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Dongbo Cai, Hao Wang, Penghui He, Chengjun Zhu, Qin Wang, Xuetuan Wei, Christopher T Nomura, Shouwen Chen
BACKGROUND: Signal peptide peptidases play an important role in the removal of remnant signal peptides in the cell membrane, a critical step for extracellular protein production. Although these proteins are likely a central component for extracellular protein production, there has been a lack of research on whether protein secretion could be enhanced via overexpression of signal peptide peptidases. RESULTS: In this study, both nattokinase and α-amylase were employed as prototypical secreted target proteins to evaluate the function of putative signal peptide peptidases (SppA and TepA) in Bacillus licheniformis...
April 24, 2017: Microbial Cell Factories
Tetsuji Takabayashi, Yoshimasa Imoto, Masafumi Sakashita, Yukinori Kato, Takahiro Tokunaga, Kanako Yoshida, Norihiko Narita, Tamotsu Ishizuka, Shigeharu Fujieda
BACKGROUND: Chronic rhinosinusitis with nasal polyps (CRSwNP) is often comorbid with asthma and resistant to therapeutic interventions. We recently reported that excessive fibrin deposition caused by impairment of fibrinolysis might play pivotal role in forming nasal polyp. Nattokinase (NK), a serine protease produced by Bacillus subtilis, has been reported to be a strong fibrinolytic enzyme. NK could be a promising drug candidate for use in the treatment of both CRSwNP and asthma. The objective of this study was to investigate the effects of NK on nasal polyp tissues from patients with CRSwNP...
April 4, 2017: Allergology International: Official Journal of the Japanese Society of Allergology
Dongbo Cai, Chengjun Zhu, Shouwen Chen
Nattokinase (EC 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...
May 2017: World Journal of Microbiology & Biotechnology
Yunqi Weng, Jian Yao, Sawyer Sparks, Kevin Yueju Wang
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...
February 28, 2017: International Journal of Molecular Sciences
Gitte S Jensen, Miki Lenninger, Michael P Ero, Kathleen F Benson
OBJECTIVE: The objective of this study is to evaluate the effects of consumption of nattokinase on hypertension in a North American hypertensive population with associated genetic, dietary, and lifestyle factors. This is in extension of, and contrast to, previous studies on Asian populations. MATERIALS AND METHODS: A randomized, double-blind, placebo-controlled, parallel-arm clinical study was performed to evaluate nattokinase (NSK-SD), a fermented soy extract nattō from which vitamin K2 has been removed...
2016: Integrated Blood Pressure Control
Sanjay Kisan Metkar, Agnishwar Girigoswami, Ramachandran Murugesan, Koyeli Girigoswami
A transition of amyloidogenic protein by alternative folding pathway under certain conditions leads to the formation of protease resistant amyloid fibrils, having predominantly cross β structure. These amyloids are related to various neurodegenerative diseases and clearance of such amyloids may be a therapeutic approach for amyloid-related diseases. Insulin, that can form amyloids, is widely used as a model amyloidogenic protein for the study of various amyloid related diseases. In this study, insulin amyloids were formed in vitro and the potential of Serratiopeptidase (SP), a fibrinolytic-like serine protease, towards the dissociation of insulin amyloids was explored...
January 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
Guoxian Wei, Na Tian, Roland Siezen, Detlef Schuppan, Eva J Helmerhorst
Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography...
September 1, 2016: American Journal of Physiology. Gastrointestinal and Liver Physiology
Jintao Cheng, Chengran Guan, Wenjing Cui, Li Zhou, Zhongmei Liu, Weijiang Li, Zhemin Zhou
Quorum-sensing related promoter srfA (PsrfA) was used to construct autoinducible expression system for production of recombinant proteins in Bacillus subtilis. PsrfA was prominent in the unique property of inducer-free activity that is closely correlated with cell density. Here, using green fluorescent protein (GFP) as the reporter protein, PsrfA was optimized by shortening its sequences and changing the nucleotides at the conserved regions of -35 -15 and -10 regions, obtaining a library of PsrfA derivatives varied in the strength of GFP production...
November 2016: Protein Expression and Purification
He Ni, Peng-Cheng Guo, Wei-Ling Jiang, Xiao-Min Fan, Xiang-Yu Luo, Hai-Hang Li
Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies...
August 10, 2016: Journal of Biotechnology
D Cai, X Wei, Y Qiu, Y Chen, J Chen, Z Wen, S Chen
AIMS: Nattokinase is an enzyme produced by Bacillus licheniformis and has potential to be used as a drug for treating cardiovascular disease due to its beneficial effects of preventing fibrin clots etc. However, the low activity and titre of this protein produced by B. licheniformis often hinders its application of commercial production. The aim of this work is to improve the nattokinase production by manipulating signal peptides and signal peptidases in B. licheniformis. METHODS AND RESULTS: The P43 promoter, amyL terminator and AprN target gene were used to form the nattokinase expression vector, pHY-SP-NK, which was transformed into B...
September 2016: Journal of Applied Microbiology
Yu Zhang, Philipp Heher, Jöns Hilborn, Heinz Redl, Dmitri A Ossipov
UNLABELLED: To strengthen the mechanical properties of a fibrin gel and improve its applicability as a scaffold for tissue engineering (TE) applications, a strategy for the in situ preparation of the interpenetrating network (IPN) of fibrin and hyaluronic acid (HA) was developed on the basis of simultaneous and orthogonal fibrinogenesis and disulfide cross-linking. The synthetic pathway included the preparation of mutually reactive HA derivatives bearing thiol and 2-dithiopyridyl groups...
