## 3 Results and Discussion
### Effects of Environmental Factors on the Activity of L-Arabinose Isomerase from K. pneumoniae  
The effects of pH and temperature on L-arabinose isomerase activity were investigated using varying pH (4.0-9.0) at a constant temperature of 35 \({}^{\circ}\)C and varying temperatures (30-60 \({}^{\circ}\)C) at a constant pH value of 8.0, respectively. The maximum activity was observed at pH 8.0 and 40 \({}^{\circ}\)C (Figure 2). The thermal stability of L-arabinose isomerase from _K. pneumoniae_ was evaluated by measuring the residual activity after incubating for 1, 2, 3, 4, and 5 h at 40 \({}^{\circ}\)C (Figure 3). The enzyme activity decreased as the time increased. The activity decreased to less than 50% of the initial activity between 3 and 4 h and dropped to less than 20% at 5 h. L-Arabinose isomerase from _K. pneumoniae_ showed lower thermostability than other mesophilic L-arabinose isomerases from _B. thermoglucasius_[19] and _L. sakei_[27], which displayed residual activities of 80% and 95% for 2 h at 40 \({}^{\circ}\)C, respectively, and than from _B. longum_[20], which had a half-life of 52 h at 45 \({}^{\circ}\)C. However, the activity of the recombinant _E. coli_ cells expressing L-arabinose isomerase from _K. pneumoniae_ was more than 60% after incubation for 5 h at 50 \({}^{\circ}\)C (Figure 3), which indicated that the recombinant cells were more stable in producing D-tagatose from D-galactose than the enzyme.  
The effects of metal ions on L-arabinose isomerase activity were evaluated at a concentration of 1 mM (Table 1). Mg\({}^{2+}\) and Mn\({}^{2+}\) enhanced the activity by 15% and 28%, respectively, and Ca\({}^{2+}\) and Co\({}^{2+}\) showed similar activity to the negative control (not treated with EDTA), whereas Ba\({}^{2+}\), Cu\({}^{2+}\), and Fe\({}^{2+}\) inhibited D-galactose isomerization. Enzyme activity was the highest when Mn\({}^{2+}\) was used, similar to most enzymes, including L-arabinose isomerases from _T. neapolitana_[33], _T. maritima_[43], _T. saccharolyticum_[31], _T. mathranii_[44], _L. fermentum_[25], _L. sakei_[27], and _G. thermodentirificans_[24].  
Figure 1: SDS-PAGE and gel filtration analyses of L-arabinose isomerase from _K. pneumoniae_. (**a**) SDS-PAGE of L-arabinose isomerase from _K. pneumoniae_. M, molecular weight size marker proteins; lane 1, pellet; lane 2, crude enzyme extract; and lane 3, purified L-arabinose isomerase from _K. pneumoniae_. (**b**) Gel filtration chromatography of L-arabinose isomerase from _K. pneumoniae_. The solution of purified L-arabinose isomerase from _K. pneumoniae_ (filled square) was applied to a Sephacryl column. The column was calibrated using the reference proteins (empty circle) conalbumin, aldolase, ferritin, and thyroglobulin, corresponding to 75, 158, 400, and 669 kDa, respectively.  
Figure 3: Thermal inactivation of l-arabinose isomerase from _K. pneumoniae_ (closed square) and _E. coli_ cells expressing the enzyme (closed circle). l-Arabinose isomerase or _E. coli_ cells were incubated at 40 \({}^{\circ}\)C or 50 \({}^{\circ}\)C, respectively, withdrawn at each time point, and assayed in 50 mM phosphate buffer (pH 8.0) containing 0.8 U/mL enzyme or 1 g/L cells and 50 mM or 20 g/L D-galactose at 40 \({}^{\circ}\)C or 50 \({}^{\circ}\)C, respectively, for 10 min. Data represent the mean of values from three experiments, and error bars represent standard deviation.  
Figure 2: Effects of pH and temperature on the activity of l-arabinose isomerase from _K. pneumoniae_. (**a**) Effect 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 50 mM D-galactose and 0.8 U/mL enzyme at 35 \({}^{\circ}\)C for 10 min. (**b**) Effect of temperature. The reactions were performed in 50 mM phosphate buffer (pH 8.0) containing 50 mM D-galactose and 0.8 U/mL enzyme 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.