Mutation Breeding And Fermentation Process Optimization Of The Strain Producting High Nattokinase Activity

Nattokinase,a serine protease produced by Bacillus subtilis natto,is found in natto food from Japan.It has been found that nattokinase has the function of dissolving thrombus,which can be used as medicine for curing and preventing thrombotic diseases.In this study,a strain with high enzymatic activity of nattokinase was screened from imported natto foods from Japan.To increase enzymatic activity,UV mutagenesis was used to screen mutant strains.The fermentation process of mutant strains was optimized to provide references for the industrial production of nattokinase.The contents and results of the study are as follows:In this study,casein plate was used for screening and fibrin plate was applied to re-sreening.Finally,5 strains were screened from the natto food produced in Hokkaido,Japan,and strain XD2 had the highest enzyme activity of 989.3 U/m L.By the 16 S r DNA molecular identification,the homology of strain XD2 and Bacillus subtilis reached 99%.According to the phylogenetic tree,the strain XD2 belonge to Bacillus subtilis.The original strain XD2 was used as the starting strain,and the mutant strain was screened according to the enzyme activity.The best UV irradiation time was determined to be 150 s,and the mortality rate was 84.09%.Under the condition,6 strains were screened,among which the mutant XD7 had the highest enzyme activity and reached1137.15 U/m L.Compared with the original strain XD2,the enzyme activity of the mutant XD7 increased by 15%.The mutant XD7 was subcultured for 5 times,and the change of enzyme activity was small,indicating its good genetic stability.It was found that nattokinase was a growth-associated metabolite from the fermentation curve,and the time of fermentation was determined to be 48 h by comprehensive analysis.With the mutant strain XD7 used for producing strains,single factor experiment and orthogonal test were carried out to determine the effects of the fermentation medium composition including carbon source,nitrogen source,inorganic salts and surfactants onenzymatic activity of nattokinase.The optimum formula of fermentation medium was as followed: glucose 1.5%,soybean peptone 1%,Na2HPO4·12H2O 0.2%,Na H2PO4 0.1%,Mg SO4 0.1%,Ca Cl2·2H2O 0.06%.Enzyme activity reached 1492 U/m L.A single factor experiment was conducted to investigate effects of the temperature,initial p H,inoculation and liquid volume.The response surface methodology was used to optimize the fermentation conditions.The best fermentation conditions were determined:the temperature was 36.63 ℃,the inoculum volume was 2.36%,the loading volume was46.71 m L,the initial p H was 7,and the rotational speed was 150 r/min.Enzyme activity reached 1598.13 U/m L.Mutant XD7,the production strain,had the highest amount of enzyme production by the optimized culture medium and fermentation conditions,and the enzyme activity reached 1598.13 U/m L.Compared with the original strain XD2,the enzyme activity of mutant XD7 was increased by 61.54%.