July 1, 2016: Acta Biomaterialia
Chengran Guan, Wenjing Cui, Jintao Cheng, Li Zhou, Zhongmei Liu, Zhemin Zhou
BACKGROUND: Bacillus subtilis, a Gram-positive organism, has been developed to be an attractive expression platform to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. We previously developed an auto-inducible expression system containing the srfA promoter (PsrfA) which was activated by the signal molecules acting in the quorum-sensing pathway for competence. The P srfA promoter exhibited the unique property of inducer-free activity that is closely correlated with cell density...
April 25, 2016: Microbial Cell Factories
Bradley J Lampe, J Caroline English
Subtilisin NAT, commonly known as "nattokinase," is a fibrinolytic enzyme produced by the bacterial strain B. subtilis var. natto, which plays a central role in the fermentation of soybeans into the popular Japanese food natto. Recent studies have reported on the potential anticoagulatory and antihypertensive effects of nattokinase administration in humans, with no indication of adverse effects. To evaluate the safety of nattokinase in a more comprehensive manner, several GLP-compliant studies in rodents and human volunteers have been conducted with the enzyme product, NSK-SD (Japan Bio Science Laboratory Co...
February 2016: Food and Chemical Toxicology
Q Shi, J Hou, C Zhao, Z Xin, J Jin, C Li, S-C Wong, J Yin
A smart core-sheath nanofiber for non-adherent cell capture and release is demonstrated. The nanofibers are fabricated by single-spinneret electrospinning of poly(N-isopropylacrylamide) (PNIPAAm), polycaprolactone (PCL) and nattokinase (NK) solution blends. The self-assembly of PNIPAAm and PCL blends during the electrospinning generates the core-sheath PCL/PNIPAAm nanofibers with PNIPAAm as the sheath. The PNIPAAm-based core-sheath nanofibers are switchable between hydrophobicity and hydrophilicity with temperature change and enhance stability in the blood...
January 28, 2016: Nanoscale
Subathra Devi Chandrasekaran, Mohanasrinivasan Vaithilingam, Ravi Shanker, Sanjeev Kumar, Swathi Thiyur, Vaishnavi Babu, Jemimah Naine Selvakumar, Suyash Prakash
BACKGROUND: Thrombolytic therapy has become a conventional treatment for acute myocardial infarction (AMI), yet currently, clinically prescribed thrombolytic drugs have problems such as delayed action and other side effects. Fibrinolytic enzymes have attracted interest as thrombolytic agents because of their efficiency in the fibrinolytic process, including plasmin activation. Nattokinase (NK) is a potent fibrinolytic agent for thrombosis therapy. OBJECTIVES: The aim of this study was to enhance the production of NK from Pseudomonas aeruginosa CMSS by media optimization and strain improvement...
October 2015: Jundishapur Journal of Microbiology
Rohit Kapoor, Harshad Harde, Sanyog Jain, Amulya Kumar Panda, Bibhu Prasad Panda
The present research work describes the downstreaming of nattokinase (NK) produced by Bacillus subtilis under solid state fermentation; and the role of efficient oral formulation of purified NK in the management of thrombotic disorders. Molecular weight of purified NK was estimated to be 28 kDa with specific activity of 504.4 FU/mg. Acid stable nattokinase loaded chitosan nanoparticles (sNLCN) were fabricated for oral delivery of this enzyme. Box-Behnken design (BBD) was employed to investigate and validate the effect of process (independent) variables on the quality attributes (dependent variables) of nanoparticles...
July 2015: Journal of Biomedical Nanotechnology
Meizhi Weng, Xiongwei Deng, Wei Bao, Li Zhu, Jieyuan Wu, Yongjun Cai, Yan Jia, Zhongliang Zheng, Guolin Zou
Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method...
September 25, 2015: Biochemical and Biophysical Research Communications
Yuko Kurosawa, Shinsuke Nirengi, Toshiyuki Homma, Kazuki Esaki, Mitsuhiro Ohta, Joseph F Clark, Takafumi Hamaoka
Our aim was to determine the quantitative effects of a single-dose of Nattokinase (NK) administration on coagulation/fibrinolysis parameters comprehensively in healthy male subjects. A double-blind, placebo-controlled cross-over NK intervention study was carried out in 12 healthy young males. Following the baseline blood draw, each subject was randomized to receive either a single-dose of 2,000 FU NK (NSK-SD, Japan Bio Science Laboratory Co., Ltd) or placebo with subsequent cross-over of the groups. Subjects donated blood samples at 2, 4, 6 and 8 hours following administration for analysis of coagulation/fibrinolysis parameters...
June 25, 2015: Scientific Reports
Marta Zapotoczna, Hannah McCarthy, Justine K Rudkin, James P O'Gara, Eoghan O'Neill
High-level resistance to antimicrobial drugs is a major factor in the pathogenesis of chronic Staphylococcus aureus biofilm-associated, medical device-related infections. Antimicrobial susceptibility analysis revealed that biofilms grown for ≤ 24 hours on biomaterials conditioned with human plasma under venous shear in iron-free cell culture medium were significantly more susceptible to antistaphylococcal antibiotics. Biofilms formed under these physiologically relevant conditions were regulated by SaeRS and dependent on coagulase-catalyzed conversion of fibrinogen into fibrin...
December 15, 2015: Journal of Infectious Diseases
Masahito Hitosugi, Katsuo Hamada, Kazutaka Misaka
The fermented soybean product natto is a popular traditional food in Japan and is considered a health supplement. NKCP(®), a natto-derived dietary food supplement whose main component is bacillopeptidase F, has antithrombotic, fibrinolytic, and blood pressure-lowering effects. We examined whether daily intake of NKCP(®) effectively improves subjective symptoms in patients with lifestyle diseases in this cross-over, double-blind study. Fermented soya extract with subtilisin NAT (nattokinase) as the main component was used as an active placebo...
2015: International Journal of General Medicine
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