海藻酸钠-聚乙烯醇-活性炭共固定化重组大肠杆菌细胞

Co-immobilized Recombinant Escherichia coli Cells with Sodium Alginate-PVA-Activated Carbon

目的:研究产酸性磷酸酶的重组大肠杆菌BL21(DE3)/p ET28b-AP/PT固定化制备条件以及固定化细胞的酶学特性。方法:比较9种细胞固定化方法,海藻酸钠-聚乙烯醇-活性炭共固定化为最佳方法;优化凝胶组成、菌体包埋量和固定化时间等条件,比较固定化细胞和游离细胞的酶学性质。结果:最适共固定化条件是:活性炭质量分数1.0%,海藻酸钠质量分数2.0%,聚乙烯醇质量分数6.0%,Ca Cl2质量分数2.0%,菌体包埋量6 g/100 m L凝胶溶液,固定化时间6 h。固定化细胞的酸性磷酸酶最适作用温度为35℃,比游离细胞提高5℃;固定化细胞与游离细胞的酸性磷酸酶最适作用p H均为5.0,固定化细胞显示出比游离细胞更宽泛的p H适应性。固定化细胞在重复使用12批后相对酶活力为54.5%,具有良好的操作稳定性。结论:海藻酸钠-聚乙烯醇-活性炭共固定化是非常适合固定化重组大肠杆菌BL21(DE3)/p ET28b-AP/PT的方法。

Objective： To investigate immobilization of acid phosphotransferase of recombinant Escherichia coli BL21（DE3）/p ET28b-AP/PT, and enzyme characteristics of immobilized cells. Methods： Suitable carrier of sodium alginate-PVA-activated carbon for the immobilization of recombinant E. coli BL21（DE3）/p ET28b-AP/PT was selected by the comparison of nine immobilization methods. The optimal immobilization conditions such as gel constitution, cells embedded and crosslinking time were determined, and the enzyme characteristics of immobilized cells and free cells were also compared. Results： The optimized preparation conditions were as follows： activated carbon concentration 1.0%, sodium alginate concentration 2.0%, polyvinyl alcohol concentration 6.0%, Ca Cl2 concentration 2.0%, embedded cell amount 6 g/100 m L gel solution, and crosslinking time 6 h. The optimum temperature of acid phosphotransferase from the immobilization cells was 35 ℃, 5 ℃ higher than free cells. The optimum p H values both immobilization cells and free cells were 5.0.Immobilized cells possessed excellent operation stability. After 12 cycles, 54.5% of activity still retained. Conclusion：sodium alginate-PVA-activated carbon is a good carrier for the immobilization of E. coli BL21（DE3）/p ET28b-AP/PT.