Enzyme immobilisation: fundamentals and application

Ulf Hanefeld

Professor Ulf Hanefeld was born in 1966 in Köln, Germany, and grew up in then (West) Berlin and London. In 1993 he received his PhD from the Georg-August-Universität zu Göttingen, having performed the research both in Göttingen (Prof. H. Laatsch) and Seattle (Prof. H. G. Floss). After postdoctoral years with Prof. C. W. Rees (Imperial College London), Prof. J. Staunton (Cambridge) and Prof. J. J. Heijnen and Dr A. J. J. Straathof (TU Delft), he received a fellowship from the Royal Netherlands Academy of Arts and Sciences (KNAW). He rose through the ranks at the Technische Universiteit Delft and his research in Delft focuses on enzymes, enzyme immobilisation and heterogeneous catalysis in organic synthesis.

Linqiu Cao

Dr Linqiu Cao studied organic chemistry and biotechnology at the Beijing Institute of Technology (BIT), and received his PhD from University of Stuttgart, Germany (Prof. Rolf D. Schmid). He then joined the group of Prof. Roger Sheldon at Delft University of Technology, the Netherlands, where he pioneered the technology for cross-linked enzyme aggregates (CLEAs). In 2000, he joined Avantium Technologies in Amsterdam, the Netherlands. Later he moved to the former DMV in Veghel, the Netherlands, where he finished writing his book “Carrier-Bound Immobilized Enzymes – Principle, Applications and Design” published by Wiley-VCH, Germany, in 2005. Currently, he is working in the newly merged dairy company Frieslandcampina, as senior research scientist and specialist for enzymes and biocatalysis.

Edmond Magner

Professor Edmond Magner studied at University College Cork (BSc) and the University of Rochester (PhD). After postdoctoral work at Imperial College and MIT, he developed electrochemical biosensors for glucose, ketones and haemoglobin at MediSense, Inc and Abbott Laboratories. He is Professor of Electrochemistry in the Department of Chemical and Environmental Sciences and a member of the Materials and Surface Science Institute at the University of Limerick. His research focuses on the immobilisation and characterisation of enzymes for applications in biocatalysis, biomaterials, biosensors and biofuel cells.

Mankind has used enzymes for many millennia in food processing. The successful application of enzymes in synthesis and industrial production of chemicals is a much more recent development. This success is due to the ever increasing number of enzymes and with them their application in a wide range of processes. However, to turn enzyme activity into industrial application, it is usually necessary to immobilise the enzymes. To date a plethora of methods have been developed for the immobilisation of enzymes. Each of these methods can influence the enzyme and its properties, i.e. its stability, activity and selectivity. The methods of enzyme immobilisation, their effect on the enzymes and their application are taught in seven tutorial reviews. Nine in depth review articles then address topics of particular importance. One review has a look at the opposite strategy, immobilising the substrates while using dissolved enzymes.

Immobilisation of an enzyme usually leads to a decrease in catalytic activity. The review by Fernández-Lafuente and co-workers focuses on an aspect of immobilisation that has received relatively little attention; how immobilisation can result in an improvement of the activity, selectivity or specificity of an enzyme (DOI: 10.1039/c2cs35231a).

The immobilisation of enzymes on mesoporous silicates has been extensively described. Magner's review (DOI: 10.1039/c2cs35450k) describes the use of these materials as enzyme supports, focusing on the ability to tailor the surface functionalisation of the silicates to suit a specific enzyme. The materials are assessed in terms of the properties required for use in large scale reactors.

This review is matched and supplemented by the review from the group of Hartmann (DOI: 10.1039/c3cs60021a). They compare different methods of immobilisation and show strengths and weaknesses of the porous silicates relative to other immobilisation methods.

The review by Secundo (DOI: 10.1039/c3cs35495d) describes a range of spectroscopic techniques to investigate the conformation of immobilised enzymes. The review then discusses how these techniques are utilised to examine protein loading, the interactions between the protein and the support, and the effect of these interactions on the structure of the enzyme.

The review by Hilterhaus and Liese (DOI: 10.1039/c3cs35511j) discusses the process of immobilisation in terms of the choice of carrier, evaluation of mass transport limitations, yield of immobilisation and the rate of deactivation of the biocatalyst. These factors are described in the context of selecting an optimal reactor system.

Gardossi and co-workers (DOI: 10.1039/c3cs35464d) describe the steps required in the immobilisation of an enzyme for a biocatalytic process, emphasising the need to develop a tailored solution for each specific enzyme. The necessity of correctly describing the conditions used for immobilisation, a particularly welcome contribution, is outlined and emphasised.

The review by Sheldon and van Pelt (DOI: 10.1039/c3cs60075k) completes the series of tutorial reviews with an overview of the use of immobilised enzymes followed by a focus on the use of cross linked enzyme aggregates (CLEAs).

The review articles then give detailed insights into topics of great importance. The special properties of lipases in organic solvents are discussed in depth by Adlercreutz (DOI: 10.1039/c3cs35446f). In particular the improved activity of the enzymes described is remarkable. His treatment forms a sound basis for the application directed review on lipases by Ansorge-Schumacher and Thum (DOI: 10.1039/c3cs35484a). Their report on the application of lipases for the cosmetics industry links lipases to everyday life and shows how closely academic and industrial research are interlinked.

The most important enzyme improvement is the stabilisation of the biocatalyst. A separate review by Bommarius and Paye focuses on this key point for the successful industrial application of enzymes (DOI: 10.1039/c3cs60137d).

Enzyme stabilisation is also examined from a very different angle, a single class of enzymes, the hydroxynitrile lyases. Hanefeld gives a critical comparison of all immobilisation methods applied to these enzymes (DOI: 10.1039/c3cs35491a).

A discussion of enzyme immobilisation from the point of view of the different feedstocks is given by Franssen, Sanders and co-workers (DOI: 10.1039/c3cs00004d). They report all recent application for the conversion of biomass. In this they describe work that builds on glucose isomerisation. The very thorough treatment of the glucose isomerisation process by DiCosimo and co-workers (DOI: 10.1039/c3cs35506c) is an excellent example of a fully optimised enzymatic process on a large scale. It is followed by other processes on still smaller scales but with equal potential.

An entirely new approach for the large scale conversion of C–O bonds by biocatalysts is introduced by Bühler and co-workers (DOI: 10.1039/c3cs60011d). Knowledge gained from the use of whole cells in ancient food processing is applied to co-factor regeneration and straightforward redox reactions without the common problems with oxidants or reducing equivalents.

By introducing elaborate nano-architecture Ariga and co-workers build up enzyme carrier structures that allow a multitude of different applications (DOI: 10.1039/c2cs35475f). Sensors are constructed and co-factors recycled in a gentle fashion, on a tiny scale, tickling the mind for further inventions.

When looking at a topic it is always good to lean back and take a completely different point of view. In this way it becomes easier to avoid tunnel vision. The contribution by Flitsch, Eyers and co-workers (DOI: 10.1039/c3cs60018a) does this beautifully, demonstrating how the immobilisation of the substrate gives new insight into the functioning of enzymes and their interaction with the substrate and the carrier.

It was a pleasure to assemble this collection of reviews for this themed issue on immobilised enzymes. We hope that you, the reader, enjoy this collection and find it stimulating for teaching and research.

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