Screening, identification , purification and characterization of alkaline phosphatase from Lactobacillus

Abstract:

[Background] Alkaline phosphatase (ALP) is a regulating enzyme involved in phosphorylation in living organisms. The properties of ALP in different species are related to its physiological functions, and the purified ALP is commonly used as a tool enzyme in genetic engineering, but there are few studies on ALP in lactic acid bacteria currently. [Objective] A strain of Lactobacillus producing ALP with potential probiotics was screened out, the enzyme was isolated and purified, and its properties were preliminarily investigated, which provided new microbial resources for the development and utilization of probiotics and the industrial production of ALP in the future. [Methods] Samples of Koumiss collected from four regions of Mongolia, and the enzyme-producing strains were screened through chromogenic reaction preliminary screening and enzyme activity detection rescreening. Strains were identified through morphological observation, physiological and biochemical identification and 16S rRNA gene sequence homology comparison. ALP was extracted by ultrasonic crushing, purified by ammonium sulfate precipitation, DEAE-52 ion exchange chromatography and Sephadex G-200 gel filtration chromatography, and the purity was determined by SDS-PAGE electrophoresis. [Results] A strain of Lactobacillus with the highest ALP-producing enzyme activity (No. Z23) was isolated and screened from 78 strains of lactic acid bacteria. The length of the 16S rRNA gene was 1 473 bp, and the identification result indicated that it was Lactobacillus rhamnosus. The specific activity of the purified enzyme was 180.27 U/mg, the purification ratio was 48.37, the enzyme activity recovery rate was 17.05%, and the relative molecular weight of the enzyme subunit was 46.7 kD. The optimum temperature of ALP produced by the strain was 37 °C, and the enzyme activity was most stable at 4 °C. The optimum pH was 9.5, and the enzyme activity stability could reach more than 90% between pH 9.0 and 10.0. Mg2+ and K+ had obvious activation effect on ALP, Ba2+ and Cu2+ had activation effect on ALP at low concentration, and inhibition effect at high concentration, Ca2+, Zn2+ and EDTA had strong inhibition effect on ALP. Using different concentrations of p-NPP as substrate, the Km value of the enzyme was 3.42 mmol/L and the Vmax was 1.24 mmol/(L?min). [Conclusion] This study has a clearer understanding of probiotic resources in Koumiss in Mongolia, which opened up a new way for screening ALP-producing bacteria and application of the enzyme in the future.

Key words:Koumiss, Lactobacillus, Alkaline phosphatase, Screening and identification, Separation and purification, Enzymatic property