基于固定化酶法合成肉桂醇糖基化衍生物的研究

doi:10.19586/j.2095-2341.2024.0157

生物技术进展 ›› 2025, Vol. 15 ›› Issue (1): 110-118.DOI: 10.19586/j.2095-2341.2024.0157

• 研究论文 • 上一篇

基于固定化酶法合成肉桂醇糖基化衍生物的研究

程歆然(), 宗卫勇, 窦文芳()

江南大学生命科学与健康工程学院，江苏无锡 214122

收稿日期:2024-09-29 接受日期:2024-11-19 出版日期:2025-01-25 发布日期:2025-03-07
通讯作者: 窦文芳
作者简介:程歆然 E-mail：954786528@qq.com;

Research on the Synthesis of Cinnamyl Alcohol Glycosylated Derivatives Based on Immobilized Enzyme Method

Xinran CHENG(), Weiyong ZONG, Wenfang DOU()

Life Sciences and Health Engineering College，Jiangnan University，Jiangsu Wuxi 214122，China

Received:2024-09-29 Accepted:2024-11-19 Online:2025-01-25 Published:2025-03-07
Contact: Wenfang DOU

摘要/Abstract

摘要：

络塞是苯丙素苷类化合物，具有多种生物活性。络塞的传统生产方式主要从植物中提取，但自然资源有限。利用来自拟南芥的糖基转移酶UGT73C5和来自大豆的蔗糖合酶GmSUS偶联催化，对2种带有His标签的酶进行固定化，偶联催化肉桂醇反应生成络塞，同时形成尿苷二磷酸葡萄糖（uridine diphosphate glucose, UDPG）的再生。对2种酶分别进行固定，确定了树脂为2 g时分别可固定5 g的UGT73C5粗酶和6 g的GmSUS粗酶，并优化固定化偶联催化反应的最适条件，测定偶联固定化酶可重复稳定反应7个批次，最后采用分批补料策略，络塞的产量可达到15.82 mmol·L^-1，转化率为97.5%。反应后进行高效液相色谱（high performance liquid chromatography, HPLC）分析，推断出反应产物为络塞。固定化酶策略将为双酶偶联合成络塞开辟新前景，并降低游离酶在大规模工业应用中的成本。

关键词: 肉桂醇, 络塞, 糖基化反应, 蔗糖合酶, 固定化酶

Abstract:

Rosin is a phenylpropanoid glycoside compound with various biological activities. The traditional way of producing rosin is through plant extraction， but natural resources are limited. This article utilized the coupling catalysis of glycosyltransferase from Arabidopsis thaliana and sucrose synthase from soybean to immobilize two enzymes with His tags， converting cinnamyl alcohol into a complex and regenerating uridine diphosphate glucose （UDPG）. Two enzymes were fixed separately， and it was determined that 5 g of UGT73C5 crude enzyme and 6 g of GmSUS crude enzyme could be fixed with 2 g of resin. The optimal conditions for the immobilized coupling catalytic reaction were optimized， and the coupling immobilized enzyme was tested to be reproducible and stable for seven batches. Finally， a batch feeding strategy was adopted， and the yield of the complex plug reached 15.82 mmol·L^-1 with a conversion rate of 97.5%. After the reaction， high-performance liquid chromatography（HPLC）was performed， and it was inferred that the reaction product is rosin. The strategy of immobilizing enzymes will open up new prospects for the development of double enzyme pairs to form rosin， and reduce the cost of free enzymes in large-scale industrial applications.

Key words: cinnamyl alcohol, rosin, glycosylation reaction, sucrose synthase, immobilized enzyme

中图分类号:

Q559

程歆然, 宗卫勇, 窦文芳. 基于固定化酶法合成肉桂醇糖基化衍生物的研究[J]. 生物技术进展, 2025, 15(1): 110-118.

Xinran CHENG, Weiyong ZONG, Wenfang DOU. Research on the Synthesis of Cinnamyl Alcohol Glycosylated Derivatives Based on Immobilized Enzyme Method[J]. Current Biotechnology, 2025, 15(1): 110-118.

图/表 11

图1 纯化蛋白UGT73C5和GmSUS的SDS-PAGE分析注：M—Marker；泳道1：UGT73C5（上清液）；泳道2：UGT73C5（沉淀）；泳道3：纯化的UGT73C5；泳道4：GmSUS（上清液）；泳道5：纯化的GmSUS。

Fig. 1 Analysis of purified UGT73C5 and GmSUS by SDS-PAGE

图2 用2 g树脂分别固定不同量菌体后洗脱的SDS-PAGE分析A: UGT73C5; B: GmSUS。M—Maker；蓝色框表示可固定粗酶的最大量。

Fig. 2 Analysis of bacterial cells eluted after immobilizing different amounts of bacteria with 2 g resin by SDS-PAGE

图3 固定蛋白饱和值分析注：方框表示固定菌体的最大量。

Fig. 3 Analysis of fixed protein saturation value

表1 树脂量、菌体量和酶量的实际对应值

Table 1 Actual corresponding values of resin content, bacterial volume, and enzyme content

酶	树脂量/g	饱和菌体量/g	蛋白量/mg
UGT73C5	1	3.0	10
GmSUS	1	2.5	8

图4 HPLC检测UGT73C5的催化活性

Fig. 4 HPLC determination of the UGT73C5 activity

图5 UGT73C5的最适条件研究A：固定化UGT73C5和游离UGT73C5的最适温度； B：固定化UGT73C5和游离UGT73C5的最适pH

Fig. 5 Research on optimal conditions of UGT73C5

图6 偶联催化反应

Fig. 6 Cascade catalysis

图7 偶联反应UGT73C5与GmSUS酶添加量优化A：固定UGT73C5酶活为 1.5 U·mL-1，增加GmSUS酶活（40~4 000 U·mL-1）； B：固定GmSUS酶活为400 U·mL-1，增加UGT73C5酶活（0.5~4 U·mL-1）

Fig. 7 Enzyme dosage optimization of UGT73C5 and GmSUS on the coupled reaction

图8 pH、温度、DMSO含量、蔗糖浓度、UDP浓度和肉桂醇浓度对偶联反应的影响

Fig. 8 Effects of pH, temperature, DMSO content, sucrose concentration, UDP concentration, and cinnamyl alcohol concentration on coupling reaction

图9 分批补料生产络塞

Fig. 9 Production of rosin by fed-batch

图10 固定化酶偶联催化重复使用能力

Fig. 10 The reusability of immobilized enzyme coupled catalysis

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基于固定化酶法合成肉桂醇糖基化衍生物的研究

Research on the Synthesis of Cinnamyl Alcohol Glycosylated Derivatives Based on Immobilized Enzyme Method

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