关键词: 烟酰胺核糖激酶1；理性设计；定点突变；热稳定性；烟酰胺单核苷酸

Abstract: In order to improve the thermal stability of nicotinamide riboside kinase 1 from Saccharomyces cerevisiae (ScNRK1), six single-point mutants of ScNRK1 were virtually designed using computer-aided technology, and their expression using site-directed mutagenesis and enzymatic characterization were carried out. Out of these, three superior mutants were selected for a second round of combined mutagenesis. The results showed that after two rounds of mutagenesis, a mutant named T136P/S209A with significantly improved thermal stability and catalytic activity was obtained. Its optimal reaction temperature was increased to 45 ℃, and its half-life at 45 ℃ was 48.98 min, which was 4.2 times as high as that of the wild-type ScNRK1. The specific enzyme activity of the purified mutant enzyme was 146.63 U/mg, which was 1.98 times as high as that of the wild type. This study is expected to provide new ideas for improving the thermal stability of enzymes through rational design, and provide a new enzyme source for efficient and low-cost production of nicotinamide mononucleotide.

Key words: nicotinamide riboside kinase 1; rational design; site-directed mutagenesis; thermal stability; nicotinamide mononucleotide