Characterization of a Metallic-Ions-Independent L-Arabinose Isomerase from Endophytic Bacillus amyloliquefaciens for Production of D-Tagatose as a Functional Sweetener

D-Tagatose is a low-calorie sugar substitute that has gained increased attention as a functional sweetener owing to its nutraceutical and prebiotic properties. Traditionally, D-tagatose is produced via the enzymatic conversion of L-galactose to D-tagatose by L-arabinose isomerase (L-AI). Nonetheless, the most reported L-AI enzymes are ion-dependent enzymes requiring Mn2+ and/or Co2+ as cofactors for their reactions, which limits their application due to safety and health concerns. Herein, we addressed the facile bioconversion of L-galactose to D-tagatose using a novel recombinant metallic-ions-independent L-AI derived from endophytic Bacillus amyloliquefaciens CAAI isolated from cantaloupe fruits. The ORF (1500 bp) of the L-arabinose isomerase gene (araA) was cloned and over-expressed in Escherichia coli. The recombinant enzyme (BAAI) was purified to homogeneity using Ni-NTA affinity chromatography, yielding a single distinct band with an apparent molecular mass of approximately 59 kDa as deduced from SDS-PAGE analysis. The purified enzyme showed optimum activity at pH and temperature of 7.5 and 45 degrees C, respectively, with obvious enzymatic activity in the presence of ethylenediaminetetraacetic acid (EDTA), indicating the metallic-ions independence from BAAI. The K-m values of BAAI for D-galactose and L-arabinose were 251.6 mM and 92.8 mM, respectively. The catalytic efficiency (k(cat)/K-m) values for D-galactose and L-arabinose were found to be 2.34 and 46.85 mM(-1) min(-1), respectively. The results revealed the production of 47.2 g/L D-tagatose from D-galactose (100 g/L) with 47.2% bioconversion efficiency in a metallic-ions-free reaction system that could be implemented in safe-production of food-grade low-calorie sweetener, D-tagatose.