초록
<P><B>Background</B></P><P>Alanine dehydrogenase (AlaDH) belongs to oxidoreductases, and it exists in several different bacteria species and plays a key role in microbial carbon and nitrogen metabolism, spore formation and photosynthesis. In addition, AlaDH can also be applied in biosynthesis of L-alanine from cheap carbon source, such as glucose.</P><P><B>Results</B></P><P>To achieve a better performance of L-alanine accumulation, system evaluation and comparison of different AlaDH with potential application value are essential. In this study, enzymatic properties of AlaDH from <I>Bacillus subtilis</I> 168 (BsAlaDH), <I>Bacillus cereus</I> (BcAlaDH), <I>Mycobacterium smegmatis</I> MC<SUP>2</SUP> 155 (MsAlaDH) and <I>Geobacillus stearothermophilus</I> (GsAlaDH) were firstly carefully investigated. Four different AlaDHs have few similarities in optimum temperature and optimum pH, while they also exhibited significant differences in enzyme activity, substrate affinity and enzymatic reaction rate. The wild <I>E. coli</I> BL21 with these four AlaDHs could produce 7.19 g/L, 7.81 g/L, 6.39 g/L and 6.52 g/L of L-alanine from 20 g/L glucose, respectively. To further increase the L-alanine titer, competitive pathways for L-alanine synthesis were completely blocked in <I>E. coli</I>. The final strain M-6 could produce 80.46 g/L of L-alanine with a yield of 1.02 g/g glucose after 63 h fed-batch fermentation, representing the highest yield for microbial L-alanine production.</P><P><B>Conclusions</B></P><P>Enzyme assay, biochemical characterization and structure analysis of BsAlaDH, BcAlaDH, MsAlaDH and GsAlaDH were carried out. In addition, application potential of these four AlaDHs in L-alanine productions were explored. The strategies here can be applied for developing L-alanine producing strains with high titers.</P><P><B>Supplementary Information</B></P><P>The online version contains supplementary material available at 10.1186/s13068-023-02373-5.</P>