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

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Mg²⁺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。

Abstract:

[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 Mg²⁺, 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 cost-effective process with the potential for industrial application.