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Enzyme immobilization inside the porous wood structure: a natural scaffold for continuous-flow biocatalysis.

ABSTRACT: Enzymes are often immobilized on solid supports to enable their recovery from reaction solutions, facilitate their reuse and hence increase cost-effectiveness in their application. Immobilized enzymes may even be used for flow-through applications in continuous processes. However, the synthesis of traditional immobilization scaffolds and immobilization techniques lack sustainability as they are often based on fuel-based materials and tedious synthesis- and immobilization approaches. Here, we present the natural material wood as a green alternative for enzyme immobilization. Its natural structure provides a mechanically stable porous scaffold with a high inner surface area that allows for directional flow-through of liquids. Enzymes were immobilized by nanoparticle-mediated adsorption, a simple, versatile and completely water-based process. The resulting wood-enzyme hybrids were intensely investigated for the model enzyme laccase. Reaction kinetics, as well as catalytic activities at various pH-values, temperatures, and ionic strengths were determined. The wood-enzyme hybrids could quickly and completely be removed from the reaction solution. Hence, they allow for multifold reusability. We show a series of 25 consecutive reaction cycles with a remaining activity in the last cycle of 90% of the maximal activity. Moreover, the anisotropic porosity of wood enabled the application of the hybrid material as a biocatalytic flow-through reactor. Flow-rate dependent productivity of a single-enzyme reaction was determined. Moreover, we show a two-step reaction cascade in continuous flow by the immobilization of the enzymes glucose oxidase and horseradish peroxidase. Therefore, the natural material wood proved to be a promising material for application in continuous-flow biocatalysis.

SUBMITTER: Goldhahn C

PROVIDER: S-EPMC9054300 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Enzyme immobilization inside the porous wood structure: a natural scaffold for continuous-flow biocatalysis.

Goldhahn Christian C Taut Josef A JA Schubert Mark M Burgert Ingo I Chanana Munish M

RSC advances 20200501 35

Enzymes are often immobilized on solid supports to enable their recovery from reaction solutions, facilitate their reuse and hence increase cost-effectiveness in their application. Immobilized enzymes may even be used for flow-through applications in continuous processes. However, the synthesis of traditional immobilization scaffolds and immobilization techniques lack sustainability as they are often based on fuel-based materials and tedious synthesis- and immobilization approaches. Here, we pre ...[more]

PMID: 35517771

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Project description:Although many different methods are known for the immobilization of enzymes on solid supports for use in flow-through applications as enzyme reactors, the reproducible immobilization of predetermined amounts of catalytically active enzyme molecules remains challenging. This challenge was tackled using a macro- and mesoporous silica monolith as a support and dendronized polymer-enzyme conjugates. The conjugates were first prepared in an aqueous solution by covalently linking enzyme molecules and either horseradish peroxidase (HRP) or bovine carbonic anhydrase (BCA) along the chains of a water-soluble second-generation dendronized polymer using an established procedure. The obtained conjugates are stable biohybrid structures in which the linking unit between the dendronized polymer and each enzyme molecule is a bisaryl hydrazone (BAH) bond. Quantitative and reproducible enzyme immobilization inside the monolith is possible by simply adding a defined volume of a conjugate solution of a defined enzyme concentration to a dry monolith piece of the desired size. In that way, (i) the entire volume of the conjugate solution is taken up by the monolith piece due to capillary forces and (ii) all conjugates of the added conjugate solution remain stably adsorbed (immobilized) noncovalently without detectable leakage from the monolith piece. The observed flow-through activity of the resulting enzyme reactors was directly proportional to the amount of conjugate used for the reactor preparation. With conjugate solutions consisting of defined amounts of both types of conjugates, the controlled coimmobilization of the two enzymes, namely, BCA and HRP, was shown to be possible in a simple way. Different stability tests of the enzyme reactors were carried out. Finally, the enzyme reactors were applied to the catalysis of a two-enzyme cascade reaction in two types of enzymatic flow-through reactor systems with either coimmobilized or sequentially immobilized BCA and HRP. Depending on the composition of the substrate solution that was pumped through the two types of enzyme reactor systems, the coimmobilized enzymes performed significantly better than the sequentially immobilized ones. This difference, however, is not due to a molecular proximity effect with regard to the enzymes but rather originates from the kinetic features of the cascade reaction used. Overall, the method developed for the controllable and reproducible immobilization of enzymes in the macro- and mesoporous silica monolith offers many possibilities for systematic investigations of immobilized enzymes in enzymatic flow-through reactors, potentially for any type of enzyme.

Project description:In recent years, 3D printing has emerged in the field of chemical engineering as a powerful manufacturing technique to rapidly design and produce tailor-made reaction equipment. In fact, reactors with complex internal geometries can be easily fabricated, optimized and interchanged in order to respond to precise process needs, such as improved mixing and increased surface area. These advantages make them interesting especially for catalytic applications, since customized structured bed reactors can be easily produced. 3D printing applications are not limited to reactor design, it is also possible to realize functional low cost alternatives to analytical equipment that can be used to increase the level of process understanding while keeping the investment costs low. In this work, in-house designed ceramic structured inserts printed via vat photopolymerization (VPP) are presented and characterized. The flow behavior inside these inserts was determined with residence time distribution (RTD) experiments enabled by in-house designed and 3D printed inline photometric flow cells. As a proof of concept, these structured inserts were fitted in an HPLC column to serve as solid inorganic supports for the immobilization of the enzyme Phenolic acid Decarboxylase (bsPAD), which catalyzes the decarboxylation of cinnamic acids. The conversion of coumaric acid to vinylphenol was chosen as a model system to prove the implementation of these engineered inserts in a continuous biocatalytic application with high product yield and process stability. The setup was further automated in order to quickly identify the optimum operating conditions via a Design of Experiments (DoE) approach. The use of a systematic optimization, together with the adaptability of 3D printed equipment to the process requirements, render the presented approach highly promising for a more feasible implementation of biocatalysts in continuous industrial processes. Graphical abstract.Supplementary informationThe online version contains supplementary material available at 10.1007/s41981-021-00163-4.

Dataset Information

Enzyme immobilization inside the porous wood structure: a natural scaffold for continuous-flow biocatalysis.

Publications

Enzyme immobilization inside the porous wood structure: a natural scaffold for continuous-flow biocatalysis.

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