β-烟酰胺单核苷酸制备用酶的共表达及其应用
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1.江南大学生物工程学院,江苏 无锡 214122;2.江南大学工业生物技术教育部重点实验室,江苏 无锡 214122;3.江南大学食品科学与资源挖掘全国重点实验室,江苏 无锡 214122

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吴敬(1969—),女,博士,教授,博士研究生导师,主要从事酶工程和发酵工程研究。Email:jingwu@jiangnan.edu.cn

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中央高校基本科研业务费项目(5812050205226750)。


Co-expression and Application of Enzymes for Preparation of β-Nicotinamide Mononucleotide
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1.School of Biotechnology, Jiangnan University, Wuxi 214122, China;2.Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China;3.State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi 214122, China

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    摘要:

    【目的】降低生产β-烟酰胺单核苷酸(β-NMN)的成本,并简化其酶催化法的生产流程。【方法】在作者所在课题组前期建立的6种酶催化合成β-NMN体系的基础上,将这6种酶基因进行两两组合,构建双酶共表达重组菌;采用共表达获得的粗酶液进行多酶级联催化反应,以合成β-NMN。【结果】当底物D-核糖浓度为10 mmol/L时,β-NMN的产率达72.90%;各双酶共表达重组菌的最佳发酵条件均为诱导温度25 ℃、IPTG诱导浓度0 mmol/L;确定了对5-磷酸核糖-1-焦磷酸合成酶(EcPrs)储存稳定性有显著提升作用的盐离子和多羟基化合物,且发现Mn2+、乳糖和山梨醇对β-NMN产率基本无影响。【结论】该研究建立的双酶共表达体系下多酶级联催化反应制备β-NMN的方法,以及对EcPrs储存稳定性的优化策略,为酶催化法合成β-NMN提供了新思路。

    Abstract:

    [Objective] This study aims to reduce the cost and simplify the production process of β-nicotinamide mononucleotide (β-NMN) by the enzyme catalytic method. [Method] On the basis of the previously established β-NMN synthesis system with six enzymes, the six enzyme genes were combined in pairs to construct double-enzyme co-expression recombinant strains. Each crude enzyme solution obtained by co-expression was used for multi-enzyme catalytic cascade reaction to synthesize β-NMN. [Result] The yield of β-NMN was as high as 72.90% when the concentration of the substrate D-ribose was 10 mmol/L. The optimal temperature and IPTG induction concentration for inducing the enzyme expression of the recombinant strains were 25 ℃ and 0 mmol/L, respectively. Finally, the salt ions and polyhydroxy compounds that significantly improved the storage stability of 5-phosphate ribose-1-pyrophosphate synthase (EcPrs) were identified. Mn2+, lactose, and sorbitol basically had no effect on the yield of β-NMN. [Conclusion] The multi-enzyme catalytic cascade reaction for the preparation of β-NMN under dual-enzyme co-expression system and the optimization for EcPrs storage stability provide a new idea for the enzyme catalytic synthesis of β-NMN.

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刘杰虎,宿玲恰,付慧娴,吴敬.β-烟酰胺单核苷酸制备用酶的共表达及其应用[J].食品与生物技术学报,2025,44(8):139-147.

LIU Jiehu, SU Lingqia, FU Huixian, WU Jing. Co-expression and Application of Enzymes for Preparation of β-Nicotinamide Mononucleotide[J]. Journal of Food Science and Biotechnology,2025,44(8):139-147.

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  • 收稿日期:2024-01-03
  • 最后修改日期:2024-03-09
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  • 在线发布日期: 2025-11-26
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