固定化吡哆醇激酶催化合成磷酸吡哆醇的研究  

Immobilized Pyridoxine Kinase Catalyzes Synthesis of Pyridoxine Phosphate

作　　者：侯学雯 李梦磊 叶蕾 邱敏 张坤晓 HOU Xuewen;LI Menglei;YE Lei;QIU Min;ZHANG Kunxiao(School of Pharmacy,Jiangsu Ocean University,Lianyungang 222005,China;Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening,Jiangsu Ocean University,Lianyungang 222005,China)

机构地区：[1]江苏海洋大学药学院,江苏连云港222005 [2]江苏海洋大学江苏省海洋药物活性分子筛选重点实验室,江苏连云港222005

出　　处：《食品与生物技术学报》2024年第12期119-126,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金青年基金项目(31601191)。

摘　　要：【目的】利用镍螯合琼脂糖亲和层析介质(Ni-NTA)与组氨酸(His)标记的蛋白质特异性结合的原理,将Ni-NTA与His标记的大肠杆菌(Escherichia coli,E.coli)来源的吡哆醇激酶(EcpdxK)固定化,形成一种固定化式反应器,并研究吡哆醇(PN)转化为磷酸吡哆醇(PNP)的最适反应条件。【方法】采用单因素分析方法探讨温度、pH、金属离子浓度和回收次数等对该催化反应的影响。【结果】固定化EcpdxK的最适反应温度为55℃,最适反应pH为5.0,最适M g2+浓度为30 mmol/L。该固定化酶在最适反应条件下,10 min内可将99%的PN转化为PNP。在采用固定化酶填充床反应器时,EcpdxK反应液柱留时间为10 min,持续反应12 h后其转化效率仍高达93%。【结论】固定化EcpdxK具有良好的温度耐受性、pH耐受性及操作稳定性,该固定化EcpdxK催化合成PNP的工艺操作简单、成本较低,具有工业化应用潜力。[Objective]According to the principle of specific binding of nickel-chelated agarose affinity chromatography medium(Ni-NTA)to histidine(His)-tagged proteins,the histidine-tagged Escherichia coli(E.coli)-derived pyridoxine kinase(EcpdxK)was immobilized with the Ni-NTA to form an immobilized reactor,and the optimal reaction conditions for the conversion of pyridoxine(PN)to pyridoxine phosphate(PNP)were investigated.[Method]The effects of temperature,pH,metal ion concentration,and recycling times on this catalytic reaction were investigated by one-way analysis of variance.[Result]The optimal reaction conditions of the immobilized EcpdxK were 55℃,pH 5.0,and 30 mmol/L Mg^(2+),under which the immobilized EcpdxK was able to convert 99%of PN into PNP within 10 min.In the immobilized EcpdxK-packed bed reactor,the column retention time of the EcpdxK reaction liquid was 10 min,and the conversion efficiency was still as high as 93%after 12 h of continuous reaction.[Conclusion]The immobilized EcpdxK has good temperature tolerance,pH tolerance,and operational stability,and the immobilized EcpdxK-catalyzed synthesis of PNP is a simple and costeffective process with the potential for industrial application.

关 键 词：吡哆醇激酶 固定化 镍螯合琼脂糖亲和层析介质 磷酸吡哆醇 

分 类 号：X70[环境科学与工程—环境工程]