| Nitrilase is a member of the nitrilase superfamily.It can directly catalyze nitrile substrates to generate corresponding carboxylic acids and ammonia.Traditionally,the nitrilase was usually transformed into E.coli for recombinant expression.However,E.coli has several defects such as requiring IPTG inducer,producing cytotoxicity and the cells are easily autolyzed,these defects limit the wide application of nitrilase in food,feed and other fields.In this study,a gram-positive bacteria B.subtilis which was certified as a food safety grade strain was used as a host to constitutively express nitrilase,through fermentation on the fermentor and magnetic immobilization to further improve the potential of enzyme production and reuse efficiency.The details were as follows:(1)In this study,we constructed a recombinant plasmid p MA5-NITR based on the nitrilase gene from Pseudomonas putida which can specifically hydrolyze 3-cyanopyridine to synthesize nicotinic acid and transformed to B.subtils for expression.Finally,we successfully constructed the constitutive expression system in B.subtilis.The recombinant B.subtilis has the highest enzyme activity was 38.03 U·m L-1 after it was cultivated for 36 hours.The study of the enzymatic properties showed that the optimal reaction temperature of the recombinant nitrilase was 30?,the optimal reaction p H was 7.2,and the half-life at 30?was 15 h.With the recombinant B.subtilis cells,12 batches of substrate 3-cyanopyridine could be completely transformed within 450 min with 200 m M of final substrate feeding concentration,and the accumulated nicotinic acid concentration was 295.46 g·L-1 in the reaction system with no residual substrate detected.(2)In order to improve the biomass and enzyme activity of the recombinant B.subtilis(p MA5-NITR),we studied different fermentation strategies for the fermentation on fermentor of the recombinant B.subtilis.In the batch fermentation,the OD600 of the recombinant B.subtilis reached a maximum of 19.27 after 36 hours,as the same time the enzyme activity also reached a maximum of 63.41 U·m L-1.On the basis of the batch fermentation,we studied the fermentation of the recombinant B.subtilis uuder the constant-rate feeding strategy.Through the constant-rate feeding strategy,the highest OD600 of the recombinant B.subtilis could reach a maximum of 55.80,which was 2.90 times of the batch fermentation;the enzyme activity of the recombinant nitrilase could reach a maximum of 125.62 U·m L-1,which was 1.98 times of the batch fermentation.In order to further improve the enzyme production capacity of the recombinant B.subtilis,we adopted a more refined p H-stat feeding strategy.The results showed that OD600 of the recombinant B.subtilis reached a maximum of 58.85 at 84 h,which was 3.05times of the batch fermentation;and the activity could reach a maxium of 167.32 U·m L-1,which was 2.64 times of the batch fermentation.(3)In order to fully exploit the applications potential of the recombinant nitrilase,we used the aminated core-shell magnetic Fe3O4 nanoparticles to immobilize the recombinant B.subtilis nitrilase.We had investigated the effects of temperature,p H,resting cell concentration,and crosslinking agent on the preparation of magnetic immobilized cells and enzyme activity,respectively.The results showed that the best magnetic immobilization condition for the recombinant B.subtilis was 5 g·L-1 of the resting cell concentration,30?and p H 7.2.The substrate tolerance and biotransformation ability of the magnetic immobilized cells were investigated by adding different substrate of concentrations.The results showed that the magnetic immobilized cells had higher tolerance to high concentrations substrates and the suitable initial substrate concentration was 200 m M for producing of nicotinic acid,which was4 times of the corresponding free cells.By batch feeding 200 m M final concentration substrate,that the magnetic immobilized cells could completely transformed 30 batches of substrate in three rounds within 450 min.The accumulated nicotinic acid concentration was 738.66 g·L-1,which was 2.5 times of the free cells,while 40%of the enzyme activity could retained after the reaction. |
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