尿苷二磷酸糖固定化酶合成法研究

doi:10.19586/j.2095-2341.2021.0146

生物技术进展 ›› 2022, Vol. 12 ›› Issue (2): 270-280.DOI: 10.19586/j.2095-2341.2021.0146

尿苷二磷酸糖固定化酶合成法研究

吕海超(), 贾哲康, 张文, 蒋丽雯, 晁玉文, 窦文芳()

江南大学药学院，江苏无锡 214122

收稿日期:2021-08-28 接受日期:2021-10-28 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 窦文芳
作者简介:吕海超 E-mail：335162058@qq.com；

Study on Uridine Diphosphate Sugar Synthesis by Immobilized Enzyme

Haichao LYU(), Zhekang JIA, Wen ZHANG, Liwen JIANG, Yuwen CHAO, Wenfang DOU()

School of Pharmaceutical Sciences，Jiangnan University，Jiangsu Wuxi 214122，China

Received:2021-08-28 Accepted:2021-10-28 Online:2022-03-25 Published:2022-03-25
Contact: Wenfang DOU

摘要/Abstract

摘要：

利用生物酶进行体外催化反应合成不同种类的尿苷二磷酸糖（uridine diphosphate sugar，UDP-糖），生物酶的重复利用率较低。为提高尿苷二磷酸糖的合成效率及增加产物种类，以镍螯合聚丙烯酸酯树脂为载体，对带有HIS标签的N-乙酰己糖胺激酶（N-acetylhexosamine kinase，NahK）和尿苷转移酶（uridine transferase，GlmU）进行固定化。以固定化NahK和固定化GlmU为催化酶，不同单糖作为底物，研究尿苷二磷酸糖的一锅法合成情况。利用Q柱对产物进行纯化，通过高效液相色谱法、质谱法、核磁共振氢谱法对反应产物进行检测。确定了镍螯合聚丙烯酸酯树脂对游离NahK和GlmU的实际载量分别为10和20 mg·g^-1。固定化酶量的最优配比为5.5 g固定化NahK和2.5 g固定化GlmU。固定化酶的最适pH和温度分别为8.0和35℃，且能在重复反应中稳定反应5个批次。葡萄糖、N-乙酰氨基葡萄糖和甘露糖可以参与一锅法反应，生成UDP-糖的相对分子质量分别为566、607、566，而葡萄糖醛酸、半乳糖和果糖在该体系下不能合成相应的UDP-糖。基于固定化酶技术，一锅法可合成UDP-葡萄糖、UDP-N-乙酰氨基葡萄糖、UDP-甘露糖。

关键词: UDP-糖固定化酶, 一锅法, UDP-葡萄糖, UDP-N-乙酰氨基葡萄糖, UDP-甘露糖

Abstract:

Using biological enzymes to synthesize more kinds of uridine diphosphate sugars（UDP sugars）， the reuse rate of biological enzymes is low. In order to synthesize more kinds of UDP sugars with high effciency， N-acetylhexosamine kinase （NahK） and uridine transferase （GlmU） with HIS tag were immobilized on nickel chelated polyacrylate resin. Using immobilized NahK and immobilized GlmU as catalytic enzymes and different monosaccharides as substrates， the one pot method synthesis of uridine diphosphate sugar was studied. The product was purified by Q sepharose. The reaction products were detected by high performance liquid chromatography、mass spectrometry and ¹H nuclear magnetic resonance. It was confirmed that the actual loading of nickel chelated polyacrylate resin on free NahK and GlmU were 10 and 20 mg·g^-1， respectively. The optimal ratio of immobilized enzyme was 5.5 g immobilized NahK and 2.5 g immobilized GlmU. The optimal pH and temperature of immobilized enzyme were 8.0 and 35 ℃， respectively. The total immobilized enzyme could stably react for 5 batches in repeated reaction. Glucose， N-acetylglucosamine and mannose could participate in the one pot method reaction. The relative molecular weights of UDP sugars were 566、607 and 566， respectively. In this system， glucuronic acid， galactose and fructose could not synthesize the corresponding UDP sugars. Based on immobilized enzyme technology， UDP glucose， UDP N-acetylglucosamine and UDP mannose were synthesized in one pot method.

Key words: UDP suger immobilized enzyme, one pot method, UDP-Glc, UDP-GlcNAc, UDP-Man

中图分类号:

吕海超, 贾哲康, 张文, 蒋丽雯, 晁玉文, 窦文芳. 尿苷二磷酸糖固定化酶合成法研究[J]. 生物技术进展, 2022, 12(2): 270-280.

Haichao LYU, Zhekang JIA, Wen ZHANG, Liwen JIANG, Yuwen CHAO, Wenfang DOU. Study on Uridine Diphosphate Sugar Synthesis by Immobilized Enzyme[J]. Current Biotechnology, 2022, 12(2): 270-280.

图/表 10

图1 合成UDP?糖的反应路线（以葡萄糖为例）

Fig. 1 Reaction route of UDP sugar synthesis (taking glucose as an example)

表1 菌体量、酶量和镍螯合聚丙烯酸酯树脂量的实际对应值

Table 1 Actual corresponding values of bacteria amount, enzyme amount and nickel chelated polyacrylate resin amount

酶	菌体量/g	酶量/mg	镍螯合聚丙烯酸酯树脂量/g
NahK	1	20	2.0
GlmU	1	30	1.5

图2 SDS?PAGE分析NahK、GlmU固定化情况注：M—Marker；1—固定前NahK；2—固定后NahK；3—固定前GlmU；4—固定后GlmU

Fig. 2 The immobilization of NahK and GlmU by SDS?PAGE analysis

图3 总固定化酶的条件优化A：固定化NahK；B：固定化GlmU

Fig. 3 Optimization of total immobilized enzyme conditions

图4 总固定化酶酶学性质A：最适pH；B：最适温度

Fig. 4 Enzymatic properties of total immobilized enzyme

图5 总固定化酶重复稳定性分析

Fig. 5 Stability analysis of total immobilized enzyme

图6 不同单糖作为底物的合成情况A:底物为N?乙酰氨基葡萄糖；B:底物为葡萄糖；C:底物为甘露糖；D：底物为葡萄糖醛酸；E:底物为半乳糖；F：底物为果糖

Fig. 6 Synthesis of different monosaccharides as substrates

图7 UDP?糖的分离纯化

Fig. 7 Separation and purification of UDP sugar

图8 UDP?糖的LC?MS分析

Fig. 8 LC?MS analysis of UDP sugar

图9 UDP?糖的1H?NMR分析

Fig. 9 1H?NMR analysis of UDP sugar

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尿苷二磷酸糖固定化酶合成法研究

Study on Uridine Diphosphate Sugar Synthesis by Immobilized Enzyme

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