蔗糖异构酶PalⅠ-Cu^(2+)纳米花固定化酶的制备及其结构、性能分析

Preparation,structure and performance of sucrose isomerase PalⅠ-Cu^(2+)nanoflower as an immobilized enzyme

目的制备蔗糖异构酶PalⅠ-Cu^(2+)纳米花固定化酶(简称蔗糖异构酶PalⅠ-Cu^(2+)纳米花),并分析其结构及性能。方法用0.5 mmol/L IPTG诱导重组菌pET28a-PalⅠE. coli BL21(DE3)表达蔗糖异构酶PalⅠ,纯化后结合Cu^(2+)制备杂化蔗糖异构酶PalⅠ-Cu^(2+)纳米花,优化制备条件[磷酸盐缓冲溶液pH(6.0、7.4、8.0及9.0)、制备温度(4及25℃)及制备时间(12、24、48、72及120 h)]。通过扫描电子显微镜(scanning electron microscope,SEM)及透射电子显微镜(tran-smission electron microscope,TEM)观察蔗糖异构酶PalⅠ-Cu^(2+)纳米花的形态结构,并分析其稳定性及循环利用性。结果经SDS-PAGE分析,纯化的蔗糖异构酶PalⅠ表达正确,其在pH 7.4的磷酸缓冲液中4℃反应48 h制备的蔗糖异构酶PalⅠ-Cu^(2+)纳米花具有最高酶活性。经SEM及TEM观察,蔗糖异构酶PalⅠ-Cu^(2+)纳米花结构为众多花瓣紧密排列的单一球状。游离的蔗糖异构酶PalⅠ于4℃保存9 d后基本失活,蔗糖异构酶PalⅠ-Cu^(2+)纳米花于4℃保存15 d后酶活性仍保留80%的初始活性,其循环利用6次后,酶活性仍余40%。结论成功制备了蔗糖异构酶PalⅠ-Cu^(2+)纳米花,其固定化结构明显提高了酶的稳定性,且具有较好的循环性能。

Objective To prepare sucrose isomerase Pal Ⅰ-Cu^(2+) nanoflower as an immobilized enzyme and analyze its structure and function. Methods Recombinant E. coli BL21(DE3) with plasmid pET28 a-PalⅠ-Cu^(2+) was induced with0. 5 mmol/L IPTG,and the expressed sucrose isomerase PalⅠwas purified and bound to copper ion to prepare hybrid sucrose isomerase PalⅠ-Cu^(2+) nanoflower. The condition for preparation,including pH value of phosphate buffer(6. 0,7. 4,8. 0 and 9. 0)as well as temperature(4 and 25 ℃)and time(12,24,48,72 and 120 h)for preparation were optimized. The morphology and structure of prepared sucrose isomerase Pal Ⅰ-Cu^(2+) nanoflower were observed under scanning electron microscope(SEM)and transmission electron microscope(TEM),while the stability and cyclic utilization were analyzed. Results SDS-PAEG proved that sucrose isomerase was expressed correctly,with which the sucrose isomerase PalⅠ-Cu^(2+) nanoflower prepared by reaction in phosphate buffer(pH 7. 4)at 4 ℃ for 48 h showed the highest enzyme activity. Under SEM and TEM,the prepared sucrose isomerase Pal Ⅰ-Cu^(2+) nanoflower was a single sphere consisting of nanoflowers in close arrangement. Free sucrose isomerase Pal Ⅰ was basically deactivated after storage at 4 ℃ for 9 d. However,sucrose isomerase PalⅠ-Cu^(2+) nanoflower remained 80% of the initial activity after storage at 4 ℃ for 15 d and 40% of the initial activity after 6 times of cyclic utilization. Conclusion Sucrose isomerase Pal Ⅰ-Cu^(2+)nanoflower was successfully prepared and showed good performance in cyclic utilization,of which the fixed structure improved the stability of isomerase.