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首页> 外文期刊>Biotechnology Progress >Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment
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Epoxy sepabeads: a novel epoxy support for stabilization of industrial enzymes via very intense multipoint covalent attachment

机译：环氧Sepabeads：一种新型的环氧载体，可通过非常强烈的多点共价连接来稳定工业酶

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Sepabeads-EP(a new epoxy support )has been utilized to immobilize-stabilize the enzyme penicillin G acylase (PGA) via multipoint covalent attachment.These supports are very robust and suitable for industrial purposes.Also,the internal geometry of the support is composed by cylindrical proes surrounded by the convex surfaces (this offers a good geometrical congruence for reaction with the enzyme),and it has a very high superficial density of epoxy groups (around 100 #mu#mo/mL).These features should permit a very intense enzyme-support interaction.However,the final stability of the immobilized enzyme is strictly dependent on the immobilization protocol.By using conventional immobilization protocols (neutral pH values,nonblockage of the support) the stability of the immobilized enzyme was quite similar to that achieved using Eupergit C to immobilize the PGA.However,when using a more sophisticated three-step immobilization/stabilization/blockage procedure,the Sepabeads derivative was hundreds-fold more stable than Eupergit C derivatives.The protocol used was as follows:(i) the enzyme was first covalently immobilized under very mild experimental conditions;(ii) the already immobilized enzyme was further incubated under more drastic conditions in order to "facilitate" the formationof new covalent linkages between the immobilized enzyme moelcule and hte suport;(iii) the remaining epoxy groups of the support were blocked with very hydrophilic compounds to stop any additional interaction between the enzyme and the support.This third point was found to be criticalfor obtaining very stable enzymes:derivatives blocked with mercaptoethanol were much less stale than derivatives blocked with glycine or other amino acids.This was attributed to the better masking of the hydorphobicity of the support by the amino acids (having two charges).
机译：Sepabeads-EP（一种新型环氧载体）已被用于通过多点共价键固定化稳定青霉素G酰基转移酶（PGA）的酶。这些载体非常坚固耐用，适用于工业用途。此外，该载体的内部几何形状也很合理通过被凸形表面包围的圆柱孔（这为与酶的反应提供了良好的几何一致性），并且它具有很高的表观环氧基密度（约100＃mu＃mo / mL）。这些特征应允许强烈的酶-支持物相互作用。然而，固定化酶的最终稳定性严格取决于固定化方案。通过使用常规固定化方案（中性pH值，载体的无阻滞），固定化酶的稳定性与所达到的稳定性非常相似使用Eupergit C固定PGA。但是，当使用更复杂的三步固定/稳定/封闭步骤时，Sepabeads衍生物的抗性很强。 eds-fold比Eupergit C衍生物稳定。使用的方案如下：（i）首先在非常温和的实验条件下将酶共价固定;（ii）将已经固定的酶在更剧烈的条件下进一步温育，以便“促进了固定化酶分子与超溶剂之间新的共价键的形成;（iii）载体的其余环氧基被亲水性很强的化合物封闭，从而阻止了酶与载体之间的任何其他相互作用。对于获得非常稳定的酶至关重要：巯基乙醇保护的衍生物比甘氨酸或其他氨基酸保护的衍生物陈旧得多，这归因于氨基酸更好地掩盖了载体的疏水性（具有两个电荷）。

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