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首页> 外文期刊>Biotechnology Progress >Development of a Thiol-Ene Based Screening Platform for Enzyme Immobilization Demonstrated Using Horseradish Peroxidase
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Development of a Thiol-Ene Based Screening Platform for Enzyme Immobilization Demonstrated Using Horseradish Peroxidase

机译：基于肌醇固定化的基于硫醇-NEE的筛选平台的研制使用辣根过氧化物酶证明

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Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster and more efficient screening, which we believe will result in a higher chance of discovery of optimal surface/enzyme interactions. The proposed system consists of a thiol-functional microplate prepared through fast photochemical curing of an off-stoichiometric thiol-ene (OSTE) mixture. Surface functionalization by thiol-ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT-FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho-HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed by fluorescence intensity (FI) measurements. Subsequently, Rho-HRP activity was measured directly on the surface. The broad range of utilized surface chemistries permits direct correlation of enzymatic activity to the surface functionality and improves the determination of promising enzyme-surface candidates. The results underline the high potential of this system as a screening platform for synergistic immobilization of enzymes onto thiol-ene polymer surfaces. (C) 2017 American Institute of Chemical Engineers
机译：在支撑表面上的酶的有效固定需要表面化学和特定酶之间的精确匹配。通常消耗多种实验中的常规树脂筛选的成功匹配通常会鉴定在测试单独的固定策略中的多个实验中。在这项研究中，我们提出了一种多功能的策略，主要扩大了单个通用平台中酶固定化的可能表面功能的数量。许多单独的表面化学品的组合并因此在一个模块化系统中固定方法允许更快，更有效的筛选，我们认为将导致发现最佳表面/酶相互作用的更高机会。所提出的系统由硫醇功能性微孔板组成，通过快速光化学固化来制备脱离化学计量硫醇-NEE（OSTE）混合物。通过硫醇-NEE化学（TEC）的表面官能化导致每个孔中的功能单层形成，而通过表面链转移自由基聚合（SCT-FRP）引入聚合物表面接枝。通过使用罗丹明标记的辣根过氧化物酶（RHO-HRP）作为模型酶来评价改性表面上的酶固定化，并且通过荧光强度（FI）测量来定性评估固定化酶的量。随后，直接测量Rho-HRP活性。广泛的利用表面化学方法允许直接相关的酶活性与表面官能团的相关性，并改善了促进酶表面候选物的测定。结果为该系统的高电位为筛选平台，用于将酶的协同固定到硫醇 - 烯醇聚合物表面上。 （c）2017美国化学工程师研究所

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