Design of tandem genes cluster for isoflavone engineering

In this paper,a universal effective novel method of constructing tandem genes cluster for isoflavone engineering was reported.A tandem genes cluster CHS-CHI-IFS(rIFS)of secondary metabolites of plant isoflavones was constructed by using the chalcone synthase(CHS),chalcone isomerase(CHI),and isoflavone synthase(IFS)(GenBank accession numbers EU526827,EU526829,EU526830)in only one recombination with the pET22b vector.The resulting expression vector pET-rIFS was heterogeneously expressed and co-incubated with barrenwort extractions,and the genistein-like com-ponent was detected.

纤维素载体固定化β-葡萄糖苷酶及其在大豆异黄酮水解中的应用

建立纤维素载体固定化β-葡萄糖苷酶系统,用于黑豆浆中大豆异黄酮去糖基化反应,并对该催化系统进行了可行性评价。结果表明,在40 cm3纤维素颗粒上可结合超过40 mgβ-葡萄糖苷酶,并且能有效将4-硝基苯-β-D-葡糖苷酸水解成对硝基苯酚,其反应最适温度为50℃。经动力学测试后可得其米氏常数Km值为(1.38±0.20)mmol/L。将该固定化酶系统用于黑豆浆中大豆异黄酮去糖基化,通过高效液相色谱检测其转化效率,表明该系统可以在30 min内利用40 cm^3含酶载体将50 mL黑豆浆中的异黄酮全部转化为去糖基化形式。该固定化酶催化系统连续化催化稳定性实验表明,在经过15次反应以及15 d之后,该固定化酶系统仍然可维持其60%的催化活性。