Liu Siyao, Höldrich Markus, Sievers-Engler Adrian, Horak Jeannie, Lämmerhofer Michael
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076, Tübingen, Germany.
Anal Chim Acta. 2017 Apr 22;963:33-43. doi: 10.1016/j.aca.2017.02.009. Epub 2017 Feb 20.
Surface-modified gold nanoparticles (GNPs) were synthesized via layer-by-layer process with alternating cationic polyallylamine and anionic poly(acrylic acid) polyelectrolyte layers leading to a highly hydrophilic biocompatible shell supporting colloidal stability. Afterwards, papain was covalently immobilized on the modified GNPs via amide coupling between the amino groups on papain and the terminal carboxylic groups of the modified GNPs by using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysulfosuccinimide sodium as coupling agents. The resultant papain-functionalized gold nanoparticles were characterized by surface plasmon resonance, dynamic light scattering and zeta potential measurements. The new technology resonant mass measurement was applied for determining the average number of papain molecules immobilized per GNP by measurement of the single nanoparticle buoyant mass in the range of femtograms. The activity of the immobilized enzyme was estimated by determination of the kinetic parameters (K, V and k) with the standard chromogenic substrate N-benzoyl-dl-arginine-4-nitroanilide hydrochloride. It was found that K of immobilized and free enzyme are in the same order of magnitude. On contrary, turnover numbers k were significantly higher for GNP-conjugated papain. Further, the gold nanobiocatalyst was applied for digestion of polyclonal human immunoglobulin G to yield protein fragments. The resultant fragment mixture was further analyzed by high-performance liquid chromatography-microelectrospray ionization-quadrupole-time-of-flight mass spectrometry, which demonstrated the applicability of the bioreactor based on papain functionalized GNPs. The immobilized papain not only has higher catalytic activity and better stability, but also can be easily isolated from the reaction medium by straightforward centrifugation steps for reuse.
通过层层组装工艺，交替沉积阳离子聚烯丙胺和阴离子聚丙烯酸聚电解质层，合成了表面改性的金纳米颗粒（GNP），形成了高度亲水性的生物相容性外壳，维持了胶体稳定性。随后，使用N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺和N-羟基磺基琥珀酰亚胺钠作为偶联剂，通过木瓜蛋白酶上的氨基与改性GNP的末端羧基之间的酰胺偶联，将木瓜蛋白酶共价固定在改性GNP上。通过表面等离子体共振、动态光散射和zeta电位测量对所得的木瓜蛋白酶功能化金纳米颗粒进行了表征。应用新技术共振质量测量法，通过测量飞克范围内单个纳米颗粒的浮力质量，来确定每个GNP固定的木瓜蛋白酶分子的平均数量。通过使用标准发色底物N-苯甲酰-dl-精氨酸-4-硝基苯胺盐酸盐测定动力学参数（K、V和k），评估固定化酶的活性。发现固定化酶和游离酶的K值处于相同的数量级。相反，GNP偶联的木瓜蛋白酶的周转数k显著更高。此外，将金纳米生物催化剂应用于多克隆人免疫球蛋白G的消化，以产生蛋白质片段。通过高效液相色谱-微电喷雾电离-四极杆-飞行时间质谱对所得的片段混合物进行进一步分析, 这证明了基于木瓜蛋白酶功能化GNP的生物反应器的适用性。固定化的木瓜蛋白酶不仅具有更高的催化活性和更好的稳定性，而且可以通过直接离心步骤轻松地从反应介质中分离出来，以便重复使用。
