Wan Wenjing, Xia Na, Zhu Siming, Liu Qiang, Gao Youcheng
School of Food Science and Engineering, South China University of Technology, Guangzhou, China.
College of Life and Geographic Sciences, Kashi University, Kashi, China.
Front Bioeng Biotechnol. 2020 Jun 10;8:608. doi: 10.3389/fbioe.2020.00608. eCollection 2020.
Hesperetin-7-O-glucoside (HMG) is a precursor for synthesizing a sweetener named neohesperidin dihydrochalcone, and the coordination toward flavonoids of metal ions tends to increase the water solubility of flavonoids. In order to achieve effective synthesis of HMG, an immobilized enzyme catalysis platform was constructed using an immobilized rhamnosidase on FeO@graphene oxide (FeO@GO), a novel reaction pathway based on the platform was designed for preparing a hesperidin complex as a soluble substrate, and ammonium hydroxide as a ligand dissociation agent to obtain HMG. The FeO@GO was characterized by Fourier transform infrared (FT-IR), X-ray diffraction (XRD), scanning electron microscope (SEM), and thermal methods (TG/DSC) analysis to evaluate the immobilization matrix properties. The enzyme activity in free and immobilized form at different pH and temperature was optimized. The reusability of immobilized enzyme was also determined. In addition, the kinetic parameters ( and ) were computed after experiment. Results indicated that rhamnosidase immobilized on FeO@GO using a green cross-linker of genipin hydrolyzed successfully and selectively the soluble hesperidin-Cu (II) complex into HMG-Cu (II), a permanent magnet helped the separation of immobilized enzyme and hydrolytes, and ammonium hydroxide was an effective ligand dissociation agent of translating HMG-Cu (II) into HMG with high purity determined by ultraviolet-visible (UV-Vis) spectra analysis and time-of-flight mass spectrometry (TOF-MS). As a result, a novel and high-effective biosynthesis pathway of HMG based on a selectively catalytic reaction platform were constructed successfully. The pathway based on the platform has great potential to produce valuable citrus monoglycoside flavonoid HMG, and the designed reaction route are feasible using the hesperidin-Cu (II) complex with good solubility as a reaction substrate and using ammonium water as a dissociation agent.
橙皮素 -7-O-葡萄糖苷（HMG）是合成甜味剂新橙皮苷二氢查耳酮的前体，金属离子与黄酮类化合物的配位作用往往会增加黄酮类化合物的水溶性。为了实现HMG的有效合成，构建了一个固定化酶催化平台，该平台使用固定在FeO@氧化石墨烯（FeO@GO）上的鼠李糖苷酶，基于该平台设计了一条新的反应途径，用于制备作为可溶性底物的橙皮苷配合物，并使用氢氧化铵作为配体解离剂来获得HMG。通过傅里叶变换红外光谱（FT - IR）、X射线衍射（XRD）、扫描电子显微镜（SEM）和热分析方法（TG/DSC）对FeO@GO进行表征，以评估固定化基质的性质。对游离和固定化形式的酶在不同pH和温度下的活性进行了优化。还测定了固定化酶的可重复使用性。此外，实验后计算了动力学参数（ 和 ）。结果表明，使用京尼平作为绿色交联剂固定在FeO@GO上的鼠李糖苷酶成功且选择性地将可溶性橙皮苷 - 铜（II）配合物水解为HMG - 铜（II），永磁体有助于固定化酶与水解产物的分离，并且氢氧化铵是一种有效的配体解离剂，通过紫外 - 可见光谱（UV - Vis）分析和飞行时间质谱（TOF - MS）测定，可将HMG - 铜（II）转化为高纯度的HMG。结果成功构建了一种基于选择性催化反应平台的新型高效HMG生物合成途径。基于该平台的途径在生产有价值的柑橘单糖苷黄酮类化合物HMG方面具有巨大潜力，并且使用具有良好溶解性的橙皮苷 - 铜（II）配合物作为反应底物并使用氨水作为解离剂所设计的反应路线是可行的。
