Our aim was to improve the catalytic efficiency of L-threonine dehydrogenase (L-TDH) on L-threonine dehydrogenation to synthesize ethyl L-2-aminoacetate. An L-TDH gene from Escherichia coli was mined and solubly expressed in E. coli BL21(DE3) through pACYCDuet-1 expression system. The expressed enzyme was purified and characterized. The results showed that high-level soluble expression of L-TDH was achieved in E. coli BL21(DE3), and the enzyme activity in the lysate was 19.13 IU/mL, which was about 79 times higher than that the level of E. coli BL21 (DE3) background expression. The specific activity of the purified L-TDH was 12.77 IU/mg; its optimal reaction temperature was 45 ℃, and its optimal reaction pH was 9.0. The residual enzyme activity was still more than 90% after being held at 35 or 40 ℃ for 120 min. In addition, the kinetic parameters of EcTDH were better than those of other reported L-TDHs, and EcTDH was superior in the synthesis of 2,5-dimethylpyrazine (2,5-DMP) by converting L-threonine. Our findings could lay a theoretical foundation for the industrial production of 2,5-DMP.

In this study, the nicotinamide riboside kinase 1 gene (ScNRK1) was cloned from Saccharomyces cerevisiae, and its soluble expression in Escherichia coli was achieved using pET28a plasmid. The enzyme activity in the fermentation broth was 14.75 IU/mL, and the specific activity the purified enzyme was 2252.59 IU/mg. In addition, the kinetic parameters of ScNRK1 were significantly higher than those of other reported nicotinamide nucleoside kinases, so ScNRK1 could be more advantageous in the enzymatic synthesis of nicotinamide mononucleotide (NMN).

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.