一株产高酶活性碱性磷酸酶解淀粉芽孢杆菌的分离及其phoD碱性磷酸酶基因的克隆与表达

Isolation of a Bacillus amyloliquefaciens strain producing high-activity alkaline phosphatase and cloning and expression of the phoD gene

摘要:

【背景】碱性磷酸酶作为工具酶被广泛应用于各个领域，在免疫学检测方面应用较多的是phoA家族的碱性磷酸酶，尚无关于phoD家族的碱性磷酸酶在免疫学检测方面的研究。【目的】筛选出一株产高酶活性phoD家族碱性磷酸酶的细菌，并将其phoD基因进行克隆表达，研究phoD的酶学性质，为phoD家族的碱性磷酸酶在免疫学检测方面的应用奠定一定的基础。【方法】采取有机质丰富的土样在有机磷平板中进行细菌分离，以4-硝基苯磷酸二钠盐(4-nitrophenyl phosphate disodium salt hexahydrate，p-NPP)为底物测定有机磷平板中单菌落的酶活性，选取酶活性高的菌株作为目的菌株，克隆其phoD基因。【结果】筛选到一株产碱性磷酸酶酶活性高的菌株S2-4，通过16s rRNA基因序列同源性比较分析，鉴定该菌株为解淀粉芽孢杆菌，克隆了其phoD基因并进行诱导表达。研究了纯化后phoD的酶学性质，phoD的最适反应温度为70 ℃；最适反应pH为9.8；phoD最适Ca²⁺浓度为3 mmol/L，Mg²⁺对phoD的酶活性有抑制作用，K⁺、Zn²⁺、Mn²⁺和Fe²⁺对phoD的酶活性没有明显影响；以p-NPP为底物，在25 ℃下测得phoD的K_m为5.94 mmol/L，V_max为31.46 μmol/(L·min)，k_cat值为103.59 s^-1，为大肠杆菌碱性磷酸酶EAP k_cat值的1.60倍。【结论】菌株S2-4能产高酶活性的碱性磷酸酶，其含有的phoD为单体酶，催化效率高于EAP，是比EAP具有更大优势的标记酶。

Abstract:

[Background] Alkaline phosphatase (ALP), a tool enzyme, is widely used in various fields. Among the ALPs, PhoA has been extensively applied in immunological detection, while the functions of PhoD in immunological detection have been rarely reported. [Objective] This paper aims to screen a bacterial strain producing high-activity PhoD, clone and express the enzyme gene phoD, and study the enzymatic properties of PhoD, which is expected to lay a foundation for the application of PhoD in immunological detection. [Methods] Soil samples rich in organic matter were used to isolate bacteria in organophosphorus plate, and 4-nitrophenyl phosphate disodium salt hexahydrate (p-NPP) was employed to detect the enzyme activity of single colonies. The strain with high enzyme activity was selected and the phoD gene was cloned. [Results] S2-4, the strain producing high-activity ALP, was screened out, which was identified as Bacillus amyloliquefaciens by 16S rRNA gene alignment. Its phoD gene was cloned and induced, and the enzymatic properties of purified PhoD are as follows:optimal reaction temperature, pH, and Ca²⁺ concentration of 70℃, 9.8, and 3 mmol/L, respectively, inhibition of Mg²⁺ on PhoD activity, no significant influence of K⁺, Zn²⁺, Mn²⁺, and Fe²⁺ on PhoD activity, Michaelis constant (K_m), maximum reaction rate (V_max), and catalytic constant (k_cat) of 5.94 mmol/L, 31.46 μmol/(L·min), and 103.59 s^-1 at 25℃ in the presence of p-NPP, separately. The k_cat was 1.60 folds that of Escherichia coli alkaline phosphatase (EAP). [Conclusion] S2-4 can synthesize alkaline phosphatase with high activity and its monomer enzyme PhoD has higher catalytic efficiency than EAP, which is more advantageous than EAP as a marker enzyme.