Enzyme immobilization on silicate glass through simple adsorption of dendronized polymer–enzyme conjugates for localized enzymatic cascade reactions

Author(s): Andreas Küchler ¹ ^, ² ^, ³ ^, ⁴ ^, ⁵ , Jozef Adamcik ⁶ ^, ⁷ ^, ⁸ ^, ³ ^, ⁹ , Raffaele Mezzenga ⁶ ^, ⁷ ^, ⁸ ^, ³ ^, ⁹ , A. Dieter Schlüter ¹ ^, ² ^, ³ ^, ⁴ ^, ⁵ , Peter Walde ¹ ^, ² ^, ³ ^, ⁴ ^, ⁵

Publication date (Electronic): 2015

Journal: RSC Advances

Publisher: Royal Society of Chemistry (RSC)

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Abstract

Conjugation of enzymes to a dendronized polymer via bis-aryl hydrazone bonds enables simple and stable enzyme immobilisation on unmodified glass.

Abstract

A methacrylate based, water soluble, polycationic second generation dendronized polymer (denpol de-PG2) was used for the preparation of de-PG2–enzyme conjugates. Aspergillus sp. glucose oxidase (GOD) and horseradish peroxidase isoenzyme C (HRP) were covalently bound to the denpol, using a UV/vis traceable bis-aryl hydrazone (BAH) linker, to form two different de-PG2–BAH–enzyme hybrid structures, each carrying several copies of either GOD or HRP on a single denpol chain (on average ≈ 50 GOD or 108 HRP bound per denpol chain). In addition, a conjugate with several copies of both types of enzymes on the same polymer chain was synthesized (≈25 GOD and 78 HRP). These denpol–BAH–enzyme conjugates were found to be useful for the immobilization of the enzymes on unmodified silicate glass surfaces via simple adsorption of the conjugates from solution in one single step. The adsorbed conjugates strongly adhered to the glass surface due to multiple interactions between the conjugates and the surface. The conjugate adsorption was characterized with the transmission interferometric adsorption sensor (TInAS) and by AFM imaging, which showed formation of a homogenous thin layer of the conjugates. Additionally, the catalytic activity and stability of the immobilized enzymes were determined and the conjugates were used for the simple fabrication of enzymatic flow reactors for catalyzing a cascade reaction which involved both enzymes, either through a sequential immobilization of the two enzymes, or through a co-immobilization. In both cases, the two enzymes remained highly active during continuous operation at room temperature for at least several hours without any desorption of the enzymes from the surface. Overall, the methodology presented can be considered as a promising platform for a desired (co-)localization of active enzymes on solid supports.

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Modifying enzyme activity and selectivity by immobilization.

Rafael C Rodrigues, Claudia Ortiz, Angel Berenguer-Murcia … (2013)

Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.