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

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

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

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°C,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(Km),maximum reaction rate(Vmax),and catalytic constant(kcat)of 5.94 mmol/L,31.46μmol/(L·min),and 103.59 s^?1 at 25°C in the presence of p-NPP,separately.The kcat 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.