Peng Feng, Shen Qi, Zou Lu-Ping, Cheng Feng, Xue Ya-Ping, Zheng Yu-Guo
Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, PR China.
Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, PR China.
J Agric Food Chem. 2024 Jun 6. doi: 10.1021/acs.jafc.4c02698.
The nicotinamide phosphoribosyltransferase (NAMPT)-catalyzed substitution reaction plays a pivotal role in the biosynthesis of nucleotide compounds. However, industrial applications are hindered by the low activity of NAMPTs. In this study, a novel dual-channel protein engineering strategy was developed to increase NAMPT activity by enhancing substrate accessibility. The best mutant (NAMPT) with a remarkable 5-fold increase in enzyme activity was obtained. By utilizing NAMPT as a biocatalyst, the accumulation of β-nicotinamide mononucleotide reached as high as 19.94 g L within 3 h with an impressive substrate conversion rate of 99.8%. Further analysis revealed that the newly generated substrate channel, formed through crack propagation, facilitated substrate binding and enhanced byproduct tolerance. In addition, three NAMPTs from different sources were designed based on the dual-channel protein engineering strategy, and the corresponding dual-channel mutants with improved enzyme activity were obtained, which proved the effectiveness and practicability of the approach.
烟酰胺磷酸核糖转移酶（NAMPT）催化的取代反应在核苷酸化合物的生物合成中起着关键作用。然而，NAMPTs的低活性阻碍了其工业应用。在本研究中，开发了一种新型的双通道蛋白质工程策略，通过提高底物可及性来提高NAMPT活性。获得了酶活性显著提高5倍的最佳突变体（NAMPT）。利用NAMPT作为生物催化剂，β-烟酰胺单核苷酸在3小时内的积累量高达19.94 g/L，底物转化率高达99.8%，令人印象深刻。进一步分析表明，通过裂纹扩展形成的新生成底物通道促进了底物结合并增强了副产物耐受性。此外，基于双通道蛋白质工程策略设计了三种不同来源的NAMPTs，并获得了相应酶活性提高的双通道突变体，证明了该方法的有效性和实用性。
