Breeding Of Nattokinase High-yield Strain And Its Isolation And Purification

Nattokinase(NK)is derived from natto,a serine protease with good thrombolytic properties,safe and no side effects,and has a good clinical application prospect.A strain of nattokinase-producing Bacillus subtilis XZI125 preserved in our laboratory is required to further increase the enzyme production capacity of the strain to the industrial fermentation production strain.In this study,the mutant strains with high yield and stable inheritance were obtained by mutagenesis breeding and genome recombination.And through the preparation of magnetic chitosan affinity adsorbent,one-step separation and purification of nattokinase crude enzyme solution is realized,and technical support for its industrial production is provided.1.Mutant strains with high yield of nattokinase were selected by UV mutagenesis of Bacillus subtilis XZI125 and mutagenesis by atmospheric pressure plasma mutagenesis(ARTP).By calculating the lethality rate and the positive mutation rate,the optimal conditions for UV mutagenesis were determined:power 20 W,irradiation distance 25 cm,mutagenic time 5 min;optimal conditions for ARTP mutagenesis:power 100 W,gas flow 10 SLM,The distance between the chip and the airflow port is 2 mm and the processing time is 90 s.Through two mutagenesis treatments combined with antibiotic resistance screening methods,a total of four mutant strains with higher NK content than the original strain were obtained,with an average increase of 24.3%.2.Optimize the preparation,regeneration,inactivation and fusion conditions of Bacillus subtilis XZI125 protoplasts,and use geome shuffling technology to breed NK high-yield strains.The results showed that the optimal conditions for protoplast preparation were:enzyme concentration 1 mg/mL,enzymatic hydrolysis time 30 min,and bacterial culture time 6 h;optimal conditions for UV inactivation were:power 20 W,irradiation distance 25 cm,irradiation time 30 min;the best conditions for heat inactivation are:100℃,the optimal conditions for protoplast fusion in water bath for 50 s are:40%PEG6000,37℃ water bath fusion for 10 min.Under the above conditions of protoplast treatment,three strains of high-yield strains of NK were screened by genome mutation,and three strains of high-yield strains G91,2G34 and 2G42 were obtained,and the nattokinase activity respectively reached 3257 IU/mL,3277IU/mL,3304 IU/mL,compared with the original strain(2490 IU/mL),the enzyme activity increased by 30.8%,31.6%,and 32.7%,respectively.3.Magnetic Fe3O4 nanoparticles were prepared by chemical co-precipitation method.Magnetic chitosan microspheres(MCTS)were prepared by emulsion cross-linking method.After activation,6-aminocaproic acid was used as spacer arm to couple ligands to aminobenzoic acid.magnetic chitosan microspheres were prepared and used for the separation and purification of nattokinase crude enzyme solution,and the kinetics of adsorption and desorption of nattokinase,enzyme purity and enzyme activity recovery were studied.By optimizing the preparation conditions of magnetic chitosan,the appropriate ligand grafting concentration was selected,so that the adsorbent could reach the adsorption equilibrium of nattokinase within 10 min,and the enzyme desorption was completed within 20 min.The recovery of enzyme activity was 62%,the purification factor was 10.6,and the purity of nattokinase was analyzed by electrophoresis as a band.