## 3 Results  
### Characteristics of the Purified Recombinant CmAP  
As described in Section 2.3, three purification steps, namely High Q anion exchange chromatography, Phenyl HP chromatography and Sephacryl(tm) S-300 gel filtration chromatography, were used. Each step was monitored by determining the enzyme activity (Table 1). The specific activity of the final enzyme was 13,133 U/mg. The SDS-PAGE result indicated that the molecular mass of one subunit is approximately 55 kDa. As shown in Figure S1, the molecular mass was higher than that of the purified ALP from _E. coli_ (prepared in our own laboratory) but lower than that of ALP from bovine intestines (Roche, Basel, Switzerland).  
Figure S2 shows the effect of pH on the activity of the purified enzyme. The maximum activity of 100% was achieved at pH 10.3. The relative activity was more than 90% when the pH values ranged between 9.5 and 10.5. When the pH was \(>\)10.7, the enzyme activity dropped sharply. Figure S3 presents the enzyme activity as a function of temperature at pH 10.3. The maximum activity of 100% was achieved at 45 \({}^{\circ}\)C. The enzyme activity increased with an increase in temperature when lower than 45 \({}^{\circ}\)C. As the temperature continued to rise, the enzyme activity gradually decreased, due to enzyme inactivation. Figure 2 presents the thermostability and storage stability of CmAP. The activity before incubation was defined as 100%. No significant change was observed after incubation for 1 h at 37 \({}^{\circ}\)C, and the relative activity was still more than 90% after treatment for 1 h at 45 \({}^{\circ}\)C. When the temperature was higher than 50 \({}^{\circ}\)C, the rate of enzyme inactivation gradually increased. The half-life time of ALP at 60 \({}^{\circ}\)C was approximately 25 min. As shown in Figure 2, the enzyme activity did not change after storage for 12 months at 4 \({}^{\circ}\)C. The enzyme activity did not change after 4 months, when stored at 25 \({}^{\circ}\)C, but it began to decline from the fifth month. Thus, CmAP was proven to have perfect stability.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Method**} & **Total Activity** & **Total Protein** & **Specific Activity** & **Yield** & **Purification** \\  & **(U)** & **(mg)** & **(U/mg)** & **(\%)** & **(fold)** \\ \hline Crude extract & 61,300 & 472 & 130 & 100 & 1 \\ High Q & 44,180 & 49 & 902 & 72 & 7 \\ Phenyl HP & 33,800 & 5.2 & 6500 & 55 & 50 \\ Sephacryl(tm) & 31,520 & 2.4 & 13,133 & 51 & 101 \\ S-300 & & & & & \\ \hline \hline \end{tabular}
\end{table}
Table 1: Purification of recombinant enzyme _Cobetia marina_ ALP (CmAP) from _E. coli_ BL21 (DE3).  
Figure 2: (a) Thermostability test of CmAP. 1 \(\mu\)g/mL CmAP solutions were incubated at temperatures ranging from 25 \({}^{\circ}\)C to 60 \({}^{\circ}\)C at pH 7.0 for 1 h. Then, the relative activity of each sample was measured at 37 \({}^{\circ}\)C, pH 10.3. The activity before incubation was defined as 100%. (b) Storage stability test of CmAP. CmAP solutions were stored at 4 \({}^{\circ}\)C and 25 \({}^{\circ}\)C for 12 months. Each sample was measured at 37 \({}^{\circ}\)C, pH 10.3. The activity before storage was defined as 100%.