Study On The Immobilization Of Nattokinase

Nattokinase has medicinal properties such as thrombolysis,lowering blood pressure,lowering blood lipids and neuroprotection.Compared with conventional drugs,nattokinase has strong substrate specificity and does not cause side effects such as local bleeding after use,and is a promising new drug for the treatment of cardiovascular diseases or neuroprotective.The most prominent advantage of nattokinase is its safety and certain characteristics of resistance to digestive degradation.Although this advantage may not be very strong,it makes it possible that the therapeutic purpose of nattokinase can be achieved in the form of oral administration.In order to minimize the degradation or inactivation of nattokinase during oral administration,improve its absorption through the gastrointestinal tract in its intact form,and enhance the therapeutic effect of nattokinase,it is necessary to improve the stability of nattokinase after oral administration.Enzyme immobilization treatment refers to the use of chemical or physical means to fix the enzyme on a suitable carrier material,to protect the spatial structure and active part of the enzyme,while improving the stability and reusability of the enzyme,while retaining the efficient catalytic ability of the enzyme and substrate specificity to the greatest extent.Chitosan has abundant sources,is cheap and readily available,safe and non-toxic,has good adsorption capacity and film-forming ability,and has good biocompatibility and biodegradability,and is a recognized safe functional material.Chitosan beads were used as a carrier to immobilize nattokinase.Through the research of this thesis,the following main results have been obtained:1.Chitosan microspheres were prepared by neutralization titration,and free nattokinase FA3 and NK（hereinafter referred to as FA3 and NK,respectively）were immobilized by covalent binding by glutaraldehyde.According to the single-factor experimental results of time,temperature,p H and glutaraldehyde concentration,nine sets of orthogonal experiments were designed according to L₉（3⁴）to optimize the fixed conditions of nattokinase,and the three factors of orthogonal experiments were temperature,p H and glutaraldehyde concentration.The results of orthogonal experiments showed that the optimal immobilization conditions for nattokinase FA3 and NK were 30℃,p H 6.0,glutaraldehyde solution at a concentration of 1.0%（V/V）;30℃,p H 6.0,glutaraldehyde solution at a concentration of 1.5%（V/V）.The specific activity（apparent）of immobilized nattokinase FA3 and NK（hereinafter referred to as i FA3 and i NK,respectively）obtained using optimal fixation conditions was 7 times and 15 times that of FA3 and NK,respectively.2.The enzymatic properties of i FA3,i NK,FA3 and NK were measured and analyzed and it was found that the optimal p H of i FA3 and FA3 was 3.0,and the immobilization treatment had little effect on the p H change adaptability of FA3,while the optimal p H of NK after immobilization had obvious acid shift,from p H 6.0 to p H 3.0,and the adaptability of i NK to p H change was significantly enhanced.The optimum temperature of FA3 and NK was 50℃,and after immobilization,their optimum temperature was increased to 60℃,and their adaptability to temperature changes was significantly enhanced.Immobilization significantly enhances the stability and thermal stability of FA3 and NK in the acidic to weak base range.The storage half-life of i FA3 was 58 days,the storage half-life of i NK,while FA3 and NK were basically lost after 80 days and 90 days of storage,respectively.After repeated use for 20 times,the enzyme activity of i FA3 and i NK can still remain above 50%of the initial enzyme activity,and their operating half-lives are 25 times and 37 times,respectively,and the immobilization treatment makes the stable reuse of nattokinase possible.Compared with FA3 and NK,i FA3 and i NK have greatly improved the digestive resistance of saliva,simulated gastric juice and simulated intestinal fluid,among which the digestive resistance of simulated gastric juice and simulated intestinal fluid has been greatly improved.i FA3 and i NK can still maintain more than 60%and65%of the initial enzyme activity after 120 min of simulated gastric digestion,respectively,and more than 60%of the initial enzyme activity after 12 h of simulated intestinal fluid digestion,which is much higher than the retention of initial enzyme activity by free enzymes.3.Use of indirect enzyme-linked immunosorbent assay（indirect ELISA）,the degradation effects of i FA3,i NK,FA3 and NK on the monomer,oligomer,and fiber（Aβ42 monomer,oligomer,and fiber abbreviated as Aβ42M/Aβ42O/Aβ42F）of extracellularβ-amyloid 1-42（Aβ42）were measured and compared and compared,and it was found that the immobilized treatment improved the degradation rate of nattokinase to Aβ42M/Aβ42O/Aβ42F.And the degradation rate of nattokinase to Aβ42M/Aβ42O/Aβ42F was significantly increased.In summary,after immobilization of nattokinase,it not only improved the enzyme activity and stability,but also accelerated the degradation rate of nattokinase to Aβ42 and the degradation level of nattokinase to Aβ42.Moreover,the immobilized enzyme has simple preparation conditions,low cost and good safety performance,which lays the foundation for the rational application of nattokinase,provides new ideas for the development of oral preparations of nattokinase,and significantly enhances the potential of nattokinase in medicine.