Screening Of High-yield Nattokinase Strains And Study Of Its Enzymatic Stability

Nattokinase is a potential thrombolytic drug.It can effectively reduce the formation of thrombus,and the thrombolytic effect is significantly higher than the same dose of urokinase,and it can also directly dissolve fibrin clot by proteolysis.In this dissertation,the high-yield Nattokinase strains isolated from the local specialty foods in Guizhou were evaluated by comprehensive performance evaluation including safety performance,probiotic performance and fermentation performance.Based on this analysis,a high-yield nattokinase strain with the best comprehensive performance was found.The crude enzyme solution was purified by the combination of acetone precipitation and reverse micelle extraction method to obtain electrophoretically pure Nattokinase.Finally,a variety of chemical modifications were combined to modify the enzyme activity of nattokinase and study its enzymatic stability.First,use the casein plate method and the agarose-fibrinogen plate method to isolate casein-producing strains from samples of local specialty foods in Guizhou,and then obtained 78 strains of nattokinase-producing strains.Through observation of the colony morphology and bacterial morphology of 15 high-yield nattokinase strains,15strains of high-yield nattokinase strains were initially identified as Bacillus.Finally,15 strains of high-producing strains were screened by ultraviolet spectrophotometry for the accurate determination of nattokinase activity.The strain GUJN05 had the highest enzyme-producing ability.The activity of nattokinase reached 141.97±0.64FU/g after solid state fermentation for 36 h.Then 15 high-yield nattokinase strains were identified by 16S rRNA molecular biology.The GULR06,GUMN16,GUTU06,GUYZ13 strains were Bacillus subtilis.The GUB01,GULC07,GUXN01,GUJN05,GUXN04,GUJZ01,GUWB03,GUBN02,GUYR02,GUWB06 strains were Bacillus amyloliquefaciens.The SN-14 strain was Bacillus velezensis.The biogenic amine content of each natto product ranged from 49.09 to 73.71 mg/kg,of which GUMN16produced the highest content of amines.By using principal component analysis,Bacillus bacillus SN-14 was selected as the producing strain of nattokinase.Combined with morphological,molecular biological and physiological and biochemical identification results,SN-14 was finally identified as Bacillus velezensis（Bacillus）.The acetone precipitation method was then used to separate and purify nattokinase from the fermentation broth.When the volume ratio of fermentation broth to acetone was 1:5,the effect of precipitation at 0℃for 6 h was best.Under this condition,the recovery of nattokinase activity was 69.7±1.3%and the purification multiple was 1.89±0.05.The reversed micelle extraction method was used to extract acetone-precipitatednattokinase solution.Theoptimal conditions for the pre-extraction were as follows:when the concentration of CTAB was 225 mM,the volume of the three cosolvents was 70%isooctane/20%n-butanol/10%N-hexanol;NaCl ion concentration in aqueous phase is 0.05 M,aqueous phase pH is 8.5;25 min extraction at 25°C,after centrifugation,an organic phase containing nattokinase is obtained.Under this condition,stripping efficiency was 84.7±1.9%.The acetone-precipitated nattokinase solution was separated and purified by reverse micelle extraction.The total reverse micelle extraction efficiency was 63.8±1.9%and the purification factor was 2.61±0.09.Nattokinase in fermentation broth was subjected to acetone precipitation and reversed micelle extraction.The purification rate of the whole process reached 4.93.The enzyme solution isolated and purified by this method was characterized by SDS-PAGE.The method was used to purify the target enzyme to electrophoresis.The amino acid sequence of the enzyme was determined by NanoLC-ESI-MS/MS.The phylogenetic tree analysis showed that the enzyme produced by the purified SN-14 strain belongs to the nattokinase family,and the enzyme was named SG14.Finally,by measuring the content of nattokinase before and after removing the metal ion SG14 NK,the activity of NK enzyme after deionization was increased by9.17%compared with that before deionization.K⁺、Ca²⁺、Mg²⁺、Ba²⁺、Fe²⁺promoted the activity of unmodified SG14 NK enzyme to a certain extent.Among them,Fe²⁺had the strongest promoting effect on nattokinase activity and the enzyme activity increased by 11.3%.Other metal ions show some degree of inhibition.When the Fe²⁺concentration was 10 mM,the activity of the SG14 NK enzyme reached a maximum,which was increased by 16.7%compared with the original use of only 5 mM to promote the activity of nattokinase,whereas it was 3.1 times higher than that of the SG14 NK before chemical modification.The enzymatic activity of SG14 NK and pPEGase modified by Fe²⁺and mPEG was the highest when mixed with equal volume of the pepsin solution.Compared with the SG14 NK modified with Fe²⁺and mPEG only,the enzyme activity was increased 1.66 times.Under the optimum reaction temperature of 37℃,the enzyme activities of SG14 NK modified with Fe2+,mPEG and pepsin were highest in turn,and its NK enzyme activity was 7.3 times that of SG14 NK without chemical modification.The optimum pH of SG14 NK was 8.5,followed by the completion of Fe²⁺,mPEG,and pepsin-modified SG14 NK.At pH 3and 12,NK enzyme activity was lost by 9%and 15.9%.Only Ca²⁺and Fe²⁺could promote the activity of Fe²⁺,mPEG-modified SG14 NK,and SG14 NK enzyme activity of Fe²⁺,mPEG and pepsin in order.After 30 days of frozen storage,the enzyme activity of non-chemically modified SG14 NK lost 60.6%,the enzyme activity of Fe²⁺-modified SG14 NK lost 56.6%,and the activity of Fe²⁺and mPEG-modified SG14 NK lost 39.1%.The loss of enzymatic activity of SG14 NK by mPEG and pepsin was 33.2%.However,the half-life of SG14 NK after multiple chemical modifications was much higher than that of unmodified SG14 NK.