Co-Production of Nattokinase and Poly (γ-Glutamic Acid) Under Solid-State Fermentation Using Soybean and Rice Husk

ABSTRACTThe aim of this work was to study the co-production of nattokinase and poly (γ-glutamic acid) by Bacillus subtilis natto with soybean and rice husk under solid-state fermentation (SSF). The results showed that the size of soybean particle and rice husk significantly improved the co-production of nattokinase and poly (γ-glutamic acid), yielding 2503.4 IU/gs and 320 mg/gs, respectively in the improved culture medium composed of 16.7% soybean flour and 13.3% rice husk with 70% water content. The yields increased by approximate 7- and 2-fold factor relative to their original ones. Thus, the co-production of nattokinase and poly (γ-glutamic acid) under SSF could be considered as an efficient method to exploit agro-residues for economical production of some higher-value products.

Nattokinase improves blood flow by inhibiting platelet aggregation and thrombus formation / 한국실험동물학회지

The effects of nattokinase on the in vitro platelet aggregation and in vivo thrombosis were investigated in comparison with aspirin. Rabbit platelet-rich plasma was incubated with nattokinase and aggregation inducers collagen and thrombin, and the platelet aggregation rate was analyzed. Nattokinase significantly inhibited both the collagen- and thrombin-induced platelet aggregations. Nattokinase also reduced thromboxane B2 formation from collagen-activated platelets in a concentration-dependent manner. Rats were orally administered with nattokinase for 1 week, and their carotid arteries were exposed. Arterial thrombosis was induced by applying 35% FeCl3-soaked filter paper for 10 min, and the blood flow was monitored with a laser Doppler probe. Nattokinase delayed the FeCl3-induced arterial occlusion in a dose-dependent manner, doubling the occlusion time at 160 mg/kg. In addition, a high dose (500 mg/kg) of nattokinase fully prevented the occlusion, as achieved with aspirin (30 mg/kg). The results indicate that nattokinase extracted from fermented soybean inhibit platelet aggregation by blocking thromboxane formation, and thereby delay thrombosis following oxidative arterial wall injury. Therefore, it is suggested that nattokinase could be a good candidate without adverse effects for the improvement of blood flow.

Expression, purification of nattokinase in Pichia pastoris and preparation of its polyclonal antibody / 中国生化药物杂志

Purpose To indicate the expression of nattokinase (NK) in Pichia pastoris , an emulsion was prepared with the purified NK to prepare polyclonal antibody. In order to establish sandwich enzyme-linked immunosorbent assay (ELISA) to assay NK in organism, furthermore to lay the foundation for researching in vivo metabolism and function of NK. Methods The NK gene was cloned into a Pichia pastoris expression vector pHBM905A to construct the recombinant plasmid pPRONK.The recipient cell of Pichia pastoris GS115 was transformed with pPRONK which had been cut by restriction enzyme Sal I , under the induction of methanol. The expressed production is purified by salting out and ultrafiltration membrane. An emulsion was prepared with the purified NK and injected into rabbits to prepare polyclonal antibody. Results NK was expressed and identified by SDS-PAGE.The molecular mass of expressed production is about 27 kD.The fibrin plate assay indicated that the NK protein can cleavage fibrin effectively. ELISA analysis indicated that the polyclonal antibody titer is about 1:8 000. Western blot demonstrated that there was a special strap nearby 27 kD. Conclusion NK was successfully expressed in Pichia pastoris , the production can cleavage fibrin effectively and it had great immunogenicity.

CLONING OF NATTOKINASE GENE AND EXPRESSION IN E. COLI / 微生物学通报

In this study, nattokinase gene was amplified by PCR using bacillus subtilis chromosomal DNA as template and cloned into expressed vector pBV220. After transforming recombinant plasmid into E.coli HB101, the recombinant strain was yielded. It was proved that expression products was secretive and expression protein was 12% of total cell protein by SDS-PAGE. Optimum culture time and inducing time was determined as 6h and 5h respectively. The plasmid stability studies showed that recombinant plasmid has excellent segregational stability but the structural stability was not good in the host cell.

Effects of nattokinase on hemorheology and platelet aggregation in rats with acute blood stasis / 中成药

AIM: To observe the effects of nattokinase from bacillas natto on hemorheology and platelet aggregation in rats with acute blood stasis model. METHODS: A model of blood stasis was established by means of giving rats an injection of adrenalin and making it swim in ice-cold water,the change of hemorheology was observed after the rats took nattokinase for 10 d.Rat platelet aggregation induced by ADP,thrombin,and arachidonic acid,and maximum aggregation were measured by turbidimetric method at different time. RESULTS: Nattokinase could markedly decrease the whole blood viscosity with low,middle,high shear rate,plasma viscosity,hematocrit,fibrinogen, erythrocyte aggregation index and electrophoresis index.Nattokinase significantly inhibited platelet aggregation induced by ADP,thrombin and arachidonic acid,and the values of ED_(50)(median effective dose) were(1 431.9 IU/kg,) 1 055.3 IU/kg and 1 390.8 IU/kg. CONCLUSION: Nattokinase could markly improve hemorheology in rats with acute blood stasis,and inhibit the platelet aggregation.

Preparation and Pharmaceutical Quality Evaluation of Nattokinase Immunoliposomes and Its Lyophilized Product / 中国药房

OBJECTIVE:To prepare nattokinase immunoliposomes and its lyophilized product and to evaluate its pharmaceutical quality. METHODS: Nattokinse liposomes were prepared by film dispersion method, then the immunoliposomes were prepared by carbodiimide method coupled anti-human fibrin D-dimer monoclone antibody SZ-63 and lyophilized and evaluated about its pharmaceutical quality. RESULTS: Lyophilized immunoliposomes were multilamellar vesicles with its mean diameter, Zeta potentials at 251.6 nm, —34.6 mV, respectively. The lyophilized product of nattokinase immunoliposomes exhibited good appearance and satisfactory redispersibility, with its loss on drying at 14.30% and critical relative humidity at 45.66%. CONCLUSION: Nattokinase immunoliposomes and its lyophilized product have been prepared successfully.

Molecular cloning, fusion expression and bioactivity of pro-nattokinase gene / 中国病理生理杂志

AIM: To construct engineered E.coli strains which can express nattokinase with fibrinolysis activity using gene engineering technology. METHODS: The pro-nattokinase (pro-NK) gene was amplified by PCR and inserted into expression vector pET3c. The recombined plasmid pENK which expressed the fusion protein of pro-NK and 22 amino acid peptide was then transferred into lysogenic host strains BL21(DE3)pLysS - and BL21(DE3)pLysS +. Both SDS-PAGE and the fibrin plate assay were used to examine the expression and the activity of the target protein. RESULTS: SDS-PAGE assay showed the fused gene encoding 42 kD fusion protein was expressed in both expression strains pENK-(DE3)pLysS - and pENK-(DE3)pLysS +, and the fibrin plate assay indicated that the expression product had fibrinolysis activity. pENK-(DE3)pLysS - exhibited the basal expression of the target gene, while fusion protein was only induced by IPTG in pENK-(DE3)pLysS +. Basal expression of the fused toxic gene in pENK-(DE3)pLysS - led to bacteriolysis and hollow lawns. CONCLUSION: A pro-NK fusion protein with fibrinolysis activity is successfully expressed in E.coli , which lay a foundation for the exploitation of nattokinase.

STUDY ON ABSORPTION AND DISTRIBUTION CHARACTERS OF NATTOKINASE IN SMALL INTESTINE OF RABBITS / 营养学报

duodenum.Conclution Nattokinase could be absorbed into blood plasm in small intestine.