烟酰胺核糖激酶(nicotinamide riboside kinase,Nrk)催化烟酰胺核糖磷酸化生成β-烟酰胺单核苷(β-nicotinamide mononucleotide,β-NMN),是目前生物酶法合成β-NMN的重要途径。该研究通过异源表达体系筛选,实现Nrk在大肠杆菌中的可溶表达,初始酶活力为2.14 U/mL。通过核糖体结合位点(ribosome binding site,RBS)序列和启动子优化,将翻译起始速率为35 000 a.u.的RBS序列与双启动子PalsR-T7组合,构建的重组菌酶活力可达5.56 U/mL,是初始水平的2.6倍。Nrk酶学性质研究表明,该酶的最适反应条件为50 ℃,pH 7.0,10 mmol/L Mg2+。最后,对产Nrk重组菌进行培养条件优化,并在5 L发酵罐中进行罐上产酶放大工艺研究,最终使得酶活力达到72.33 U/mL,是摇瓶发酵水平的13倍。该研究显著提升了Nrk在大肠杆菌中的异源表达水平,为β-NMN的生物酶法合成奠定了基础。
Nicotinamide riboside kinase (Nrk) catalyzes the phosphorylation of nicotinamide riboside to generate β-nicotinamide mononucleotide (β-NMN), serving as a crucial route for the enzymatic synthesis of β-NMN.An efficient heterologous expression system was used in this research for the expression of Nrk in Escherichia coli with an initial enzyme activity of 2.14 U/mL.Through ribosome binding site (RBS) and promoter optimization, this study combined an RBS sequence with a translation initiation rate of 35 000 a.u.with the dual promoter PalsR-T7, and the recombinant strain demonstrated an enzyme activity of 5.56 U/mL, 2.6 times the initial level.The enzymatic properties of Nrk revealed optimal reaction conditions at 50 ℃, pH 7.0, and 10 mmol/L Mg2+.Finally, cultivation conditions for the Nrk-producing recombinant strain were optimized and the fermentation was scaled up to a 5 L bioreactor, achieving an enzyme activity of 72.33 U/mL, 13 times the shake flask fermentation level.This research significantly improved the heterologous expression of Nrk in Escherichia coli, laying the foundation for the enzymatic synthesis of β-NMN.
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