三种芸香糖苷酶的底物特异性和酶学特征  

Substrate Specificity and Enzymatic Characteristics of Three Rutinosidases

作　　者：肖枫伟 孙江 叶家影 李利君[1,2,3] 倪辉 XIAO Fengwei;SUN Jiang;YE Jiaying;LI Lijun;NI Hui(College of Food and Biological Engineering,Jimei University,Xiamen 361021,China;Fujian Provincial Key Laboratory of Food Microbiology and Enzyme Engineering,Xiamen 361021,China;Research Center of Food Biotechnology of Xiamen,Xiamen 361021,China;School of Marine Biology,Xiamen Ocean Vocational and Technical College,Xiamen 361021,China)

机构地区：[1]集美大学海洋食品与生物工程学院,福建厦门361021 [2]福建省食品微生物与酶工程重点实验室,福建厦门361021 [3]厦门市食品生物工程技术研究中心,福建厦门361021 [4]厦门海洋职业技术学院海洋生物学院,福建厦门361021

出　　处：《食品工业科技》2025年第2期25-34,共10页Science and Technology of Food Industry

基　　金：国家自然科学基金项目(32170056)。

摘　　要：芸香糖苷类黄酮化合物的水解产物槲皮素、柚皮素、橙皮素具有丰富的生物活性和应用前景。为了探讨不同芸香糖苷酶水解制备槲皮素、柚皮素和橙皮素的特性,选择了Aspergillus niger CBS 513.88的芸香糖苷酶AnRut,Acremonium sp. DSM 24697的αRβD Ⅰ和αRβD Ⅱ进行底物特异性和酶学性质的比较研究。结果表明,三种芸香糖苷酶均只能水解α-1,6连接的芸香糖苷类黄酮化合物,对α-1,2连接的新橙皮糖苷类黄酮化合物无作用。AnRut偏好水解3-O连接的芦丁,αRβD Ⅰ偏好水解7-O连接的芸香柚皮苷和橙皮苷,αRβD Ⅱ对两种类型底物都具有良好的水解活性。分子对接结果表明三种酶与芦丁、芸香柚皮苷和橙皮苷的结合模式不同,三种酶与底物的苷元结构的相互作用差异影响了底物特异性。AnRut最适温度50℃,最适pH4.0,10 mmol/L的β-ME和DTT对AnRut酶活有明显促进作用,相对酶活是未处理酶的223%和242%。αRβD Ⅰ最适温度70℃,最适pH4.0,在酸性条件下对芸香柚皮苷和橙皮苷具有良好的水解活性。αRβD Ⅱ最适温度40℃,最适pH6.0,较适用于中性条件下对三种底物的水解。本研究为应用不同芸香糖苷酶制备槲皮素、柚皮素、橙皮素以及芸香糖苷酶构效关系的后续研究提供了实验和理论依据。Quercetin,naringenin,and hesperetin are the hydrolysis products of rutinoside flavonoids,known for their multiple biological activities and potential applications.To explore the characteristics of different rutinosidases in preparing quercetin,naringenin,and hesperetin,the rutinosidase AnRut from Aspergillus niger CBS 513.88,andαRβD I andαRβD II from Acremonium sp.DSM 24697 were selected for comparative studies on substrate specificity and enzymatic properties.The results showed that,all three rutinosidases could only hydrolyze flavonoid compounds withα-1,6-linked rutinosides,but had no effect on flavonoid compounds withα-1,2-linked neohesperidosides.AnRut primarily hydrolyzed 3-O-linked rutin,αRβD I mainly hydrolyzed 7-O-linked narirutin and hesperidin,whileαRβD II showed no significant difference in hydrolytic activity towards both types of substrates.Molecular docking results indicated that there were distinct binding modes within the three rutinosidases with rutin,narirutin,and hesperidin,and the substrate specificities of the three rutinosidases were influenced with variations in their interactions with the glycoside structures rutinoside flavonoids.The optimal temperature for AnRut was 50℃,and the optimal pH was 4.0.Additionally,10 mmol/Lβ-ME and DTT significantly enhanced AnRut's enzymatic activity,increasing the relative activity to 223%and 242%of the wild type,respectively.The optimum temperature and pH ofαRβD I was 70℃and 4.0,demonstrating efficient hydrolysis of narirutin and hesperidin under acidic conditions.αRβD II,meanwhile,had an optimal temperature of 40℃and an optimal pH of 6.0,indicating its suitability for hydrolyzing rutinoside flavonoids under neutral conditions.This study would provide experimental and theoretical references for the preparation of quercetin,naringenin,and hesperetin using rutinosidases and lay the groundwork for future research on the structure-activity relationship of rutinosidases.

关 键 词：芸香糖苷酶 芸香糖苷类黄酮 底物特异性 酶学特征 分子对接 

分 类 号：Q814.9[生物学—生物工程]