Study On The Synthesis Of Vitamin B3 By Recombinant Nitrilase

Niacin,as a vitamin drug,is an indispensable nutrient in the human body.The development of biocatalytic method for green and economical synthesis of niacin has important research significance.In this thesis,the process of hydrolyzing 3-cyanopyridine to form nicotinic acid was studied using E.coli recombinant nitrilase derived from Agile acidophilus 72W as a catalyst.A recombinant strain E.coli pRSF-NIT2 capable of expressing nitrilase in two sites was constructed.Through research on culture conditions,reaction system optimization,and cell immobilization,immobilized cells with high activity and stability were obtained,and high-efficient biosynthesis of niacin was achieved in the bioreactor.The main research contents of this article include:First,the recombinant strain E.coli pRSF-NIT2 was constructed.On the basis of the existing E.coli p ET21a-NIT,the nitrilase gene was ligated to the double-cloning expression site of plasmid pRSFDuet plasmid by PCR technology and digestion ligation experiment,and the formed plasmid pRSF-NIT2 was transformed into the E.coli BL21（DE3）host strain.Based on this,the culture medium and the conditions for the production of nitrilase were systematically optimized.It was found that E.coli pRSF-NIT2 can achieve the expression of nitrilase without adding an inducer.Based on this,the fermentation enzyme-producing medium and the conditions for the fermentation of this engineered bacteria were systematically optimized.It was found that E.coli pRSF-NIT2 can achieve the expression of nitrilase without the need for an inducer.The optimal culture medium components were the following（g/L）:soluble starch 20;yeast powder 15;sodium chloride 5;K₂HPO₄4;Mg SO₄·H₂O 1;optimal enzyme production conditions and results are:shaking culture at30℃and 200 rpm for 16 h,and the cell mass and enzyme activity can reach 4.0 g_dcw/L and2846 U/L,respectively.On the basis of the shaker level,the recombinant bacteria were scaled up in a 5 L fermenter,and the fermentation was cultured at 30℃and 300 rpm for 12 hours.The cell mass and enzyme activity were 5.38 g_dcw/L and 3058 U/L,respectively.Secondly,the hydrolysis of 3-cyanopyridine catalyzed by whole cells of recombinant bacteria was investigated,and a series of conditions for the reaction were optimized.The optimum reaction pH is 7.0 and the optimum temperature is 30℃.When a single concentration optimization is performed,the reaction can be completely converted within 4hours when the substrate concentration between 100-800 m M.When the substrate concentration is 1 M,it takes 6 hours for complete conversion;after the substrate concentration continues to increase to 1.6 M,the conversion rate is only about 20%after 8 h.When the initial substrate concentration is 1 M and the batch fed concentration is 200 m M,the feeding can be continued for 10 times,and the conversion rate of 100%can still be reached,and the final product concentration is 3 M.Finally,several different methods of immobilizing cells were examined,and it was found that the method of sodium alginate/glutaraldehyde/polyethyleneimine immobilization was the best.The conditions of immobilization and the properties of the immobilized cells were investigated.Finally,the optimal immobilization conditions were determined as follows:E.coli concentration was 15 g/L,sodium alginate concentration was 2%,glutaraldehyde concentration was 2%,and PEI concentration was 3%.The catalytic properties of the immobilized cells were studied.The optimum reaction temperature and pH were 30℃and Tris-HCl 8.0.The thermal stability and pH stability of the immobilized cells were improved.The immobilized cells were stored for 60 days after storage.The residue enzyme activity of immobilized cells was kept 82%after 60 days storage,while it was only 15%for free cells.Using immobilized cells as a biocatalyst,a semi-continuous biocatalytic reaction is performed in a packed bed reactor.The substrate flow rate,substrate concentration,and reusability in the reactor were studied.The results showed that the highest space-time yield of1576 g/L/d was obtained when the substrate concentration was 0.8 M and the substrate solution flow rate was 2 m L/min.The reactor can be recycled for 41 batches and the conversion remained 100%at the same condition,and about 6 kg of dry weight cells were needed to produce 1 ton of niacin.