聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

Immobilization of recombinant NAD kinase in E. coli by polyvinyl alcohol-gelatin composite membrane

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。

The NAD kinase was immobilized on the composite membrane to improve its stability. Polyvinyl alcohol （PVA） , polylactic acid （PLA） , sodium alginate （SA） and gelatin （GEL） membrane were used as immobilized carrier. Based on the single-factor tests, the optimal immobilization conditions were determined as follows PVA： GEL = 4：1 , enzyme concentration of 0. 6 mL, immobilization time of 6 h, immobilization temperature of 35 ℃. Under the optimal immobilization conditions, the activity recovery was up to 84%. Properties of the immobilized NAD kinase were investigated and were compared with those of the free enzyme. After immobilization, the optimum reaction temperature was increased from 50℃ to 55℃ , the optimum reaction pH was decreased from 8. 0 to 7. 0, the pH stability and thermal stability of immobilized enzyme were superior to those of free enzyme, but the affinity of the immobilized enzyme was decreased. After the immobilized enzyme was reused for 6 times, the enzyme activity could maintain more than 75% of the initial enzyme activity. The results showed that the PVA-GEL composite membrane immobilized enzyme had good operation stability.