Abstract: In this study, the acetolactate synthase-encoding genes (BsALS and PpALS) of two high-yield acetoin-producing strains obtained in our laboratory, Bacillus subtilis JN17 and Pediococcus pentosaceus JXQ20, were cloned and heterologously expressed and their enzymatic properties and structural characteristics were systematically characterized. The results showed that B. subtilis JN17 produced 18.158 1 g/L of acetoin, significantly higher than 0.044 2 g/L produced by P. pentosaceus JXQ20. The activities of BsALS and PpALS were 184.87 and 2.57 U/mg, respectively. Their optimal temperatures were 50 and 30 ℃, and optimal pH were 7 and 6, respectively, with BsALS exhibiting superior thermal and pH stability. Cs+ showed the strongest inhibition on BsALS, while Cu2+ significantly enhanced PpALS activity, and sodium dodecyl sulfate (SDS) completely inactivated both enzymes. BsALS retained 76.03% of its initial activity at 20% (V/V) ethanol, while PpALS lost all activity at 15% (V/V) ethanol. In the presence of 5% NaCl, BsALS maintained 52.42% of its original activity, compared with only 21.04% for PpALS. The Michaelis constants (Km) for BsALS and PpALS were 77.54 and 255.98 mmol/L, respectively, with maximum reaction rates (Vmax) of 595.14 and 19.04 U/mg, respectively. Structural analysis indicated the two ALSs differed in key amino acid residues that bind to thiamine diphosphate (ThDP) and Mg2+ as well as the number of formed hydrogen bonds, possibly explaining the significant differences in their catalytic performance and stability. These findings offer a theoretical foundation for the rational modification of acetolactate synthase and its application in food fermentation and industrial biosynthesis.

Key words: acetolactate synthase; Bacillus subtilis; Pediococcus pentosaceus; heterologous expression; enzymatic properties