## 3 Results and Discussion
### Substrate Specificity of L-Arabinose Isomerase from K. pneumoniae  
The substrate specificity of L-arabinose isomerase from _K. pneumoniae_ was investigated using various pentoses such as L-arabinose, D-xylose, L-ribulose, and D-xylulose and hexoses such as D-galactose, D-glucose, D-tagatose, and D-fructose (Table 2). The enzyme activity was the highest for L-arabinose, followed by D-galactose, L-ribulose, D-xylulose, D-xylose, D-tagatose, and D-glucose. The activity was generally higher for pentose than hexose except for D-galactose; the activity for L-arabinose was 3.15 times higher than that for D-galactose. However, the activity against D-tagatose, a product of D-galactose, was only 4.3% of that of D-galactose. Optimization of Reaction Conditions Using E. coli Cells Expressing L-Arabinose Isomerase from K. pneumoniae  
To produce D-tagatose from D-galactose, reaction conditions such as pH, temperature, and cell concentration were optimized using recombinant _E. coli_ cells expressing L-arabinose isomerase from _K. pneumoniae_. The maximum activity of the recombinant _E. coli_ cells was observed at pH 8.0, the same as the enzyme, and 50 \({}^{\circ}\)C (Figure 4), 10 \({}^{\circ}\)C higher than that of the enzyme (Figure 2). After incubation for 5 h at 50 \({}^{\circ}\)C, the activity of the recombinant _E. coli_ cells was more than 60%, which was approximately three-fold higher than that of the enzyme after 5 h at 40 \({}^{\circ}\)C (Figure 3). Therefore, the optimal temperature for producing D-tagatose from D-galactose using recombinant _E. coli_ cells was determined to be 50 \({}^{\circ}\)C.  
The production of D-tagatose from D-galactose was further optimized by varying the concentration of the recombinant cells (Figure 5a). The production increased with an increase in the concentration of cells up to 4 g/L and reached a plateau above that concentration, indicating that the optimum concentration for the recombinant _E. coli_ cells was 4 g/L. \begin{table}
\begin{tabular}{c c} \hline
**Metal Ion** & **Relative Activity (\%)** \\ \hline Ba\({}^{2+}\) & 82 \\ Ca\({}^{2+}\) & 101 \\ Co\({}^{2+}\) & 105 \\ Cu\({}^{2+}\) & 56 \\ Fe\({}^{2+}\) & 94 \\ Mg\({}^{2+}\) & 115 \\ Mn\({}^{2+}\) & 128 \\ EDTA & 6 \\ Negative control & 100 \\ \hline \end{tabular}
\end{table} 
Table 1: Effects of metal ions on the activity of L-arabinose isomerase from _K. pneumoniae_. \begin{table}
\begin{tabular}{c c c c} \hline  & **Substrate** & **Product** & **Relative Activity (\%)** \\ \hline Pentose & L-Arabinose & L-Ribulose & 315.6 \\  & D-xylose & D-xylulose & 10.2 \\  & L-Ribulose & L-Arabinose & 30.2 \\  & D-xylulose & D-xylose & 27.6 \\ Hexose & D-Galactose & D-Tagatose & 100.0 \\  & D-Glucose & D-Fructose & 2.4 \\  & D-Tagatose & D-Galactose & 4.3 \\  & D-Fructose & D-Glucose & ND \\ \hline \end{tabular} ND, not detected.  
\end{table} 
Table 2: Substrate specificity of L-arabinose isomerase from _K. pneumoniae_. Figure 4: Investigation of pH and temperature to optimize the production of d-tagatose from d-galactose by _E. coli_ cells expressing l-arabinose isomerase from _K. pneumoniae_. (**a**) Investigation of pH. The reactions were performed in 50 mM citrate (pH 4.0–6.0), phosphate (pH 6.0–8.0), and Tris-HCl (pH 8.0–9.0) buffers containing 20 g/L d-galactose and 1 g/L cells at 40 \({}^{\circ}\)C for 10 min. (**b**) Investigation of temperature. The reactions were performed in 50 mM phosphate buffer (pH 8.0) containing 20 g/L d-galactose and 1 g/L cells at different temperatures, ranging from 30 \({}^{\circ}\)C to 60 \({}^{\circ}\)C for 10 min. Data represent the mean of values from three experiments, and error bars represent standard deviation. Figure 5: Production of d-tagatose from d-galactose by _E. coli_ cells expressing l-arabinose isomerase from _K. pneumoniae_. (**a**) Effect of the concentration of _E. coli_ cells expressing l-arabinose isomerase from _K. pneumoniae_ on d-tagatose production.