黑曲霉β-葡萄糖苷酶An-bgl3的重组表达及东莨菪苷的转化  

Expression of β-glucosidase An-bgl3 from Aspergillus niger for conversion of scopolin

作　　者：于坤朋 彭程 林燕玲 李利君[1,2,3,4] 倪辉 李清彪[1,2,3] YU Kunpeng;PENG Cheng;LIN Yanling;LI Lijun;NI Hui;LI Qingbiao(College of Ocean Food and Biological Engineering,Jimei University,Xiamen 361021,Fujian,China;Fujian Provincial Key Laboratory of Food Microbiology and Engineering,Xiamen 361021,Fujian,China;Research Center of Food Biotechnology of Xiamen City,Xiamen 361021,Fujian,China;Collaborative Innovation Center of Seafood Deep Processing,Dalian 116034,Liaoning,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]海洋食品精深加工关键技术省部共建协同创新中心,辽宁大连116034

出　　处：《生物工程学报》2023年第3期1232-1246,共15页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(31871765)。

摘　　要：东莨菪素是一种香豆素类化合物,有消肿抗炎、镇痛、抗菌、杀螨等生物活性。从植物中提取东莨菪素由于东莨菪苷及其他成分的干扰常导致提取率低、纯化困难。本研究对黑曲霉(Aspergillus niger)来源的β-葡萄糖苷酶基因An-bgl3进行异源表达、纯化及表征,分析了该酶与东莨菪苷的构效关系,并研究其对植物粗提物中东莨菪苷的转化作用。结果表明,纯化后β-葡萄糖苷酶An-bgl3的比活力为15.22 IU/mg;表观分子量约为120 kDa;最适反应温度和pH分别为55℃和4.0。10 mmol/L的金属离子Fe^(2+)和Mn^(2+)可使该酶活力活分别提高1.74倍和1.20倍;10 mmol/L的Tween-20、Tween-80和Triton X-100对于该酶的抑制效果均为30%;能够耐受10%浓度的甲醇和乙醇溶剂。东莨菪苷与An-bgl3具有较好的亲和作用。该酶处理丁公藤(Erycibe obtusifolia Benth)粗提物,能够专一性地将东莨菪苷水解为东莨菪素,使东莨菪素含量增加47.8%。本研究表明黑曲霉β-葡萄糖苷酶An-bgl3对东莨菪苷具有专一性,且酶学性质良好。该研究为使用β-葡萄糖苷酶资源从植物材料中提取东莨菪素提供了参考。Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However,interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding β-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and β-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified β-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe^(2+) and Mn^(2+) increased the enzyme activity by 1.74-fold and 1.20-fold, respectively.A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10%methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the β-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.

关 键 词：东莨菪素 东莨菪苷 Β-葡萄糖苷酶 黑曲霉 丁公藤粗提物 

分 类 号：R284.1[医药卫生—中药学]