一种基于儿茶素的多酚氧化酶交联聚集体制备及其高效催化合成茶黄素-3,3ʹ-双没食子酸酯研究

doi:10.13305/j.cnki.jts.2023.03.005

茶叶科学 ›› 2023, Vol. 43 ›› Issue (3): 377-388.doi: 10.13305/j.cnki.jts.2023.03.005

一种基于儿茶素的多酚氧化酶交联聚集体制备及其高效催化合成茶黄素-3,3ʹ-双没食子酸酯研究

周晶辉^1,2,3,4,5, 刘昌伟^1,2,3,4, 张盛^1,2,3,4, 许岗⁵, 胥伟⁶, 黄建安^1,2,3,4, 刘仲华^1,2,3,4,*

1.湖南农业大学茶学教育部重点实验室,湖南长沙 410128;
2.国家植物功能成分利用工程技术研究中心,湖南长沙 410128;
3.植物功能成分利用省部共建协同创新中心,湖南长沙 410128;
4.农业农村部园艺作物基因资源评价利用重点实验室,湖南长沙 410128;
5.医药工业用酶技术国家地方联合工程研究中心湖南福来格生物技术有限公司,湖南长沙 410100;
6.精制川茶四川省重点实验室,四川宜宾 644000

收稿日期:2023-02-14 修回日期:2023-04-06 出版日期:2023-06-15 发布日期:2023-06-29
通讯作者: *larkin_liu@163.com
作者简介:周晶辉,男,博士研究生,主要从事生物催化方面的研究,340310899@qq.com。
基金资助:
国家重点研发计划项目（2022YFE0111200）、国家茶叶产业技术体系（CARS-19）、湖南省产业创新链项目（2021NK1020-3）、精制川茶四川省重点实验室开放基金（SCTOF202201）

Preparation of a Cross-linked Polyphenol Oxidase Aggregate Based on Catechins and Its Efficient Catalytic Synthesis of Theaflavin-3,3ʹ-digalate

ZHOU Jinghui^1,2,3,4,5, LIU Changwei^1,2,3,4, ZHANG Sheng^1,2,3,4, XU Gang⁵, XU Wei⁶, HUANG Jian′an^1,2,3,4, LIU Zhonghua^1,2,3,4,*

1. Key Laboratory of Tea Science of Ministry of Education, Hunan agricultural university, Changsha 410128, China;
2. National Research Center of Engineering and Technology for Utilization of Botanical Functional Ingredients, Changsha 410128, China;
3. Co-Innovation Center of Education Ministry for Utilization of Botanical Functional Ingredients, Changsha 410128, China;
4. Key Laboratory for Evaluation and Utilization of Gene Resources of Horticultural Crops, Ministry of Agriculture and Rural Affairs of China, Changsha 410128, China;
5. National and local joint Engineering Research Center of Enzyme Technology for Pharmaceutical Industry, Hunan Flag Bio-technology Co., Ltd., Changsha 410100, China;
6. Tea Refining and Innovation Key Laboratory of Sichuan Province, Yibin 644000, China

Received:2023-02-14 Revised:2023-04-06 Online:2023-06-15 Published:2023-06-29

摘要/Abstract

摘要： 为了更加高效低成本地制备茶黄素,以儿茶素作为多酚氧化酶蛋白的交联试剂,对巨大芽孢杆菌（Bacillus megaterium）来源的多酚氧化酶进行了酶交联聚集体的制备并应用于茶黄素-3,3ʹ-双没食子酸酯的合成。通过对酶交联参数优化以及交联前后的酶催化性能研究,结果表明,在pH 4.0,EGCG为0.5 mg·mL^-1,多酚氧化酶活性200 U·mL^-1,交联时间50 min时,可以获得交联酶的最佳酶活回收率。与游离酶相比,交联酶表现出更优异的催化性能（热稳定性、有机溶剂耐受性、底物耐受性）。交联酶被用于合成茶黄素-3,3ʹ-双没食子酸酯时,产物质量浓度可达800 μg·mL^-1,且交联酶能被重复循环使用至少3个批次,利用该方法制备茶黄素,可以显著降低酶的应用成本,具有潜在工业化应用价值。

关键词: 酶交联聚集体, 酪氨酸酶, 制备, 茶黄素

Abstract: In order to prepare theaflavin more efficiently and economically, catechins were used as the cross-linking reagent in this study, and a polyphenol oxidase (tyrosinase) crossed-linked aggregate derived from Bacillus megaterium was prepared and used for the synthesis of theaflavin-3,3ʹ-digalate. Through optimization of enzyme cross-linking parameters and the catalytic performance before and after cross-linking, the results show that the optimal enzyme activity recovery rate of cross-linked enzyme (200 U·mL^-1) could be obtained at pH 4.0, EGCG 0.5 mg·mL^-1 and cross-linking time 50 min. Compared with free enzymes, cross-linked enzymes showed better catalytic performance (thermal stability, organic solvent tolerance, substrate tolerance). When the cross-linked enzyme was used to synthesize theaflavin-3,3′-digallate, the concentration of the product could reach 800 μg·mL^-1, and the cross-linked enzyme could be reused for at least three batches. This method could significantly reduce the application cost of theaflavin, which has potential industrial application value.

Key words: enzyme cross-linked aggregates, tyrosinase, preparation, theaflavins

中图分类号:

周晶辉, 刘昌伟, 张盛, 许岗, 胥伟, 黄建安, 刘仲华. 一种基于儿茶素的多酚氧化酶交联聚集体制备及其高效催化合成茶黄素-3,3ʹ-双没食子酸酯研究[J]. 茶叶科学, 2023, 43(3): 377-388. doi: 10.13305/j.cnki.jts.2023.03.005.

ZHOU Jinghui, LIU Changwei, ZHANG Sheng, XU Gang, XU Wei, HUANG Jian′an, LIU Zhonghua. Preparation of a Cross-linked Polyphenol Oxidase Aggregate Based on Catechins and Its Efficient Catalytic Synthesis of Theaflavin-3,3ʹ-digalate[J]. Journal of Tea Science, 2023, 43(3): 377-388. doi: 10.13305/j.cnki.jts.2023.03.005.

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一种基于儿茶素的多酚氧化酶交联聚集体制备及其高效催化合成茶黄素-3,3ʹ-双没食子酸酯研究

Preparation of a Cross-linked Polyphenol Oxidase Aggregate Based on Catechins and Its Efficient Catalytic Synthesis of Theaflavin-3,3ʹ-digalate

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