Breeding Nattokinase-producing Strainsby Genome Shufflingon The Fermentation Of Rapeseed Meal

The purpose of this study was to use ultraviolet and microwave mutation of Bacillus natto strains, obtained positive mutation UV mutagenesis strains and microwave mutation strains as genome shuffling breeding parent, for parental strains by genome shuffling technology integration, taking nattokinase vigor for the appraisal indicator, on the basic nitrogen source preparation fermentation medium with grease byproduct of rapeseed meal, repeated screening excellent strains of nattokinase producing stable performance.1. Bacillus subtilis natto Z1was treated byUV radiation, selecting74mutant strainsnamed U1～U49. After a series of screening and rescreening, get positive mutant Bacillus natto strain U49, the relative activity of nattokinase value was1329.62IU/mL,100.34%higher than the original strain Zl. The mutant strain U49was passaged5times, the activity of nattokinase basically stable, has good genetic stability.2. Bacillus subtilis natto Z1was treated by microwave, selecting72mutant strains namedW1～W72. After screening and rescreening, getting the mutant W51, its relative activity of nattokinase was735.19IU/mL,42.69%higher than the original strain Z1. The mutant strain W51was passaged5times, the activity of nattokinase basically stable, has good genetic stability. The mutanted sample W34、W51and W65respectively by casein plate method, Folin-phenol method, agarose-fibrin plate assay, the data collected two two correlation curve, a good correlation can draw the Folin-phenol method and agarose-fibrin plate method between, and according to the the final formula X3=10{2log[0.28461n (X1)+17.874]-1.659}/0.2789, namely Folin-phenol method measured the OD value of X1can be directly calculated to obtain the relative activity of nattokinase value X3, bring great convenience for future screening.3. By genome shuffling of mutant strains U49and W51protoplast fusion parents, screened a strain of the highest enzyme activity of F22from50strains of reorganization strains obtained from, U49、W51、F22and original strain Z1enzyme activity was measured at the same time, three strains of enzyme activity increased rates were:101.49%、46.58%、265.82%. After passage, fusant strain F22was cultured the Nattokinase Activity stability. The results show that, good stability, has reached the basic requirements of industrial production, can be used for the production of nattokinase.4. Through the colony morphology and microscopic observation, mutant strains U49and W51with fusion strains of F22in the cell morphology was changed. Through the RAPD test results, the similar genetic coefficients of four strains, F22and the existence of differences in the starting strain genetic coefficient.