纳豆激酶的研究现状与展望

纳豆激酶是从日本传统食品纳豆中提取的一种枯草杆菌蛋白酶,具有高效的抗血栓作用.与其他传统溶栓剂相比,纳豆激酶具有安全性好、成本低、易被人体吸收、作用直接迅速、作用持续时间长等优点,因此极有可能开发为新一代的溶栓剂.概括了纳豆激酶的分子结构、理化性质、溶栓作用机理、活性检测方法,介绍了其分子生物学研究进展,并对其开发应用进行了展望,证明纳豆激酶具有广阔的市场应用前景.     

纳豆芽孢杆菌菌种纯化及不同氮源对其产纳豆激酶的影响

纳豆激酶(nattokinase, NK)是一种由纳豆芽孢杆菌发酵产生的丝氨酸蛋白酶,具有良好的纤溶活性。本研究从wako Nattokinase中分离纯化出高品质的纳豆芽孢杆菌,旨在探究最适宜该菌产纳豆激酶的发酵培养基氮源。研究人员选择了6种氮源对其进行发酵实验,通过连续测定发酵液的菌量、pH和纤溶活性以观察不同氮源对纳豆芽孢杆菌产纳豆激酶的影响。研究结果表明:最优氮源为乳清蛋白,在以此为氮源的培养基中发酵培养120 h后,纳豆激酶的纤溶活性高达1 757.79 U/mL。以乳清蛋白发酵培养基对纳豆芽孢杆菌进行发酵,不仅可以得到高活性的纳豆激酶,还可为纳豆激酶应用于食品、保健品领域提供思路。     

纳豆激酶基因工程研究进展

纳豆激酶是由纳豆芽孢杆菌(Bacillussubtillisnatto)分泌的一种具有纤溶作用的碱性丝氨酸蛋白酶。对纳豆激酶基因的克隆与表达,基因与蛋白质结构以及基因工程纳豆激酶的特性和功能进行了综述。     

纳豆激酶的研究与应用

纳豆激酶是一种枯草芽孢杆菌产生的丝氨酸蛋白酶 ,具有强烈的纤溶活性 ,有望开发成为新型口服溶栓药。综述了纳豆激酶的生化特性、生物学活性、结构与功能及开发应用前景等。     

药食两用纳豆激酶的研究进展

本文从血栓栓塞疾病的形成机制为依据,综述了纳豆激酶的药理作用。介绍了国内外产纤溶酶菌株的筛选方法,纳豆激酶的分离、纯化常用技术,纳豆激酶的生理生化特性、活性测定方法。并说明了影响纳豆激酶稳定的4个因素,和增加纳豆激酶产量的有效方法。通过药食两用纳豆激酶的研究进展,希望对纳豆激酶的研究提供理论基础。     

一株高产纳豆激酶菌株的鉴定与分析

鉴定高产纳豆激酶菌株Td,分析纳豆激酶的分子特征。利用菌体形态、生理生化特征、以及分子生物学方法对菌株Td进行鉴定;并采用MALDI-TOF质谱测定与分析、SDS-PAGE和纤溶活性测定等方法检测纳豆激酶特性,利用PCR方法扩增纳豆激酶的基因全长。结合菌体形态、生理生化特征和16S r DNA、gyr A基因序列、DNA-DNA杂交率等实验结果,鉴定菌株Td为枯草芽孢杆菌枯草亚种(Bacillus subtilis subsp.subtilis);菌株Td发酵产生的纳豆激酶产量可达300 mg/L以上,占发酵液总蛋白的40%以上;纤溶活性达230 U/m L以上;氨基酸序列与subtilisin E的序列相似性最高;基因全长序列为1 143 bp。枯草芽孢杆菌枯草亚种Td是一株高产、高活性纳豆激酶的产生菌,具有优良的工业化开发价值。     

液态发酵豆粕制备纳豆激酶方法的优化

纳豆激酶是一种丝氨酸蛋白酶,具有很强的纤溶活性,由于具有安全性好、作用迅速持久、成本低等优点,适合用于开发新一代的溶栓剂或保健食品,具有广阔的市场前景。本研究探讨了以豆粕为原料液态发酵豆粕生产纳豆激酶的发酵方法。首先通过单因素实验发现影响产酶的主要因素有接菌量、发酵时间、培养基pH及豆粕含量,再由正交实验得到最优组合为接菌量1%,豆粕含量2%,pH为7.0,发酵时间48h,该条件下发酵酶活力最高达到4 429.6U/mL。本研究确定了以豆粕为原料制备纳豆激酶的最佳条件,为豆粕的合理使用和纳豆激酶的工业化生产提供了实验依据。     

