催化学报

催化学报 ›› 2016, Vol. 37 ›› Issue (4): 584-595.DOI: 10.1016/S1872-2067(15)61045-2

• 论文 • 上一篇    下一篇

基于聚多巴胺的磁性纳米生物催化剂高效催化二氢杨梅素酰化

邓啸a,c, 曹诗林b,c, 李宁c, 吴虹c, Thomas J. Smithdd, 宗敏华a,b, 娄文勇a,c   

  1. a. 华南理工大学制浆造纸工程国家重点实验室, 广东广州 510640;
    b. 华南理工大学化学与化工学院, 广东广州 510640;
    c. 华南理工大学食品科学与工程学院应用生物催化实验室, 广东广州 510640;
    d. 谢菲尔德哈勒姆大学生物分子科学研究中心, 谢菲尔德, S1 1WB, 英国
  • 收稿日期:2015-11-01 修回日期:2016-01-04 出版日期:2016-03-30 发布日期:2016-03-30
  • 通讯作者: Thomas J. Smith, Wenyong Lou
  • 基金资助:

    国家自然科学基金(21336002; 21222606; 21376096); 广东省自然科学基金(S2013020013049); 中央高校基本科研业务费专项资金(2015PT002; 2015ZP009); 制浆造纸工程国家重点实验室项目(2015C04).

A magnetic biocatalyst based on mussel-inspired polydopamine and its acylation of dihydromyricetin

Xiao Denga,c, Shilin Caob,c, Ning Lic, Hong Wuc, Thomas J. Smithd, Minhua Zonga,b, Wenyong Loua,c   

  1. a State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China;
    b School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China;
    c Laboratory of Applied Biocatalysis, School of Food Science and Technology, South China University of Technology, Guangzhou 510640, Guangdong, China;
    d Biomolecular Sciences Research Centre, Sheffield Hallam University, Sheffield, S1 1WB, UK
  • Received:2015-11-01 Revised:2016-01-04 Online:2016-03-30 Published:2016-03-30
  • Contact: Thomas J. Smith, Wenyong Lou
  • Supported by:

    This work was supported by the National Natural Science Foundation of China(21336002, 21222606, 21376096), the Key Program of Guangdong Natural Science Foundation(S2013020013049), the Fundamental Research Funds for the Chinese Universities(2015PT002, 2015ZP009), and the Program of State Key Laboratory of Pulp and Paper Engineering(2015C04).

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摘要：

脂肪酶是一种三酰基甘油水解酶, 目前广泛用于油脂化学、食品、有机合成和生物医药等领域. 但是, 游离脂肪酶在有机反应体系中容易失活, 难以从反应体系中回收, 导致其循环利用困难和生产成本增加. 因此, 需要对游离脂肪酶进行固定化, 提高酶的稳定性和重复使用性, 使其能够大规模用于工业生产.
磁性四氧化三铁纳米粒子(MNPs)具有其超顺磁性和大比表面积等性质, 但MNPs需表面修饰才能进一步应用. 近年来, 仿生矿化法制备的聚多巴胺纳米材料受到人们关注. 在仿生矿化过程中, 单体多巴胺经自聚合作用后形成聚多巴胺, 该反应活性高, 能对各类有机和无机纳米材料进行表面修饰. 而且, 聚多巴胺表层中的活性基团能与含有氨基和巯基的生物大分子发生迈克尔加成或席夫碱反应, 从而将生物大分子固定在材料表面.
本文利用聚多巴胺表面修饰MNPs, 对所得聚多巴胺表面修饰的四氧化三铁纳米粒子 (PD-MNPs) 进行了结构表征. 结果表明, PD-MNPs 尺寸在 14 nm 左右. 同时, 成功将黑曲霉脂肪酶 (ANL) 固定在 PD-MNPs上, 结果显示在pH = 8、固定化时间为 12 h 条件下, 酶负载量为 138 mg/g, 酶活回收率达到 83.6%, 而且固定化酶的 pH 稳定性及热稳定性、储藏稳定性都优于游离酶. 动力学研究表明, 固定化酶 Km值 (63.2 mmol/L) 低于游离酶 (74.5 mmol/L), 固定化酶的底物亲和性增强. 进一步研究了固定化酶和游离酶在乙腈、二甲基亚砜、乙醇和[HMIm]BF4 这四种溶剂中的溶剂耐受性, 结果显示固定化酶的耐受性均强于游离酶. 采用红外光谱对游离酶和固定化酶二级结构的分析表明, 游离黑曲霉脂肪酶经固定化后, α-螺旋和β-折叠含量分别增加了 0.84% 和 2.74%, 使得固定化后α-螺旋和β-折叠中存在的氢键能够更好地保持酶结构刚性, 避免因结构改变而引起酶失活, 增强了固定化酶在溶剂中的耐受性.
二氢杨梅素是一种具有类黄酮结构的天然产物, 具有抗氧化、抗菌、抗肿瘤和保护肝脏等作用, 但其脂溶性很差, 很难透过细胞膜被人体吸收. 本课题组曾首次以乙酸乙烯酯为酰基供体, 采用游离脂肪酶生物催化方法成功将二氢杨梅素酰化. 本文考察了PD-MNPs固定化脂肪酶在二氢杨梅素酰化反应中的应用. 结果表明, 与游离酶相比, 固定化酶在反应介质二甲基亚砜中的耐受性更强, 反应 48 h 后其催化二氢杨梅素酰化的转化率接近 80%, 明显好于游离酶 (69%). 固定化酶催化二氢杨梅素酰化的最适底物摩尔比、温度和酶量分别为 10:1(乙酸乙烯酯:二氢杨梅素)、45 ℃, 和40 U. 此外, 固定化酶在外界磁场作用下能迅速从反应混合物中分离, 从而可回收利用, 在重复使用 10 次后, 其活性仍保持在初始活性的 55% 以上, 具有良好的工业应用前景.

关键词: 磁性四氧化三铁, 纳米粒子, 聚多巴胺, 黑曲霉脂肪酶, 二氢杨梅素, 酰化

Abstract:

A support made of mussel-inspired polydopamine-coated magnetic iron oxide nanoparticles (PD-MNPs) was prepared and characterized. The widely used Aspergillus niger lipase (ANL) was immobilized on the PD-MNPs (ANL@PD-MNPs) with a protein loading of 138 mg/g and an activity recovery of 83.6% under optimized conditions. For the immobilization, the pH and immobilization time were investigated. The pH and thermal and storage stability of the ANL@PD-MNPs significantly surpassed those of free ANL. The ANL@PD-MNPs had better solvent tolerance than free ANL. The secondary structure of free ANL and ANL@PD-MNPs was analyzed by infrared spectroscopy. A kinetic study demonstrated that the ANL@PD-MNPs had enhanced enzyme-substrate affinity and high catalytic efficiency. The ANL@PD-MNPs was applied as a biocatalyst for the regioselective acylation of dihydromyricetin (DMY) in DMSO and gave a conversion of 79.3%, which was higher than that of previous reports. The ANL@PD-MNPs retained over 55% of its initial activity after 10 cycles of reuse. The ANL@PD-MNPs were readily separated from the reaction system by a magnet. The PD-MNPs is an excellent support for ANL and the resulting ANL@PD-MNPs displayed good potential for the efficient synthesis of dihydromyricetin-3-acetate by enzymatic regioselective acylation.

Key words: Magnetic iron oxide, Nanoparticle, Polydopamine, Aspergillus niger lipase, Dihydromyricetin, Acylation