Study on Uridine Diphosphate Sugar Synthesis by Immobilized Enzyme

doi:10.19586/j.2095-2341.2021.0146

Current Biotechnology ›› 2022, Vol. 12 ›› Issue (2): 270-280.DOI: 10.19586/j.2095-2341.2021.0146

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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

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

通讯作者: 窦文芳
作者简介:吕海超 E-mail：335162058@qq.com；

Abstract

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

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

CLC Number:

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.

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.

Figures/Tables 10

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

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

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

Fig. 3 Optimization of total immobilized enzyme conditions

Fig. 4 Enzymatic properties of total immobilized enzyme

Fig. 5 Stability analysis of total immobilized enzyme

Fig. 6 Synthesis of different monosaccharides as substrates

Fig. 7 Separation and purification of UDP sugar

Fig. 8 LC?MS analysis of UDP sugar

Fig. 9 1H?NMR analysis of UDP sugar

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Study on Uridine Diphosphate Sugar Synthesis by Immobilized Enzyme

尿苷二磷酸糖固定化酶合成法研究

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