M.natoriense TNJL143-2菌的D-氨基酰化酶的固定化研究  

Study on the immobilization of D-aminoacylase from M. natoriense TNJL143-2

作　　者：贾超然 于笑晨 刘蔚 孙唯轩 陈丹阳 刘建[1] JIA Chaoran;YU Xiaochen;LIU Wei;SUN Weixuan;CHEN Danyang;LIU Jian(School of Life Science and Technology,Changchun University of Science and Technology,Changchun 130022,China;China-Japan Union Hospital,Jilin University,Changchun 130033,China)

机构地区：[1]长春理工大学生命科学技术学院,长春130022 [2]吉林大学中日联谊医院,长春130033

出　　处：《生命的化学》2020年第3期424-432,共9页Chemistry of Life

基　　金：吉林省科技发展计划项目(20100950)。

摘　　要：D-氨基酰化酶可用于D-氨基酸的生产,本研究利用来源于Microbacterium natoriense TNJL143-2的D-氨基酰化酶,分别通过琼脂糖包埋、介孔二氧化硅MCM-41和SBA-15吸附,制备了三种固定化酶,并对三种固定化酶的固定化条件、酶学性质、活性保持时间、重复使用次数、米氏常数等参数进行了研究。结果表明,MCM-41载体固定化酶的蛋白固定率为91.6%,SBA-15载体固定化酶的蛋白固定率为88.0%,琼脂糖包埋法蛋白固定率为79.5%。MCM-41、SBA-15以及琼脂糖三种载体固定化酶最适反应pH均为7.0,最适反应温度范围均为37℃。在固定化酶的活性保持时间以及重复利用活性方面,SBA-15固定化酶同样优于其他两种固定化酶。以D型苯丙氨酸(D-Phe)为底物时,琼脂糖包埋固定化酶的Km为28.8 mol/L,SBA-15固定化酶的Km为25.9 mol/L,MCM-41固定化酶的Km为25.0 mol/L。同时本文还探索了三种固定化酶的pH使用范围及酸碱稳定性、温度使用范围及热稳定性,结果显示,SBA-15作为固定化载体均表现出较广的适用范围及较高的稳定性。在不同条件的反应体系中,SBA-15固定化酶的蛋白损失率始终小于其他两种固定化酶。D-aminoacylase can be used for the production of D-amino acids. In this study, D-aminoacylase derived from Microbacterium natoriense TNJL143-2 was used to agarose-embedded, mesoporous resin MCM-41 and SBA-15 adsorption, respectively. Three kinds of immobilized enzymes were prepared, and the immobilization conditions, enzymatic properties, activity retention time, number of repeats, Mie constants and other parameters of the three immobilized enzymes were investigated. The results showed that the protein immobilization rate of MCM-41 carrier immobilized enzyme was 91.6%, the protein immobilization rate of SBA-15 carrier immobilized enzyme was 88.0%, and the protein immobilization rate by agarose embedding method was 79.5%. MCM-41, SBA-15 and agarose, three carrier-immobilized enzymes, had an optimum reaction pH range of 7.0 and an optimum reaction temperature range of 37 ℃. In different reaction systems, the protein loss rate of SBA-15 immobilized enzyme was always lower than that of the other two immobilized enzymes. Resin SBA-15 immobilized enzyme is also superior to the other two immobilized enzymes in terms of retention time of immobilized enzyme activity and reuse activity. When D-Phe was used as substrate, the Km of the agarose-immobilized enzyme was 28.8 mol/L, the Km of SBA-15-immobilized enzyme was 25.9 mol/L, and the Km of MCM-41-immobilized enzyme was 25.0 mol/L. Meanwhile, the optimal pH range and pH stability of the three immobilized enzymes, the optimal temperature range and stability of the three immobilized enzymes were investigated. The results showed that SBA-15 as an immobilized carrier showed a wider range of application and higher stability. In reaction systems with different conditions, the protein loss rate of the resin SBA-15 immobilized enzyme was always smaller than that of the other two immobilized enzymes.

关 键 词：D-氨基酰化酶 Microbacterium natoriense TNJL143-2 固定化酶 

分 类 号：Q814.2[生物学—生物工程]