CBM介导的蔗糖异构酶的固定化研究  

CBM Mediated Immobilization of Sucrose Isomerase

作　　者：李思彭 吕奕霏 李宪臻[1] 郭小宇[1] LI Si-peng;LYU Yi-fei;LI Xian-zhen;GUO Xiao-yu(Schl.of Bio-Engineering,Dalian Polytech.Uni.,Dalian 116033)

机构地区：[1]大连工业大学生物工程学院,辽宁大连116033

出　　处：《微生物学杂志》2024年第3期12-23,共12页Journal of Microbiology

基　　金：国家自然科学基金项目(31771907)。

摘　　要：利用碳水化合物结构域CBM17将来源于Klebsiella sp.LX3的蔗糖异构酶PalI固定于微晶纤维素表面,以获得一种经济环保、稳定性高、可循环使用的固定化蔗糖异构酶。通过无限制克隆法构建融合CBM17标签的PalI表达载体,基于CBM17与微晶纤维素自发结合的特性,制备该酶的固定化酶;采用3,5-二硝基水杨酸比色定糖法测定该酶的酶活力;比较游离状态与固定化状态酶的酶学性质,衡量固定化酶的催化性能;测定固定化酶的可循环使用次数、可储存时间以对其稳定性进行评价。成功构建融合CBM17标签的PalI,并固定于微晶纤维素表面;固定化与游离形式的PalI表现出相似的最适温度及pH,分别为45℃、pH 6.0,而其温度稳定性及pH稳定性较游离酶均有提高,最高酶活力为(11.04±0.02)U/mg微晶纤维素(Avicel PH101);固定化酶与底物蔗糖亲和力增强,K_(m)为(79.94±9.56)mmol/L;该固定化酶具有良好的操作稳定性及储存稳定性,连续反应12次,酶活力仍保持在初始酶活力的80%左右,在4℃下储存至56 d时,酶活力降至60%以下。本研究成功制备了一种环境友好、经济可行、稳定性高的固定化蔗糖异构酶,这种固定化策略为蔗糖异构酶在工业中的应用提供了一种新的思路。CBM17 is used to immobilize the sucrose isomerase PalI originated from Klebsiella sp.LX3 on the surface of microcrystalline cellulose to obtain a kind of economic,environmental friendly,high stability and recyclable immobilized sucrose isomerase.The fusion CBM17 tagged PalI expression vector was constructed by Restriction-Free cloning,and the immobilized enzyme was prepared based on the property that CBM17 binds spontaneously to microcrystalline cellulose;The enzyme activity of the enzyme was determined by 3,5-dinitrosalicylic acid colorimetric glycometry;Comparing the enzymatic properties of enzymes in the free state with the immobilized state,a measure of the catalytic performance of the immobilized enzyme;The number of recyclable uses and the storage time of the immobilized enzyme were measured to evaluate its stability.CBM17 tagged PalI was successfully constructed and immobilized on the surface of microcrystalline cellulose;The immobilized and free forms of PalI showed similar optimum temperature and pH,respectively 45℃and pH 6.0,and their temperature stability and pH stability were improved compared with those of free enzymes.The highest enzyme activity was(11.04±0.02)U/mg Avicel PH101.The immobilized enzyme showed an enhanced affinity for the substrate sucrose with K_(m) at(79.94±9.56)mmol/L;The immobilized enzyme showed fine operational and storage stability,the enzyme activity remained at about 80% of the initial enzyme activity for 12 consecutive reactions,decreased to less than 60% when stored until 56 days at 4℃.In this study,an environmentally benign,economically feasible,and highly stable immobilized sucrose isomerase was successfully prepared,and this immobilization strategy provides a new way of thinking for the application of sucrose isomerase in industry.

关 键 词：异麦芽酮糖 蔗糖异构酶 碳水化合物结构域 固定化酶 酶学性质 纤维素 

分 类 号：Q936[生物学—微生物学]