嘌呤核苷磷酸化酶/嘧啶核苷磷酸化酶共表达及固定化催化合成阿糖核苷  

Co-expression of purine nucleoside phosphorylase and uridine phosphorylase in Escherichia coli for the synthesis of arabinoside

作　　者：刘玉雪 张震 胡晓静 王振宇[1] 刘国生[1] LIU Yuxue;ZHANG Zhen;HU Xiaojing;WANG Zhenyu;LIU Guosheng(Henan Engineering Laboratory for Bioconversion Technology of Functional Microbes,College of Life Science,Henan Normal University,Xinxiang 453007,China)

机构地区：[1]河南师范大学生命科学学院功能微生物绿色转化技术河南省工程实验室,河南新乡453007

出　　处：《生物加工过程》2024年第2期139-146,共8页Chinese Journal of Bioprocess Engineering

基　　金：河南省高等学校重点科研计划(20A180033);河南省自然科学基金(222300420201)。

摘　　要：将嘌呤核苷磷酸化酶(PNP)和尿苷磷酸化酶(UP)进行共表达和双酶固定化,提高生物法合成阿糖核苷过程中底物转化率和催化剂稳定性。首先,构建大肠杆菌内源(PNP)和(UP)过表达工程菌,考察工程菌催化不同阿糖核苷之间转化的效率。将PNP和UP双酶固定化,并对固定化双酶进行回收利用,考察重复使用过程中固定化双酶的催化效率。结果表明:工程菌催化阿糖尿苷和腺嘌呤生成阿糖腺苷的反应最高转化率可达93.6%。固定化双酶催化合成阿糖2,6-二氨基嘌呤核苷的最高转化率可达99.8%。重复回收使用固定化双酶19次后对底物的转化率达到60.6%,累计有效催化时长可达684 h。PNP和UP的双酶耦合体系能高效催化阿糖核苷之间的转化,固定化修饰有利于延长酶的使用寿命,为生物法生产核苷类似物提供重要科学参考。Biocatalysis has demonstrated great potential in the synthesis of nucleoside analogues due to its mild reaction conditions,environmental friendliness and sustainability.As frequently used enzymes in biocatalytic approaches,nucleoside phosphorylases attracted great attention in recent years.Herein,the co-expression of purine and pyrimidine nucleoside phosphorylases was achieved in an engineered Escherichia coli(E.coli),which could overcome the limitation and reversibility of substrates in the transformation catalyzed by single nucleoside phosphorylase.The engineered E.coli strain,which was constructed through overexpression of both endogenous genes deoD and udp,was used for biosynthesis of different nucleoside analogues.As a result,the co-expression engineered strain could effectively catalyze the conversion of different arabinosides,with excellent preference for purine over pyrimidine.In addition,the immobilized double enzymes could further improve the substrate conversion rate and prolong the service life of the enzyme,notably with the cumulative effective catalytic time up to 684 h.Our research provided valuable information for the biosynthesis of nucleoside analogues.

关 键 词：嘌呤核苷磷酸化酶 嘧啶核苷磷酸化酶 阿糖核苷 固定化酶 生物催化 

分 类 号：Q814[生物学—生物工程]