12): 1121-1123.
[22] Li Y, Tang X, Chen L, et al.Improvement of the fibrinolytic activity, acid resistance and thermostability of nattokinase by surface charge engineering[J]. Int J Biol Macromol, 2023, 253(Pt 6): 127373.
[23] Liu Z, Zhao H, Han L, et al.Improvement of the acid resistance, catalytic efficiency, and thermostability of nattokinase by multisite-directed mutagenesis[J]. Biotechnol Bioeng, 2019, 116(8): 1833-1843.
[24] Weng M, Zheng Z, Bao W, et al.Enhancement of oxidative stability of the subtilisin nattokinase by site-directed mutagenesis expressed in Escherichia coli[J]. Biochim Biophys Acta, 2009, 1794(11): 1566-1572.
[25] Luo J, Song C, Cui W, et al.Counteraction of stability-activity trade-off of Nattokinase through flexible region shifting[J]. Food Chem, 2023, 423: 136241.
[26] Ni H, Guo P C, Jiang W L, et al.Expression of nattokinase in Escherichia coli and renaturation of its inclusion body[J]. J Biotechnol, 2016, 231: 65-71.
[27] Liang X, Zhang L, Zhong J, et al.Secretory expression of a heterologous nattokinase in Lactococcus lactis[J]. Appl Microbiol Biotechnol, 2007, 75(1): 95-101.
[28] Chen P T, Chiang C J, Chao Y P.Strategy to approach stable production of recombinant nattokinase in Bacillus subtilis[J]. Biotechnol Prog, 2007, 23(4): 808-813.
[29] Wei X, Zhou Y, Chen J, et al.Efficient expression of nattokinase in Bacillus licheniformis: host strain construction and signal peptide optimization[J]. J Ind Microbiol Biotechnol, 2015, 42(2): 287-295.
[30] Cui W, Han L, Suo F, et al.Exploitation of Bacillus subtilis as a robust workhorse for production of heterologous proteins and beyond[J]. World J Microbiol Biotechnol, 2018, 34(10): 145.
[31] Han L, Chen Q, Luo J, et al.Development of a Glycerol-Inducible Expression System for High-Yield Heterologous Protein Production in Bacillus subtilis[J]. Microbiol Spectr, 2022, 10(5): e0132222.
[32] Guan C, Cui W, Cheng J, et al.Development of an efficient autoinducible expression system by promoter engineering in Bacillus subtilis[J]. Microb Cell Fact, 2016, 15: 66.
[33] Han L, Cui W, Suo F, et al.Development of a novel strategy for robust synthetic bacterial promoters based on a stepwise evolution targeting the spacer region of the core promoter in Bacillus subtilis[J]. Microb Cell Fact, 2019, 18(1): 96.
[34] Liu Z, Zheng W, Ge C, et al.High-level extracellular production of recombinant nattokinase in Bacillus subtilis WB800 by multiple tandem promoters[J]. BMC Microbiol, 2019, 19(1): 89.
[35] Cai D, Wei X, Qiu Y, et al.High-level expression of nattokinase in Bacillus licheniformis by manipulating signal peptide and signal peptidase[J]. J Appl Microbiol, 2016, 121(3): 704-712.
[36] Sheng Y, Yang J, Wang C, et al.Microbial nattokinase: from synthesis to potential application[J]. Food Funct, 2023, 14(6): 2568-2585.
[37] Urano T, Ihara H, Umemura K, et al.The profibrinolytic enzyme subtilisin NAT purified from Bacillus subtilis Cleaves and inactivates plasminogen activator inhibitor type 1[J]. J Biol Chem, 2001, 276(27): 24690-24696.
[38] Hsia C H, Shen M C, Lin J S, et al.Nattokinase decreases plasma levels of fibrinogen, factor VII, and factor VIII in human subjects[J]. Nutr Res, 2009, 29(3): 190-196.
[39] Jang J Y, Kim T S, Cai J, et al.Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation[J]. Lab Anim Res, 2013, 29(4): 221-225.
[40] Chiu H W, Chou C L, Lee K T, et al.Nattokinase attenuates endothelial inflammation through the activation of SRF and THBS1[J]. Int J Biol Macromol, 2024, 268(Pt 2): 131779.
[41] Zhou Y, Fu Z, Dou P, et al.Decoding nattokinase efficacy: From digestion and absorption to lipid pathway modulation in high-fat diet-induced atherosclerosis[J]. Food Bioscience, 2025, 66: 106203.
[42] Muric M, Nikolic M, Todorovic A, et al.Comparative Cardioprotective Effectiveness: NOACs vs Nattokinase-Bridging Basic Research to Clinical Findings[J]. Biomolecules, 2024, 14(8):956.
[43] Kim J Y, Gum S N, Paik J K, et al.Effects of nattokinase on blood pressure: a randomized, controlled trial[J]. Hypertens Res, 2008, 31(8): 1583-1588.
[44] Yoo H J, Kim M, Kim M, et al.The effects of nattokinase supplementation on collagen-epinephrine closure time, prothrombin time and activated partial thromboplastin time in nondiabetic and hypercholesterolemic subjects[J]. Food Funct, 2019, 10(5): 2888-2893.
[45] Wu H, Wang H, Xu F, et al.Acute toxicity and genotoxicity evaluations of Nattokinase, a promising agent for cardiovascular diseases prevention[J]. Regul Toxicol Pharmacol, 2019, 103: 205-209.
[46] Guo H, Ban Y H, Cha Y, et al.Comparative anti-thrombotic activity and haemorrhagic adverse effect of nattokinase and tissue-type plasminogen activator[J]. Food Sci Biotechnol, 2019, 28(5): 1535-1542.
[47] Wu H, Wang H, Li W, et al.Nattokinase-heparin exhibits beneficial efficacy and safety-an optimal strategy for CKD patients on hemodialysis[J]. Glycoconj J, 2019, 36(2): 93-101.
[48] Chen H, Chen J, Zhang F, et al.Effective management of atherosclerosis progress and hyperlipidemia with nattokinase: A clinical study with 1,062 participants[J]. Front Cardiovasc Med, 2022, 9: 964977.
[49] Law D, Zhang Z.Stabilization and target delivery of Nattokinase using compression coating[J]. Drug Dev Ind Pharm, 2007, 33(5): 495-503.
[50] Priya V, Samridhi, Singh N, et al.Nattokinase Encapsulated Nanomedicine for Targeted Thrombolysis: Development, Improved in Vivo Thrombolytic Effects, and Ultrasound/Photoacoustic Imaging[J]. Mol Pharm, 2024, 21(1): 283-302.
[51] Xie M, Yang M, Wang Z, et al.Preserving the activity of attenuating atherosclerosis of nattokinase in gastrointestinal system by NK/SA/CS microcapsules[J]. Food Bioscience, 2024, 59: 103949.
[52] Zhang X, Lyu X, Tong Y, et al.Chitosan/casein based microparticles with a bilayer shell-core structure for oral delivery of nattokinase[J]. Food Funct, 2020, 11(12): 10799-10816.
[53] Shi Y, Liu C, Gui Y, et al.A Nattokinase-Loaded Nanozyme for Alleviating Acute Myocardial Infarction via Thrombolysis and Antioxidation[J]. Adv Healthc Mater, 2025, 14(8): e2402763.
[54] Gao R, Zhang W, Chen X, et al.Dual Frequency-Regulated Magnetic Vortex Nanorobots Empower Nattokinase for Focalized Microvascular Thrombolysis[J]. ACS Nano, 2024, 18(43): 29492-29506.