Expression and production optimization of alanine dehydrogenase from Arthrobacter ureafaciens CZ31

Abstract:

[Objective] Alanine dehydrogenase gene (ald) of Arthrobacter ureafaciens CZ31 was cloned and transformed into Escherichia coli Rosetta (DE3) to construct engineering bacteria CZR07 with soluble expression of ald, and the conditions of alanine dehydrogenase(AlaDH) production were optimized.[Methods] Genomic DNA of Arthrobacter ureafaciens CZ31 was extracted; A pair of specific primers was designed to obtain the ald gene, and then subcloned into expression plasmid, pET-28a-ald, and expressed in E. coli Rosetta. The recombinant protein, AlaDH, was purified by Ni²⁺ chromatography. Response surface methodology was used to optimize fermentation conditions.[Results] The length of ald gene was 1119 bp, encoding 372 amino acid residues, molecular weight of the target protein was 40 kDa according to SDS-PAGE analysis. Specific enzyme activity of the recombinant enzyme was 2.65 U/mg. The optimal induction conditions were:22℃, isopropyl-β-D-thiogalactoside 0.7 mmol/L, induction time 8 h. The specific activity of the enzyme was 15.23 U/mg under optimized conditions, about 5.75 times of the initial.[Conclusion] We optimized the induction conditions of the recombinant enzyme production through Box-Benhnken Design, and achieved the desired results, which provided a reference for the optimization of other genetic engineering bacteria.