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食品与发酵工业  2021, Vol. 47 Issue (16): 128-134    DOI: 10.13995/j.cnki.11-1802/ts.027565
  生产与科研应用 本期目录 | 过刊浏览 | 高级检索 |
腈水解酶重组菌的培养工艺及磁性固定化研究
商玉婷1, 龚劲松2, 王顺治1, 陆震鸣1, 李恒2, 史劲松2, 许正宏1*
1(江南大学 生物工程学院,粮食发酵工艺与技术国家工程实验室,江苏 无锡,214122)
2(江南大学 药学院,糖化学与生物技术教育部重点实验室,江苏 无锡,214122)
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)
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摘要 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。
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商玉婷
龚劲松
王顺治
陆震鸣
李恒
史劲松
许正宏
关键词:  腈水解酶  烟酸  生物转化  发酵  固定化    
Abstract: 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.
Key words:  nitrilase    nicotinic acid    bioconversion    fermentation    immobilization
收稿日期:  2021-04-06      修回日期:  2021-04-27                发布日期:  2021-09-10      期的出版日期:  2021-08-25
基金资助: 国家重点研发计划(2018YFC1603800;2018YFC1603802);国家自然基金面上项目(21676121);江苏省重点研发社会发展面上项目(BE2018622)
作者简介:  硕士研究生(许正宏教授为通讯作者,E-mail:zhenghxu@jiangnan.edu.cn)
引用本文:    
商玉婷,龚劲松,王顺治,等. 腈水解酶重组菌的培养工艺及磁性固定化研究[J]. 食品与发酵工业, 2021, 47(16): 128-134.
SHANG Yuting,GONG Jinsong,WANG Shunzhi,et al. Culture conditions and magnetic immobilization of nitrilase-producing strain[J]. Food and Fermentation Industries, 2021, 47(16): 128-134.
链接本文:  
http://sf1970.cnif.cn/CN/10.13995/j.cnki.11-1802/ts.027565  或          http://sf1970.cnif.cn/CN/Y2021/V47/I16/128
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