基于糖基转移酶与蔗糖合酶级联催化合成紫云英苷的研究

doi:10.19586/j.2095-2341.2022.0212

生物技术进展 ›› 2023, Vol. 13 ›› Issue (2): 247-256.DOI: 10.19586/j.2095-2341.2022.0212

• 研究论文 • 上一篇

基于糖基转移酶与蔗糖合酶级联催化合成紫云英苷的研究

贾哲康(), 程歆然, 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:2022-12-23 接受日期:2023-03-06 出版日期:2023-03-25 发布日期:2023-04-07
通讯作者: 窦文芳
作者简介:贾哲康 E-mail: jiazhekang456@qq.com；

Cascade Catalysis of Glycosyltransferase and Sucrose Synthase to Produce Astragalin

Zhekang JIA(), Xinran CHENG, Wenfang DOU()

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

Received:2022-12-23 Accepted:2023-03-06 Online:2023-03-25 Published:2023-04-07
Contact: Wenfang DOU

摘要/Abstract

摘要：

紫云英苷属于黄酮类3-O-糖苷类化合物，具有抗氧化、抗病毒、抗菌等多种药理学活性。利用来自葡萄柚中的糖基转移酶和大豆中的蔗糖合酶级联催化，将山奈酚转化为紫云英苷，同时将糖基化反应副产物二磷酸尿苷（uridine diphosphate， UDP）再生为UDP-葡萄糖，实现了UDP的循环再生。探究了级联催化反应的最适条件，并通过分批补料操作，紫云英苷的产量达到了1 550.1 mg·L^-1，转化率为86.5%，UDP循环了34.8次。对分离纯化后的物质进行鉴定，判断反应产物为紫云英苷。研究提出的紫云英苷合成方法极大地降低了反应成本，为工业化生产紫云英苷提供了新思路。

关键词: 糖基化反应, 蔗糖合酶, 紫云英苷, 山奈酚, UDP-葡萄糖

Abstract:

Astragalin is a flavonoid 3-O-glycoside compound with various pharmacological activities such as antioxidant， antiviral and antibacterial. In this study， glucosyltransferase from grapefruit and sucrose synthase from soybean were used to convert kaferol to astragalin， while the glycosylation byproduct uridine diphosphate （UDP） was regenerated to UDP-glucose. Through fed-batch operation， the yield of astragalin reached 1 550.1 mg·L^-1， the conversion rate was 86.5%， and the UDP cycle was 34.8 times. After isolation and purification， the results were basically consistent with the literature， and the reaction product was indeed astragalin. The synthesis method of astragalin proposed in this study greatly reduced the reaction cost and provided an new idea for the industrial production of astragalin.

Key words: glycosylation, sucrose synthase, astragalin, kaempferol, UDP-glucose

中图分类号:

Q555

贾哲康, 程歆然, 窦文芳. 基于糖基转移酶与蔗糖合酶级联催化合成紫云英苷的研究[J]. 生物技术进展, 2023, 13(2): 247-256.

Zhekang JIA, Xinran CHENG, Wenfang DOU. Cascade Catalysis of Glycosyltransferase and Sucrose Synthase to Produce Astragalin[J]. Current Biotechnology, 2023, 13(2): 247-256.

图/表 12

图1 纯化蛋白的SDS-PAGE分析注：M—蛋白Marker；1—纯化的FGT；2—纯化的GmSUS。

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

图2 HPLC检测FGT的催化活性A：紫云英苷标准品；B：山奈酚标准品；C：反应前；D：反应后

Fig. 2 HPLC determination of the glycosyltransferase activity

图3 pH、温度以及DMSO、UDP、果糖和蔗糖浓度对FGT酶活的影响

Fig. 3 Effects of pH, temperature and concentration of DMSO, sucrose, fructose, and UDP on the FGT activity

图4 HPLC检测GmSUS的催化活性A：GmSUS催化反应后；B：没有GmSUS催化的反应；C：UDP-葡萄糖标准品；D：UDP标准品

Fig. 4 HPLC determination of the sucrose synthase activity

图5 级联催化反应A：仅FGT参与的催化；B：仅GmSUS参与的催化；C：级联催化

Fig. 5 Cascade catalysis

表1 酶浓度对紫云英苷产率的影响

Table 1 Effect of enzyme concentration on the yield of astragaloside

序号	FGT浓度/（mU·mL^-1）	GmSUS浓度/（mU·mL^-1）	紫云英苷产量/（mmol·L^-1）
1	3.68	166	0.063 1
2	18.4	166	0.153 1
3	36.8	166	0.246 7
4	55.2	166	0.235 6
5	73.6	166	0.259 3
6	36.8	830	0.339 9
7	36.8	1 660	0.417 6
8	36.8	2 490	0.456 1
9	36.8	3 320	0.460 2

图6 pH、温度及DMSO、UDP、蔗糖和山奈酚浓度对级联催化反应的影响

Fig. 6 Effects of pH, temperature and concentration of DMSO, UDP, sucrose and kaempferol on the cocatalytic reaction

图7 分批补料生产紫云英苷

Fig. 7 Production of astragalin by fed-batch

图8 纯化产物的HPLC分析

Fig. 8 HPLC analysis of purified product

图9 纯化产物的LC-MS分析

Fig. 9 LC-MS analysis of purified product

图10 纯化产物的核磁分析A:核磁共振氢谱。氢谱解析的数据为：1H NMR(400 MHz,DMSO-d6)δ 12.62(s,1H),10.86(s,1H),10.18(s,1H),8.09-8.01(m,2H),6.93-6.85(m,2H),6.44(d,J=2.0Hz,1H),6.21(d,J=2.0Hz,1H),5.47(d,J=7.3Hz,1H),5.35(d,J=4.5 Hz,1H),5.06(d,J=4.6 Hz,1H),4.98-4.92(m,1H),4.26(t,J=5.6 Hz,1H),3.57(dd,J =11.6,5.5 Hz,1H),3.35-3.28(m,1H),3.19(dtd,J=13.6,8.6,4.7 Hz,2H),3.09(q,J=4.1,3.4 Hz,2H)。B:碳谱检测。碳谱解析的数据为：13C NMR(101 MHz,DMSO-d6)δ177.91,164.55,161.66,160.38,156.81,156.68,133.61,131.32,121.33,115.54,104.45,101.31,101.24,99.11,94.08,77.94,76.85,74.65,70.32,61.27。

Fig. 10 NMR analysis of purified product

图11 FGT和GmSUS级联反应合成紫云英苷

Fig. 11 FGT and GmSUS reacted cascade ally to produce astragalin

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[1]	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.
[2]	何丽丽,窦文芳,吕海超,薛欣欣,张文,江南. 基于双酶偶联法合成尿苷二磷酸葡萄糖醛酸的研究[J]. 生物技术进展, 2021, 11(3): 393-402.

基于糖基转移酶与蔗糖合酶级联催化合成紫云英苷的研究

Cascade Catalysis of Glycosyltransferase and Sucrose Synthase to Produce Astragalin

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