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首页> 外文期刊>Biotechnology Progress >A Novel Enzymatic Microreactor with Aspergillus oryzae β-Galactosidase Immobilized on Silicon Dioxide Nanosprings
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A Novel Enzymatic Microreactor with Aspergillus oryzae β-Galactosidase Immobilized on Silicon Dioxide Nanosprings

机译：米曲霉β-半乳糖苷酶固定在二氧化硅纳米弹簧上的新型酶微反应器

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The use of silicon dioxide (SiO2) nanosprings as supports for immobilized enzymes in a continuous microreactor is described. A nanospring mat (2.2 cm~2 x 60 μm thick) was functional-ized with y-aminopropyltriethoxysilane, then treated with N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) and dithiothreitol (DTT) to produce surface thiol (SH) groups. SPDP-modified β-galactosidase from Aspergillus oryzae was immobilized on the thiolated nanosprings by reversible disulfide linkages. The enzyme-coated nanospring mat was placed into a 175-μm high microchannel, with the mat partially occluding the channel. The kinetics and steady-state conversion of hydrolysis of o-nitrophenyl β-D-galactosylpyranoside at various substrate flow rates and concentrations were measured. Substantial flow was observed through the nanosprings, for which the Darcy permeability k ≈ 3 x 10~(-6) cm~2. A simple, one-parameter numerical model coupling Navier-Stokes and Darcy flow with a pseudo-first-order reaction was used to fit the experimental data. Simulated reactor performance was sensitive to changes in k and the height of the nanospring mat. Permeabilities lower than 10~(-8) cm~2 practically eliminated convective flow through the nanosprings, and substantially decreased conversion. Increasing the height of the mat increased conversion in simulations, but requires more enzymes and could cause sealing issues if grown above channel walls. Preliminary results indicate that in situ regeneration by reduction with DTT and incubation with SPDP-modified β-ga-lactosidase is possible. Nanosprings provide high solvent-accessible surface area with good permeability and mechanical stability, can be patterned into existing microdevices, and are amenable to immobilization of biomolecules. Nanosprings offer a novel and useful support for enzymatic microreactors, biosensors, and lab-on-chip devices.
机译：描述了使用二氧化硅（SiO2）纳米弹簧作为连续微反应器中固定化酶的载体。将纳米弹簧垫（2.2 cm〜2 x 60μm厚）用y-氨基丙基三乙氧基硅烷官能化，然后用N-琥珀酰亚胺基-3-（2-吡啶基二硫代）-丙酸酯（SPDP）和二硫苏糖醇（DTT）处理以产生表面硫醇（SH）组。来自米曲霉的SPDP修饰的β-半乳糖苷酶通过可逆的二硫键固定在硫醇化的纳米弹簧上。将涂有酶的纳米弹簧垫放入175μm高的微通道中，该垫部分遮盖通道。测量了在各种底物流速和浓度下邻硝基苯基β-D-半乳糖基吡喃糖苷水解的动力学和稳态转化率。通过纳米弹簧观察到大量流动，达西渗透率k≈3 x 10〜（-6）cm〜2。一个简单的单参数数值模型将Navier-Stokes和Darcy流与拟一级反应耦合在一起，用于拟合实验数据。模拟的反应堆性能对k和纳米弹簧垫高度的变化很敏感。低于10〜（-8）cm〜2的磁导率实际上消除了通过纳米弹簧的对流，并大大降低了转化率。增加垫子的高度会增加模拟中的转化率，但需要更多的酶，并且如果在通道壁上方生长，则可能导致密封问题。初步结果表明，通过DTT还原和SPDP修饰的β-ga-乳糖苷酶进行原位再生是可能的。纳米弹簧可提供高溶剂可及的表面积，并具有良好的渗透性和机械稳定性，可被图案化到现有的微设备中，并适合生物分子的固定化。纳米弹簧为酶促微反应器，生物传感器和芯片实验室设备提供了新颖而有用的支持。

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