Cloning,purification,and characterization of branched-chain α-keto acid decarboxylases from Lactococcus lactis strains with different 3-methylbutanal production abilities

Lactococcus lactis is an important food-grade microorganism that has been successfully applied as a starter to increase the level of 3-methylbutanal produced during the ripening of cheese.Three variants of branched-chain α-keto acid decarboxylase (KADC) were discovered in L.lactis strains with different 3-methylbutanal production abilities.Three genes encoding KADCs of varying lengths (KADC-long,KADC-middle,and KADC-short) were cloned and heterologously expressed into Escherichia coli.KADC activity was only detected in the E.coli cloned with the KADC-long-encoding gene.Homology modeling of the three KADC recombination proteins showed that an active-site residue (Glu462) and an S-pocket structure were necessary for the ability to catalyze substrates.KADC-long showed maximum activity at pH 7.0 and 30 ℃.The substrate hydrolysis and kinetic parameters demonstrated that KADC-long efficiently produces 2-methylbutanal and 3-methylbutanal.The heterologous expression of the full-length kdcA in low-3-methylbutanal-yield L.lactis strains increased their production yields.The results of this study demonstrate the function of the complete KADC in 3-methylbutanal production.