A novel l-arabinose isomerase from Lactobacillus fermentum CGMCC2921 for d-tagatose production: Gene cloning, purification and characterization

The araA gene encoding l-arabinose isomerase (l-AI) from the acidophilus bacterium Lactobacillus fermentum CGMCC2921 was cloned and over-expressed in Escherichia coli. The open reading frame of the l-AI consisted of 1425 nucleotides encoding 474 amino acid residues. The molecular mass of the enzyme was estimated to be approximately 53 kDa on SDS-PAGE. The purified recombinant enzyme showed maximum activity at 65 °C and pH 6.5, which were extremely suitable for industrial applications. It required divalent metal ions, either Mn²⁺ or Co²⁺, for enzymatic activity and thermostability improvement at higher temperatures. The enzyme was active and stable at acidic pH, it exhibited 83% of its maximal activity at pH 6.0 and retained 88% of the original activity after incubation at pH 6.0 for 24 h. Kinetic parameter study showed that the catalytic efficiency was relatively high, with a k _cat/K_m of 9.02 mM^-1 min^-1 for d-galactose. The purified L. fermentum CGMCC2921 l-AI converted d-galactose into d-tagatose with a high conversion rate of 55% with 1 mM Mn²⁺ after 12 h at 65 °C, suggesting its excellent potential in d-tagatose production.