High-Level Expression of Cold-Adapted α-acetolactate Decarboxylase in Bacillus subtilis

Abstract:

α-Acetolactate decarboxylase(ALDC) gene alsD was cloned from Bacillus subtilis L520 and constructed into pMA5.The recombinant plasmid pMA5-alsD-His was transformed into B.subtilis WB600.SDS-PAGE indicated that alsD gene was successfully expressed in recombinant B.subtilis and the molecular mass of ALDC was 32×103.Recombinant protein was purified by Ni-NTA affinity chromatography.The specific activity and total yield of the purified enzyme were 272.3 U/ mg and 89.8%,respectively.The optimum temperature of purified ALDC was 25 ℃.And the enzyme showed high activity between 20 ℃ and 35 ℃.The enzyme activity was activated by Ni2+ and totally inhibited by Fe3+.When cultured in shake-flask,the ALDC activity of the recombinant strain was approximately 70-fold higher than that of original strain.The result of scaled-up experiment showed that the enzyme activity was 75.9 U/mL in 5 L fermentor.