霍氏肠杆菌β-胡萝卜素9,10'双加氧酶的制备及其应用

以霍氏肠杆菌（Enterobacter hormaechei）基因组DNA为模板，通过聚合酶链式反应（PCR）法扩增β-胡萝卜素9,10'双加氧酶基因，构建重组质粒，在大肠杆菌（Escherichia coli）BL21（DE3）中表达，并采用高效液相色谱（HPLC）法检测9,10'双加氧酶活性。结果表明，通过镍柱亲和层析和分子筛Sephacryl TM S-200，得到纯化重组β-胡萝卜素9,10'双加氧酶，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）结果表明，该酶分子质量约57 kDa，最适反应温度为40 ℃，最适反应pH值为8.5；当底物β-胡萝卜素质量浓度为500 mg/L，β-胡萝卜素9,10'双加氧酶酶活为0.4 U/mL时，β-紫罗兰酮产量为142.3 mg/L，产率达到79.4%。

Preparation and application ofβ-carotene-9,10’-dioxygenase from Enterobacter hormaechei

Using the genome DNA of Enterobacter hormaechei as template, the gene of β-carotene-9,10'-dioxygenase was amplified by polymerase chain reaction (PCR), the recombinant plasmid was constructed to express in Escherichia coli BL21(DE3), and the enzyme activity was detected by HPLC. The results showed that the purified recombinant β-carotene 9,10' dioxygenase was obtained by Ni-chelating affinity chromatography and molecular sieve Sephacryl TM S-200. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the molecular mass of the enzyme was approximately 57 kDa, and the optimum reaction temperature and pH of the enzyme were 40 ℃ and 8.5, respectively. When the substrate β-carotene mass concentration was 500 mg/L and β-carotene 9,10' dioxygenase activity was 0.4 U/ml, the β-ionone yield was 142.3 mg/L and the yield reached 79.4%.