Culture conditions and magnetic immobilization of nitrilase-producing strain
SHANG Yuting1, GONG Jinsong2, WANG Shunzhi1, LU Zhenming1, LI Heng2, SHI Jinsong2, XU Zhenghong1*
1(School of Biotechnology, National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China) 2(School of Pharmaceutical Sciences, Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China)
Abstract: Previously, a recombinant Bacillus subtilis pMA5-NITR harboring nitrilasewas constructed in our laboratory, which could catalyze 3-cyanopyridine into nicotinic acid. In this study, the recombinant B. subtilis pMA5-NITR was cultured by different cultivation strategies including batch fermentation, constant-rate feeding, and pH-stat feeding, to achieve resting cells with high nitrilase activity. In order to enhance the stability of nitrilase in cells, the aminated core-shell magnetic Fe3O4 nanoparticles was used to immobilize the recombinant B. subtilis nitrilase with immobilization conditions optimized. In addition, the effects of temperature stability, substrate tolerance and initial substrate concentration on the transformation efficiency of magnetically immobilized cells were examined to determine the process conditions for further enhancing the nicotinic acid production capacity of recombinant nitrilase. The results showed that pH-stat feeding strategy produced the highest nitrilase activity. Under the pH-stat feeding strategy, the activity of nitrilase could reach 167.32 U/mL, which was 2.64 times that of the batch fermentation. The magnetically immobilized cells of recombinant nitrilase were more resistant to high concentrations of 3-cyanopyridine. Thirty batches of 3-cyanopyridine could be completely converted within 450 min, and the cumulative concentration of nicotinic acid reached 738.66 g/L, which was 2.5 times higher than that of free cells. And it was also the highest yield of nicotinic acid produced by nitrilase derived from B. subtilis. In summary, different culture conditions and the magnetic immobilization strategy could improve the ability of the recombinant nitrilase to produce nicotinic acid from 3-cyanopyridine, and the recombinant nitrilase had great potential in the industrial production of nicotinic acid.
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2021, Vol. 47 
