吸附-交联法固定D-泛解酸内酯水解酶的研究

Immobilization of D-lactonohydrolase by adsorption-crosslinking method

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为:加酶量6 U/g树脂、吸附pH 7.5、吸附时间4 h、吸附温度30℃、交联剂戊二醛终浓度0.1%、交联时间2 h。实验表明在此条件下制得的固定化酶有很好的稳定性:固定化酶在连续20次的底物水解反应后,剩余酶活达到71%。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60%以上。游离酶的pH稳定性范围为pH 7～8,而固定化酶为pH 6.5～8.5。

D-lactonohydrolase was immobilized on six kinds of industrial absorption and ion exchange resins. Among them, D380 showed an excellent result as the carrier for D-laetonohydrolase immobilization. In this experiment, the Dlactonohydrolase was adsorbed firstly, and then cross-linked with glutaraldehyde. The optimum immobilization conditions were as followed： the amount of enzyme 6 U/g resin, adsorption pH 7.5, adsorption time 4 h, adsorption temperature 30 ℃, the final concentration of glutaraldehyde 0.1%, eros,slinking time 2 h. Under this condition, the immobilized enzyme showed an excellent stability： The long-term batch reactions were carried out with immobilized enzyme. After 20 times batch reaction, the residual activity of immobilized enzyme was 71%. When the temperature reached 80℃, the free enzyme lost almost all of its activity, but the immobilized enzyme has maintained more than 36% of its initial activity. The pH range of free enzyme stability was pH 7- 8, while the immobilized enzyme was pH 6.5 - 8.5.