该实验室前期构建了1株高产重组腈水解酶的Bacillus subtilis pMA5-NITR,可以转化3-氰基吡啶生产烟酸。该研究分别采取分批培养、恒速补料培养和恒pH培养等策略对产腈水解酶重组菌进行发酵培养,以期获得具有高催化活性的腈水解酶静息细胞。为进一步提高细胞中腈水解酶的稳定性,采用氨基化核壳结构磁性Fe3O4纳米粒子对重组腈水解酶进行固定化并对制备条件进行优化。此外,通过考察磁性固定化细胞的温度稳定性、底物耐受性和初始底物浓度对转化效率的影响,确定磁性固定化细胞转化3-氰基吡啶为烟酸的工艺条件,以进一步增强重组腈水解酶的烟酸生产能力。结果表明,恒pH培养策略效果最佳,重组腈水解酶活力可达167.32 U/mL,是分批培养的2.64倍。重组酶的磁性固定化细胞具有更高的耐受性,在450 min内可以完全转化30批次底物,累积生成烟酸质量浓度达到738.66 g/L,是游离细胞的2.5倍,酶活力和烟酸转化产量均是目前报道的枯草芽孢杆菌来源腈水解酶的最高记录。该研究通过对不同培养方式的探索及磁性固定化策略的应用,提高了重组腈水解酶的生物转化潜力,在工业生产烟酸上具有良好的应用价值。
Previously, a recombinant Bacillus subtilis pMA5-NITR harboring nitrilase was 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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