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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...
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...
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
Maqsood M Elahi, Charles H Choi, Subbareddy Konda, Jay G Shake
This report describes a patient's self-substitution of nattokinase for the vitamin K antagonist warfarin after aortic valve replacement with a mechanical prosthesis. Nattokinase is an enzyme derived from a popular fermented soybean preparation in Japan (natto), which has fibrinolytic properties and is gaining popularity in nontraditional health journals and nonmedical health websites as an over-the-counter thrombolytic. After nearly a year of use of nattokinase without warfarin, the patient developed thrombus on the mechanical valve and underwent successful repeat valve replacement...
January 2015: Proceedings of the Baylor University Medical Center
Xuetuan Wei, Yinhua Zhou, Jingbang Chen, Dongbo Cai, Dan Wang, Gaofu Qi, Shouwen Chen
Nattokinase (NK) possesses the potential for prevention and treatment of thrombus-related diseases. In this study, high-level expression of nattokinase was achieved in Bacillus licheniformis WX-02 via host strain construction and signal peptides optimization. First, ten genes (mpr, vpr, aprX, epr, bpr, wprA, aprE, bprA, hag, amyl) encoding for eight extracellular proteases, a flagellin and an amylase were deleted to obtain B. licheniformis BL10, which showed no extracellular proteases activity in gelatin zymography...
February 2015: Journal of Industrial Microbiology & Biotechnology
Hongrui Ji, Liang Yu, Keyu Liu, Zhigang Yu, Qian Zhang, Fengjuan Zou, Bo Liu
In vivo, the level of cyclic Adenosine Monophosphate (cAMP) and the pathway of the Janus Kinase1/Signal Transducers and Activators of Transcription1 (JAK1/STAT1) were studied. In vitro, the Ca(2+) mobilization in human platelet stimulated by thrombin was observed. In addition, vasomotion of vascular smooth muscle was measured by adding KCl or norepinephrine(NE) under the Ca(2+) contained bath solutions. The effect induced by NE in the presence of N-nitro-L-arginine methyl ester (L-NAME) or indometacin (Indo) was also detected...
December 15, 2014: European Journal of Pharmacology
Fatemeh Dabbagh, Manica Negahdaripour, Aydin Berenjian, Abdolazim Behfar, Fatemeh Mohammadi, Mozhdeh Zamani, Cambyz Irajie, Younes Ghasemi
Nattokinase (NK, also known as subtilisin NAT) (EC is one of the most considerable extracellular enzymes produced by Bacillus subtilis natto. The main interest about this enzyme is due to its direct fibrinolytic activity. Being stable enough in the gastrointestinal tract makes this enzyme a useful agent for the oral thrombolytic therapy. Thus, NK is regarded as a valuable dietary supplement or nutraceutical. Proven safety and ease of mass production are other advantages of this enzyme. In addition to these valuable advantages, there are other applications attributed to NK including treatment of hypertension, Alzheimer's disease, and vitreoretinal disorders...
November 2014: Applied Microbiology and Biotechnology
Yan Jia, Xinhua Cao, Yu Deng, Wei Bao, Changyan Tang, Hanjing Ding, Zhongliang Zheng, Guolin Zou
Subtilisin propeptide functions as an intramolecular chaperone that guides precursor folding. Nattokinase, a member of subtilisin family, is synthesized as a precursor consisting of a signal peptide, a propeptide, and a subtilisin domain, and the mechanism of its folding remains to be understood. In this study, the essential residues of nattokinase propeptide which contribute to precursor folding were determined. Deletion analysis showed that the conserved regions in propeptide were important for precursor folding...
November 2014: Acta Biochimica et Biophysica Sinica
Yi Wang, Zhenmin Chen, Ruili Zhao, Tingting Jin, Xiaoming Zhang, Xiangdong Chen
BACKGROUND: Bacillus subtilis is widely used in agriculture and industrial biotechnology; however, cell autolysis significantly decreases its yield in liquid cultures. Numerous factors mediate the lysis of B. subtilis, such as cannibalism factors, prophages, and peptidoglycan (PG) hydrolases. The aim of this work was to use molecular genetic techniques to develop a new strategy to prevent cell lysis and enhance biomass as well as the production of recombinant proteins. RESULTS: Five genes or genetic elements representing three different functional categories were studied as follows: lytC encoding PG hydrolases, the prophage genes xpf and yqxG-yqxH-cwlA (yGlA), and skfA and sdpC that encode cannibalism factors...
2014: Microbial Cell Factories
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