亚牛磺酸对南美白对虾多酚氧化酶活性及酶构象的影响

doi:10.7506/spkx1002-6630-20191203-040

食品科学 ›› 2021, Vol. 42 ›› Issue (4): 100-106.doi: 10.7506/spkx1002-6630-20191203-040

亚牛磺酸对南美白对虾多酚氧化酶活性及酶构象的影响

周雅琪，黄佳茵，陈美玉，葛雨珺，李苑，胡亚芹

（1.浙江大学生物系统工程与食品科学学院，馥莉食品研究院，智能食品加工技术与装备国家（地方）联合实验室，农业农村部农产品产后处理重点实验室，农业农村部农产品营养功能评价实验室，浙江省农产品加工技术研究重点实验室，浙江?杭州 310058；2.浙江大学宁波研究院，浙江?杭州 315100）

出版日期:2021-02-25 发布日期:2021-02-25
基金资助:
宁波市公益计划重点项目（2019C10083）；国家自然科学基金面上项目（31871868）

Effects of Hypotaurine on the Activity and Conformation Polyphenol Oxidase in Penaeus vannamei

ZHOU Yaqi, HUANG Jiayin, CHEN Meiyu, GE Yujun, LI Yuan, HU Yaqin

(1. National Engineering Laboratory of Intelligent Food Technology and Equipment, Key Laboratory for Agro-products Postharvest Handling, Ministry of Agriculture and Rural Affairs, Key Laboratory for Agro-products Nutritional Evaluation, Ministry of Agriculture and Rural Affairs, Zhejiang Key Laboratory for Agro-food Processing, Fuli Institute of Food Science, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China; 2. Ningbo Research Institute, Zhejiang University, Hangzhou 315100, China)

Online:2021-02-25 Published:2021-02-25

摘要/Abstract

摘要： 通过研究不同质量浓度（1、5、10、15、20 g/L）亚牛磺酸（hypotaurine，HTU）对南美白对虾多酚氧化酶（polyphenol oxidase，PPO）活性的影响，判断HTU的抑制类型并测定抑制常数，同时通过测定PPO蛋白的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）、圆二色谱、表面疏水性以及内源荧光光谱研究HTU对PPO构象的影响。结果表明HTU能抑制PPO活性，当其质量浓度为20 g/L时，能抑制79.38%的酶活性，最大半数抑制浓度（half-maximal inhibitory concentration，IC50）为16.09 g/L。Lineweaver-Burk双倒数作图的结果显示Vmax不变，Km增大，推测HTU是竞争性抑制剂的一种，其抑制常数KI为0.445 mmol/L。随着HTU质量浓度的增大，SDS-PAGE结果显示酶蛋白的分子质量未发生明显变化；圆二色谱和表面疏水性的结果表明其α-螺旋与β-折叠向β-转角和无规卷曲发生转变，酶蛋白发生部分去折叠，使得疏水性氨基酸残基暴露，导致蛋白表面疏水性增强；内源荧光光谱结果显示HTU能够使PPO的荧光强度发生猝灭，最大发射波长发生红移。因此推测HTU可能是通过改变PPO的空间结构影响酶的活性。

关键词: 南美白对虾；多酚氧化酶；亚牛磺酸；酶活性；酶构象

Abstract: In this research, we studied the inhibitory effects of different concentrations (1, 5, 10, 15, and 20 g/L) of hypotaurine (HTU) on the activity of polyphenol oxidase (PPO) from Penaeus vannamei, and further we determined the inhibition type and inhibition constant. Meanwhile, the effects of HTU on PPO conformation were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD) spectroscopy, surface hydrophobicity measurement and endogenous fluorescence spectroscopy. The results showed that HTU could inhibit 79.38% of PPO activity when its concentration was 20 g/L, and the half-maximum inhibition concentration (IC50) value was 16.09 g/L. The Lineweaver-Burk double reciprocal plot showed that the Vmax remained unchanged while the Km increased. It was presumed that HTU was a competitive inhibitor with an inhibition constant KI of 0.445 mmol/L. SDS-PAGE results showed that the molecular mass of the enzyme did not change significantly with increasing HTU concentration. The results of CD spectroscopy and surface hydrophobicity showed that the secondary structure of the enzyme was transformed from α-helix and β-sheet to β-turn and random coil, and partial unfolding occurred, leading to the exposure of hydrophobic amino acid residues and enhanced surface hydrophobicity. HTU could quench the endogenous fluorescence of PPO and result in a red shift in the maximum emission wavelength. Therefore, it is speculated that HTU may affect the enzymatic activity by changing the spatial structure of PPO.

Key words: Penaeus vannamei; polyphenol oxidase; hypotaurine; enzymatic activity; enzyme conformation

中图分类号:

TS254.1

周雅琪，黄佳茵，陈美玉，葛雨珺，李苑，胡亚芹. 亚牛磺酸对南美白对虾多酚氧化酶活性及酶构象的影响[J]. 食品科学, 2021, 42(4): 100-106.

ZHOU Yaqi, HUANG Jiayin, CHEN Meiyu, GE Yujun, LI Yuan, HU Yaqin. Effects of Hypotaurine on the Activity and Conformation Polyphenol Oxidase in Penaeus vannamei[J]. FOOD SCIENCE, 2021, 42(4): 100-106.

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亚牛磺酸对南美白对虾多酚氧化酶活性及酶构象的影响

Effects of Hypotaurine on the Activity and Conformation Polyphenol Oxidase in Penaeus vannamei

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