纳豆激酶在血管性疾病防治中的研究进展

血管性疾病是疾病谱中最主要的死亡原因之一，血栓栓塞是血管性疾病中的重要组成部分，而目前针对其预防和治疗手段非常有限。纳豆激酶（nattokinase，NK）是从纳豆中提取的活性成分，具有多种对心血管有益作用，如较强的纤溶活性、降压、降脂及抗凝血等。本文综述了纳豆激酶的生化特性、防治心血管疾病的功效及应用前景，通过总结最新文献重点完善了纳豆激酶在心血管疾病方面的药理作用，表明纳豆激酶可以作为预防和治疗血管性疾病的理想膳食补充剂，其药品开发同样前景广阔。     

纳豆激酶的液体发酵条件优化

以纳豆素中分离的纳豆芽孢杆菌进行纳豆激酶发酵试验,考察装液量、接种量、摇床转速等因素对生物量和纳豆激酶纤溶活性的影响,揭示产酶规律并确定最优产酶条件。发酵培养基配方为2%葡萄糖、2%蛋白胨、0.05%Mg SO4、0.01%Ca Cl2、0.6%Na2HPO4、0.4%Na H2PO4。最终确定的发酵条件为:p H 6.9,2%接种量,45 m L/250 m L的装液量,33.5℃,摇床转速为165 r/min,在此条件下发酵33~34 h为产酶高峰期,纳豆激酶的纤溶活性达到1 004 U/m L,结果也表明纳豆激酶是一种生长关联型代谢产物,纤溶活性与生物量变化趋势高度一致。     

纳豆激酶基因的克隆及其在毕赤酵母中的表达

纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。     

Microbial production of nattokinase: current progress,challenge and prospect

Nattokinase (EC 3.4.21.62) is a profibrinolytic serine protease with a potent fibrin-degrading activity, and it has been produced by many host strains. Compared to other fibrinolytic enzymes (urokinase, t-PA and streprokinase), nattokinase shows the advantages of having no side effects, low cost and long life-time, and it has the potential to be used as a drug for treating cardiovascular disease and served as a functional food additive. In this review, we focused on screening of producing strains, genetic engineering, fermentation process optimization for microbial nattokinase production, and the extraction and purification of nattokinase were also discussed in this particular chapter. The selection of optimal nattokinase producing strain was the crucial starting element for improvement of nattokinase production. Genetic engineering, protein engineering, fermentation optimization and process control have been proved to be the effective strategies for enhancement of nattokinase production. Also, extraction and purification of nattokinase are critical for the quality evaluation of nattokinase. Finally, the prospect of microbial nattokinase production was also discussed regarding the recent progress, challenge, and trends in this field.     

纳豆激酶基因的克隆与表达

利用ＰＣＲ方法从分泌纳豆激酶的枯草杆菌基因组ＤＮＡ 中扩增得到了纳豆激酶基因,并测定其核苷酸序列．利用基因重组技术构建了纳豆激酶基因的表达载体,并在大肠杆菌中进行了表达．ＳＤＳ－聚丙烯酰胺凝胶电泳表明,表达蛋白占菌体蛋白的１５．２％ ,琼脂糖－纤维蛋白平板法测出表达产物具有溶解血栓活性．     

A novel nattokinase produced by Pseudomonas sp. TKU015 using shrimp shells as substrate

A nattokinase was purified from the culture supernatant of Pseudomonas sp. TKU015 with shrimp shell wastes as the sole carbon/nitrogen source. The molecular masses of TKU015 nattokinase determined by SDS-PAGE and gel filtration were approximately 21 and 24 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU015 nattokinase were 7, 50 °C, pH 4–11, and less than 50 °C, respectively. TKU015 nattokinase was inhibited completely by PMSF, indicating that the TKU015 nattokinase was serine protease. The results of peptide mass mapping showed that two tryptic peptides of the nattokinase were identical to a chitin binding protein from Bacillus cereus ATCC 14579 (GenBank accession number gi30020946) with 23% sequence coverage. With this method, Pseudomonas sp. TKU015 produces a nattokinase/fibrinolytic enzyme and may be considered as a new source for thrombolytic agents.     

