Expression of nattokinase in Escherichia coli and renaturation of its inclusion body

Ni, He; Guo, Peng-Cheng; Jiang, Wanling; Fan, Xiao; Luo, Xiang-Yu; Li, Haihang

doi:10.1016/j.jbiotec.2016.05.034

Cited by 35 publications

(21 citation statements)

References 30 publications

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“…In order to increase NK yields and simplify the downstream purification process, the NK gene has been cloned and expressed in various microbial host systems, including Escherichia coli [43,44,45], B. subtilis [46,47,48], and Lactococcus lactis [49]. E. coli has been extensively investigated as the easiest and cheapest host system to produce recombinant NK.…”

Section: Recombinant Nattokinase Production Via Genetic Engineeringmentioning

confidence: 99%

“…E. coli has been extensively investigated as the easiest and cheapest host system to produce recombinant NK. Although NK can be expressed in E. coli , large amounts of recombinant protein aggregate results in the formation of insoluble and inactive inclusion bodies [43,44]. Recovery of bioactive NK from inclusion bodies is challenging.…”

Section: Recombinant Nattokinase Production Via Genetic Engineeringmentioning

confidence: 99%

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Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

Weng

Yao

Sparks

et al. 2017

IJMS

132

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Natto, a fermented soybean product, has been consumed as a traditional food in Japan for thousands of years. Nattokinase (NK), a potent blood-clot dissolving protein used for the treatment of cardiovascular diseases, is produced by the bacterium Bacillus subtilis during the fermentation of soybeans to produce Natto. NK has been extensively studied in Japan, Korea, and China. Recently, the fibrinolytic (anti-clotting) capacity of NK has been recognized by Western medicine. The National Science Foundation in the United States has investigated and evaluated the safety of NK. NK is currently undergoing a clinical trial study (Phase II) in the USA for atherothrombotic prevention. Multiple NK genes have been cloned, characterized, and produced in various expression system studies. Recombinant technology represents a promising approach for the production of NK with high purity for its use in antithrombotic applications. This review covers the history, benefit, safety, and production of NK. Opportunities for utilizing plant systems for the large-scale production of NK, or for the production of edible plants that can be used to provide oral delivery of NK without extraction and purification are also discussed.

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Section: Recombinant Nattokinase Production Via Genetic Engineeringmentioning

confidence: 99%

Section: Recombinant Nattokinase Production Via Genetic Engineeringmentioning

confidence: 99%

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

Weng

Yao

Sparks

et al. 2017

IJMS

132

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“…It is a potential thrombolytic drug exhibiting strong fibrinolytic activity by directly cleaving crosslinked fibrins, catalyzing the conversion of plasminogen to plasmin or inactivating the fibrinolysis inhibitor. [6][7][8] Most importantly, NK has high safety, low cost, simple production process and low side effects 9 as compared to several other thrombolytic drugs. [10][11][12] Despite these positive attributes, NK is sensitive to variations in harsh external environment and easy to lose the biological activity, thus leading to high dose requirements.…”

Section: Introductionmentioning

confidence: 99%

“…Natto. It is a potential thrombolytic drug exhibiting strong fibrinolytic activity by directly cleaving crosslinked fibrins, catalyzing the conversion of plasminogen to plasmin or inactivating the fibrinolysis inhibitor . Most importantly, NK has high safety, low cost, simple production process and low side effects as compared to several other thrombolytic drugs .…”

Section: Introductionmentioning

confidence: 99%

Multiarm‐polyethylene glycol‐polyglutamic acid peptide dendrimer: Design, synthesis, and dissolving thrombus

Zhang

Lü

Gao

et al. 2018

J Biomedical Materials Res

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Thrombotic events affect many individuals in a number of ways, all of which can cause significant morbidity and mortality. Nattokinase (NK), as a novel thrombolytic drug, has been used for thrombolytic therapy. It not only possesses plasminogen activator activity, but also directly digests fibrin through limited proteolysis. However, it may undergo inactivation and denaturation in the harsh external environment. In this study, a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer was fabricated and used as a carrier for NK protection and delivery. Different arm numbers of polyethylene glycol-polyglutamic acid peptide dendrimers (x-PEG(G ) , x = 2, 4, 6, 8) were designed, prepared, and characterized by H NMR and FTIR. Then, x-PEG(G ) were loaded with NK to form nanocomposites. Their size and morphology were determined by dynamic light scattering and transmission electron microscopy. Enzyme activity was evaluated via UV-Vis absorbance spectra, fluorescence spectra, circular dichroism spectra, and zeta potential measurements. The study reveals that the obtained x-PEG(G ) /NK nanocomposites possess high enzyme activity. In addition, the nanocomposites show increased viability of rat macrophage cells, and excellent thrombolysis ability in vitro and in vivo. This work establishes a multiarm-polyethylene glycol-polyglutamic acid peptide dendrimer with potential application in NK carrier and thrombolytic therapy. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1687-1696, 2018.

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“…Scientists have also attempted to express the gene encoding this enzyme in various hosts such as Escherichia coli (Ni et al, 2016), Bacillus lincheniformis (Cai et al, 2016;Wei et al, 2015), Spodoptera frugiperda (Li et al, 2007), protease-deficient B. subtilis strain such as WB800 (Nguyen et al, 2013), and engineered B. subtilis strain (Wang et al, 2014). These hosts have been shown to be able to produce recombinant nattokinase in good quantities and few of them can be directly used in functional food production.…”

Section: Introductionmentioning

confidence: 99%

Extrachromosomal expression of nat05 gene encoding an alkaline serine protease from Bacillus subtilis N05

Thư

Chau

Thien

et al. 2018

bta

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Nattokinase, the fibrinolytic serine protease, has been shown to be beneficial in preventing strokes caused due to blood clots. Therefore, synthesis of recombinant nattokinase in a host system, which can be both safe for human consumption as well as demonstrate good productivity and amenable to the downstream processing, will have a great socioeconomic significance. A nat05 gene encoding nattokinase-the most important serine protease secreted by the Bacillus subtilis natto-was cloned from B. subtilis strain N05 and expressed using pHT43 plasmid in another B. subtilis strain (BD170). Enzymatic assays, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymogram, were employed to assess the efficiency of the production of nattokinase using a relatively novel recombinant DNA technique. Among the five recombinant bacterial clones (R1-R5), R4 was found to secrete high amounts of nattokinase into the culture medium, as evidenced by the total proteolytic enzyme assay, fibrinolytic enzyme assay, SDS-PAGE, and zymogram. A preliminary analysis of the biochemical properties of the crude enzyme showed characteristics that are typical of nattokinase as reported by other studies. Thus, in this study, we were able to successfully produce nattokinase in B. subtilis strain (BD170) using recombinant DNA approach. The R4 recombinant clone that was found to secrete nattokinase can be used as a source of recombinant protein that can be further purified for various therapeutic applications, or the strain could be used as a probiotic bacterium in functional foods.

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Expression of nattokinase in Escherichia coli and renaturation of its inclusion body

Cited by 35 publications

References 30 publications

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

Nattokinase: An Oral Antithrombotic Agent for the Prevention of Cardiovascular Disease

Multiarm‐polyethylene glycol‐polyglutamic acid peptide dendrimer: Design, synthesis, and dissolving thrombus

Extrachromosomal expression of nat05 gene encoding an alkaline serine protease from Bacillus subtilis N05

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