Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases
Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases
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DOI：
10.1016/j.aca.2005.12.066
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发表时间：
2006-03-30
影响因子：
6.2
通讯作者：
Brennan, JD
中科院分区：
文献类型：
--
作者：
Besanger, TR;Hodgson, RJ;Brennan, JD
我们小组最近报告了蛋白质掺杂整体硅胶柱在固定化酶反应器色谱中的应用，该色谱可以使用质谱法检测筛选混合物中存在的酶抑制剂。该酶通过包埋在由生物相容性溶胶-凝胶工艺路线制备的双峰介孔/大孔二氧化硅材料内来固定化。虽然此类柱被证明对于蛋白质-配体相互作用的筛选等应用很有用，但由于材料中大孔比例很高，大量包埋的蛋白质从柱中浸出。在这里，我们描述了一个详细的研究影响蛋白质掺杂的生物亲和柱的形态的因素，并证明，特定的pH值和浓度的聚（乙二醇）可以用来制备基本上介孔柱，保留超过80%的初始加载酶的活性和可访问的形式，但仍然保留足够的孔隙率，以允许压力驱动的流量在低μ L/min的范围。使用的酶γ-谷氨酰转肽酶（γ-GT），我们进一步评估的催化常数截留在毛细管柱与不同的二氧化硅形态的流量和背压的函数，使用酶反应器测定模式的酶。结果发现，酶的表观活性是最高的中孔柱，保留高水平的酶。在这样的柱中，由于k（cat）的增加和K-M的降低，酶活性随着在色谱活性测定期间产生的流速（从250至1000 nL/Mm）和背压（从500至2100 psi）的增加而增加了约2倍，在约100 ℃下从扩散控制切换到反应控制条件。2000 psi。这些结果表明，具有最小大孔体积（< 5%）的柱对于用于生物亲和和生物反应器层析的可溶性蛋白质的截留是有利的。(c)2006 Elsevier B. V.保留所有权利。
Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low mu L/min range. Using the enzyme gamma-glutamyl transpeptidase (gamma-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by similar to 2-fold with increases in both flowrate (from 250 to 1000 nL/Mm) and backpressure generated (from 500 to 2 100 psi) during the chromatographic activity assay owing to increases in k(cat) and decreases in K-M, switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (< 5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography. (c) 2006 Elsevier B.V. All rights reserved.