Thermostable L‐arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D‐tagatose production: gene cloning, purification and characterisation

Abstract BACKGROUND: D ‐Tagatose, as one of the rare sugars, has been found to be a natural and safe low‐calorie sweetener in food products and is classified as a GRAS substance. L ‐Arabinose isomerase (L‐AI, EC 5.3.1.4), catalysing the isomerisations of L ‐arabinose and D ‐galactose to L ‐ribulose and D ‐tagatose respectively, is considered to be the most promising enzyme for the production of D ‐tagatose. RESULTS: The araA gene encoding an L‐AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli . The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L‐AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 °C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn 2+ for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D ‐galactose to D ‐tagatose by the purified L‐AI after 12 h at 65 °C reached 36%. CONCLUSION: The purified L‐AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D ‐tagatose production. Copyright © 2010 Society of Chemical Industry