阴离子交换树脂固定化核酸酶P1的研究  

Immobilization of nuclease P1 by anion exchange resin

作　　者：王天赐 徐康 侯亚利 杨忠华 WANG Tianci;XU Kang;HOU Yali;YANG Zhonghua(School of Chemistry and Chemical Engineering,Wuhan University of Science and Technology,Wuhan 430081,China)

机构地区：[1]武汉科技大学化学与化工学院,湖北武汉430081

出　　处：《生物加工过程》2024年第1期33-41,共9页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金(21376184);湖北省教育厅科研基金重点项目(d20121108)。

摘　　要：5′-核苷酸及其衍生物因具有较高的营养保健价值和药用价值,市场需求不断增大。核酸酶P1是目前5′-核苷酸工业生产的关键酶,但游离酶存在难以重复利用、消耗量过大等缺点。为了解决上述问题,提高核酸酶的利用效率,笔者利用不同类型树脂对核酸酶P1进行固定化。以核酸酶P1的吸附量和固定化酶的酶活为评价标准,研究发现阴离子交换树脂1(AER1)固定化时效果最佳,优化后的最佳固定化条件为酶量3 644.7 U/mL(2.2 mL)、戊二醛质量分数0.25%、交联时间1.5 h、固定化时间10.0 h、pH 5.1。该条件下制备的核酸酶P1的比酶活为47 980.95 U/g。在此最佳条件下,固定化酶的热稳定性有一定程度的提高;固定化酶的酸碱稳定性大幅度提高;固定化酶在4℃下保存6周后,酶活保留51.8%,是游离酶的1.6倍。The market demand for 5′-nucleotides and their derivatives keeps increasing because of their high nutritional and health care value and broad medical application.Nuclease P1 is currently the key enzyme in the industrial production of 5′-nucleotides,but the free enzyme form still suffers from limitations such as difficulty in reuse and excessive consumption.In order to solve the above problems and improve its utilization efficiency,different types of resins were evaluated for the immobilization of nuclease P1 in this study.Based on the adsorption amount of nuclease P1 and the enzymatic activity of the immobilized enzyme,the anion exchange resin 1(AER1)offered the best immobilization effects.The optimal immobilization conditions were described as follows:the amount of enzyme was 3644.7 U/ml(2.2 mL),the mass ratio of glutaraldehyde was 0.25%,the cross-linking time was 1.5 h,the immobilization time was 10.0 h,and the pH was 5.1.The specific activity of such immobilized nuclease P1 was 47980.95 U/g.More importantly,the immobilized enzyme not only displayed the improvement of thermal stability to a certain extent,but also demonstrated much better acid-base stability.Compared with the free enzyme,the immobilized nuclease P1 also showed excellent storage stability,with 51.8%retention of its enzymatic activity after it was stored at 4℃for 6 weeks.

关 键 词：核酸酶P1 树脂 固定化酶 交联 

分 类 号：Q556.1[生物学—生物化学]