###### Abstract  
D-Tagatose, a functional sweetener, is converted from D-galactose by L-arabinose isomerase, which catalyzes the conversion of L-arabinose to L-ribulose. In this study, the _araA_ gene encoding L-arabinose isomerase from _Klebsiella pneumoniae_ was cloned and expressed in _Escherichia coli_, and the expressed enzyme was purified and characterized. The purified L-arabinose isomerase, a soluble protein with 11.6-fold purification and a 22% final yield, displayed a specific activity of 1.8 U/mg for D-galactose and existed as a homohexamer of 336 kDa. The enzyme exhibited maximum activity at pH 8.0 and 40 \({}^{\circ}\)C in the presence of Mn\({}^{2+}\) and relative activity for pentoses and hexoses in the order L-arabinose > D-galactose > L-ribulose > D-xylulose > D-tagatose > D-glucose. The thermal stability of recombinant _E. coli_ cells expressing L-arabinose isomerase from _K. pneumoniae_ was higher than that of the enzyme. Thus, the reaction conditions of the recombinant cells were optimized to pH 8.0, 50 \({}^{\circ}\)C, and 4 g/L cell concentration using 100 g/L D-galactose with 1 mM Mn\({}^{2+}\). Under these conditions, 33.5 g/L D-tagatose was produced from D-galactose with 33.5% molar yield and 67 g/L/h productivity. Our findings will help produce D-tagatose using whole-cell reactions, extending its industrial application.  
Klebsiella pneumoniae; L-arabinose isomerase; D-tagatose; D-galactose; recombinant Escherichia coli +
Footnote †: journal: Applied Sciences  
0000-0002-0001-9999-22499-  
can catalyze the conversion of D-galactose to D-tagatose because of its broad substrate specificity toward similarly configured substrates [9]. To date, D-galactose has been converted to D-tagatose using L-arabinose isomerases from various microorganisms, including _Aicidothermus cellulolyticus_[10], _Alicyclobacillus acidocaldarius_[11], _Arthobacter_ sp. [12], _Anoxyobacillus flavithermus_[13], _Bacillus coagululans_[14], _Bacillus halodurans_[15], _Bacillus licheniformis_[16], _Bacillus stearothermophilus_[17], _Bacillus subtilis_[18], _Bacillus thermoglucasidasius_[19], _Bifidobacterium longum_[20], _Escherichia coli_[21], _Geobacillus kaustophilus_[22], _Geobacillus stearothermophilus_[23], _Geobacillus thermodenitrificians_[24], _Lactobacillus fermentum_[25], _Lactobacillus lus plantarum_[26], _Lactobacillus sakei_[27], _Pediococcus pentosaceus_[28], _Shevanella_ sp. [29], _Thermoanaerobacter anthranii_[30], _Thermoanaerobacterium saccarolyticum_[31], _Thermotoga maritima_[32], _Thermotoga neapolitana_[33], and _Thermus_ sp. [34].  
L-arabinose isomerases from thermophilic microorganisms, such as _A. cellulolyticus, A. acidocaldarius, B. thermoglucasius, G. kaustophilus, G. sterathermophilus, G. thermodenitr  
were harvested and resuspended in 50 mM of phosphate buffer (pH 7.0) containing 300 mM NaCl, 10 mM imidazole, and 1 mg/mL lysozyme. The resuspended cells were disrupted by sonication on ice for 25 min. The unbroken cells and cell debris were removed by centrifugation at 13,000\(\times\)\(g\) at 4 \({}^{\circ}\)C for 20 min. The cell-free supernatant was loaded on a His-Trap HP affinity chromatography column equipped with a fast protein liquid chromatography system (Bio-Rad Laboratories). The bound protein was eluted at 1 mL/min using 50 mM phosphate buffer (pH 7.0) containing 250 mM imidazole at 4 \({}^{\circ}\)C. The eluted active fractions were dialyzed against 50 mM phosphate buffer (pH 7.0) without other components at 4 \({}^{\circ}\)C for 16 h. The obtained protein was used as purified l-arabinose isomerase.