Production of nattokinase by high cell density fed-batch culture of Bacillus subtilis

Bacillus subtilis was cultivated to high cell density for nattokinase production by pH-stat fed-batch culture. A concentrated mixture solution of glucose and peptone was automatically added by acid-supplying pump when culture pH rose above high limit. Effect of the ratio of glucose to peptone in feeding solution was investigated on cell growth and nattokinase production by changing the ratio from 0.2 to 5 g glucose/g peptone. The highest cell concentration was 77 g/L when the ratio was 0.2 g glucose/g peptone. Cell concentration decreased with increasing the ratio of glucose to peptone in feeding solution, while the optimum condition existed for nattokinase production. The highest nattokinase activity was 14,500 unit/mL at a ratio of 0.33 g glucose/g peptone, which was 4.3 times higher than that in batch culture.     

Secretory Expression of Nattokinase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> YF38 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Nattokinase producing bacterium, B. subtilis YF38, was isolated from douchi, using the fibrin plate method. The gene encoding this enzyme was cloned by polymerase chain reaction (PCR). Cytoplasmic expression of this enzyme in E. coli resulted in inactive inclusion bodies. But with the help of two different signal peptides, the native signal peptide of nattokinase and the signal peptide of PelB, active nattokinase was successfully expressed in E. coli with periplasmic secretion, and the nattokinase in culture medium displayed high fibrinolytic activity. The fibrinolytic activity of the expressed enzyme in the culture was determined to reach 260 urokinase units per micro-liter when the recombinant strain was induced by 0.7 mmol l⁻¹ isopropyl-β-D- thiogalactopyranoside (IPTG) at 20°C for 20 h, resulting 49.3 mg active enzyme per liter culture. The characteristic of this recombinant nattokinase is comparable to the native nattokinase from B. subtilis YF38. Secretory expression of nattokinase in E. coli would facilitate the development of this enzyme into a therapeutic product for the control and prevention of thrombosis diseases.     

Rapid Hydrolysis Pathway for a Tertiary Alcohol Ester through Intramolecular Transesterification in Rats

A specific and sensitive enzyme-linked immunosorbent assay (ELISA) was developed for nattokinase, a subtilisin-like fibrinolytic enzyme in the fermented soybean food, natto. The assay method was developed using a combination of a murine anti-nattokinase monoclonal antibody coated on the microtiter well as a catching antibody and rabbit anti-nattokinase polyclonal antibody as a peroxidase-conjugated detecting antibody. The assay sensitivity was 0.1 ng/ml and cross-reactivity with subtilisin BPN′ and subtilisin Carlsberg was 0.0002% and 0.00002%, respectively. The ELISA value reflected the fibrinolytic activity of nattokinase. The correlation coefficient between nattokinase measured by a fibrinolytic assay and by the ELISA in 14 commercially available natto extracts was 0. 967, suggesting that nattokinase is the only fibrinolytic enzyme in natto.     

Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth

An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.     

Secretory expression of a heterologous nattokinase in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Nattokinase has been reported as an oral health product for the prevention of atherosclerosis. We developed a novel strategy to express a nattokinase from Bacillus subtilis in a live delivery vehicle, Lactococcus lactis. Promoter P _nisZ and signal peptide SP_Usp were used for inducible and secretory expression of nattokinase in L. lactis. Western blotting analysis demonstrated that nattokinase was successfully expressed, and about 94% of the enzyme was secreted to the culture. The recombinant nattokinase showed potent fibrinolytic activity, equivalent to 41.7 urokinase units per milliliter culture. Expression and delivery of such a fibrinolytic enzyme in the food-grade vehicle L. lactis would facilitate the widespread application of nattokinase in the control and prevention of thrombosis diseases.     

Directed evolution improves the fibrinolytic activity of nattokinase from Bacillus natto

Nattokinase (subtilisin NAT, NK) is a relatively effective microbial fibrinolytic enzyme that has been identified and characterized from Bacillus natto. In the current report, DNA family shuffling was used to improve the fibrinolytic activity of nattokinase. Three homologous genes from B. natto AS 1.107, Bacillus amyloliquefaciens CICC 20164 and Bacillus licheniformis CICC 10092 were shuffled to generate a mutant library. A plate-based method was used to screen the mutant libraries for improved activity. After three rounds of DNA shuffling, one desirable mutant with 16 amino acid substitutions was obtained. The mutant enzyme was purified and characterized. The kinetic measurements showed that the catalytic efficiency of the mutant NK was approximately 2.3 times higher than that of the wild-type nattokinase. In addition, the molecular modeling analysis suggested that the mutations affect the enzymatic function by changing the surface conformation of the substrate-binding pocket. The current study shows that the evolution of nattokinase with improved fibrinolytic activity by DNA family shuffling is feasible and provides useful references to facilitate the application of nattokinase in thrombolytic therapy.