Purification and characterization of alkaline phosphatase from lactic acid bacteria

Abstract

Alkaline phosphatase (ALP) excreted from lactic acid bacteria (LAB) showed the ability to degrade organophosphorus pesticides. This study reported the first purification and characterization of ALP from LAB. The molecular weight of ALP was estimated to be 43 kDa measured by SDS-PAGE. The activity of purified enzyme was determined with the binding of p-nitrophenyl phosphate as the substrate. The results showed that the optimal temperature for ALP activity was 37 °C, and the optimal pH was 8.5. But ALP was stable at temperatures below 32 °C. The ALP activity remained at 80% when the pH was 8-9.5. The enzyme activity could be activated by Mg²⁺, Ca²⁺, and inhibited by Cu²⁺, Zn²⁺, and EDTA. The Michaelis-Menten constant was 6.05 mg kg^-1 with dimethoate as the substrate according to the Lineweaver-Burk plots. These results highlight an important potential use of ALP from LAB for the cleanup of pesticide pollution in raw materials for the food industry.

Fig. 1. Purification of the ALP from lactic acid bacteria (A) anion exchange chromatography (DEAE-FF); (B) gel filtration molecular sieve Superdex 75.

Fig. 2. SDS-PAGE (12%) pattern of purified ALP from lactic acid bacteria. lane 1: marker; lane 2: crude enzyme; lane 3: the supernatant after precipitation by ammonium sulfate; lane 4: the sample after DEAE Sepharose Fast Flow; lane 5: the sample after Superdex 75.

Fig. 3. Effect of pH and temperature on the activity of the ALP from lactic acid bacteria. (a) The optimal pH determined by the enzyme assay at varied pH; (b) the pH stability determined by the residual activity of ALP after incubating the enzyme with buffers at different pH (6–11) for 1 h; (c) the optimal temperature for ALP by performing the enzyme assay at 22–67 °C; (d) the thermal stability determined by the residual activity of ALP after incubating the enzyme at different temperature (22–67 °C) for 3 h.

Fig. 4. Lineweaver–Burk plot for the Michaelis–Menten constant (K_m) and the maximum velocity (V_max) for the ALP with p-NPP as substrate.

Fig. 5. Typical GC profiles of dimethoate for a standard solution.

Fig. 6. Lineweaver–Burk plot for the Michaelis–Menten constant (K_m) and the maximum velocity (V_max) for the ALP with dimethoate as substrate.