新型交联试剂三羟甲基磷固定α-葡萄糖苷酶

Immobilization of α-glucosidase with a Novel Coupling Reagent-tris(hydroxymethyl)phosphine

用一种新型的交联剂-三羟甲基磷(THP)和壳聚糖载体固定α-葡萄糖苷酶。其最佳条件为:壳聚糖、THP和α-葡萄糖苷酶(0.1U/ml)的量分别为70mg、250mg和1μl;壳聚糖的颗粒要过120目筛,脱乙酰度要高,为93%;壳聚糖-THP的制备8min完成;在没氮气保护情况下,整个THP及壳聚糖-THP的制备在1h完成;酶与壳聚糖-THP在pH6.0下交联2h;除酶与壳聚糖-THP交联在5℃下进行,其他过程都在室温下进行。该固定化酶的酶活性OD值达到0.229,酶活力回收率为41.2%,是采用戊二醛作为交联剂活性的9倍,另外其酸碱稳定性、耐有机溶剂性、热稳定性及贮存稳定性与游离的酶和用戊二醛作交联剂固定的该酶相比较有较大提高。

The α-glucosidase was immobilized on chitosan with a novel coupling reagent-tris(hydroxymethyl)phosphine (THP). The optimum condition of immobilization was investigated, and the results showed that: the quantity of chitosan, THP, and α-glucosidase(0.1U/ml) was 70mg,250mg, and 1μl respectively; the pretreated chitosan could sieve into a particle size of 120 mesh and it's deacetylation was 93%; synthesis of chitosan-THP was achieved in 8 min;synthesis THP and chitosan-THP must be completed in 1h without N protection, the coupling reaction between α-glucosidase and chitosan-THP was done in 2 the condition of pH6.0 for 2h; all the immobilization process was accomplished in room temperature except the coupling reaction between α-glucosidase and chitosan-THP at 5℃. The experiment showed that the activity of the immobilized α- glucosidase was 0.229(absorbance at 400nm), 9 times than the activity of the immobilized one used glutaraldehyde as coupling reagent, and it's immobilization yield was 41.2%; in addition, the stability of immobilized α-glucosidase for enduring pH changing, organic solvent, heat, and long-time storing was much better than the nature one's and the stable of immobilized one's with glutaraldehyde as coupling reagent.