| Nattokinase(NK)is a serine protease produced by Bacillus natto,which has the function of disintegrating thrombus.At present,it has been developed into a new generation of functional health food and drugs for the prevention and treatment of cardiovascular and cerebrovascular diseases,with the advantages of high safety,oral administration and low cost.In the industrial production process of nattokinase,the production capacity of the strain is limited and the enzyme activity is low.In order to solve these problems,it is necessary to cultivate industrial high-yield strains.In this study,mutated bacteria with high enzyme activity are screened by mutagenesis breeding for genome reshuffling and high-yield fusion,and then nattokinase expression gene NK is restructured by DNA shuffling technology to construct engineering bacteria.The specific research contents are as follows:(1)Bacillus natto ZYPND2019 was used as the primary bacterium,and four strains of Bacillus natto with relatively high circle diameter were obtained through preliminary screening.The standard curve was drawn using urokinase standard as the control,and the protein fiber plate method was used for re-screening.The strain Bacillus natto W2 with high enzyme activity was obtained,and the initial enzyme activity was 673±39.1 U/m L.After optimization of fermentation parameters,the optimal fermentation conditions were obtained as follows: fermentation time was24 h,temperature was 36℃,initial p H was 7.3,shaking speed was 210 r/min.Finally,the enzyme activity of W2 after optimization was 974 U/m L,which increased by 44.7% compared with that before optimization.(2)Bacillus natto W2 was mutated by UV and ARTP to screen high-yielding bacteria.Three strains with high enzyme activity were obtained after UV mutagenesis,and the enzyme activity was 1324±29.3,1455±10.2 and 1180±16.5U/m L.Compared with the original strain W2,it increased by 35.9%,49.4% and21.1%,respectively.After ARTP mutagenesis,two strains with higher enzyme activity were obtained,and the enzyme activity was 1281±21.8 and 1410±15.6U/m L,which were 31.5% and 44.8.% higher than that of the original strain W2,respectively.Protoplasts were prepared from the high-yielding bacteria screened by the two methods as the parents of the protoplasts.The inactivation conditions were uv irradiation distance of 30 cm for 6 min and heat inactivation time of 55℃ for 10 min,respectively.The fusion was induced by chemical method with PEG6000 concentration of 40% as a melting aid.The optimal fusion conditions were determined as PEG6000 concentration of 40%,p H of 8,temperature of 30℃ and fusion time of 10 min.After verification,the fusion rate reached 21.5%.After two rounds of recursive fusion,two rounds of forward mutant G2-1 and G2-2 were obtained.After 20 passages,the enzyme activities were 2285 U/m L and 2345U/m L,respectively,which were increased by 134.6% and 140.8% compared with the starter bacteria,respectively.(3)G2-1,G2-2 target gene apr N is cloned.After two rounds of DNA shuffling,p ET26b(+)-m NK-2 recombinant plasmid is constructed and transformed into E.coli BL21 competent cells,and a strain of E.coli m NK-2 with high enzyme activity is screened.E.coli m NK-2 was successfully expressed as danattokinase by SDS-PAGE with molecular weight of 28.2 k Da.The enzyme activity was 1546U/m L by fibrin plate method.Compared with the original strain W2,the temperature stability of E.coli m NK-2 enzyme was slightly improved,and it was more stable than the wild type under alkaline conditions.After sequencing comparison,a total of 8 amino acid residues in the primary structure of the mutant protein were mutated,which affected the spatial conformation of the molecule to a certain extent. |
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