Enzymes are versatile catalysts in the laboratory and on an industrial scale. To broaden their applicability in the laboratory and to ensure their (re)use in manufacturing the stability of enzymes can often require improvement. Immobilisation can address the issue of enzymatic instability. Immobilisation can also help to enable the employment of enzymes in different solvents, at extremes of pH and temperature and exceptionally high substrate concentrations. At the same time substrate-specificity, enantioselectivity and reactivity can be modified. However, most often the molecular and physical-chemical bases of these phenomena have not been elucidated yet. This tutorial review focuses on the understanding of enzyme immobilisation.

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A three-dimensional, mesoporous, silicate containing zirconium, Zr-TUD-1, was synthesized by a direct hydrothermal treatment method with triethanolamine as a complexing and templating reagent to ensure that zirconium was incorporated as isolated atoms. The mesoporosity of Zr-TUD-1 was confirmed by X-ray diffraction (XRD), N(2) sorption and high-resolution transmission electron micrograph (HR-TEM) studies. The nature and strength of the Lewis acid sites present in Zr-TUD-1 were evaluated by FTIR studies of pyridine adsorption and temperature-programmed desorption of ammonia. FTIR, X-ray photoelectron spectroscopic (XPS) and UV/Vis spectroscopic studies showed that, at Si/Zr ratios of 25 and higher, all the zirconium was tetrahedrally incorporated into the mesoporous framework, while at low Si/Zr ratios, a small part of the zirconium was present as ZrO(2) nanoparticles. Zr-TUD-1 is a Lewis acidic, stable and recyclable catalyst for the Meerwein-Ponndorf-Verley (MPV) reaction and for the Prins reaction.

The production of renewable chemicals is gaining attention over the past few years. The natural resources from which they can be derived in a sustainable way are most abundant in sugars, cellulose and hemicellulose. These highly functionalized molecules need to be de-functionalized in order to be feedstocks for the chemical industry. A fundamentally different approach to chemistry thus becomes necessary, since the traditionally employed oil-based chemicals normally lack functionality. This new chemical toolbox needs to be designed to guarantee the demands of future generations at a reasonable price. The surplus of functionality in sugars and glycerol consists of alcohol groups. To yield suitable renewable chemicals these natural products need to be defunctionalized by means of dehydroxylation. Here we review the possible approaches and evaluate them from a fundamental chemical aspect.

Biocatalysis is one of the most promising technologies for the sustainable synthesis of molecules for pharmaceutical, biotechnological and industrial purposes.

Relatively simple considerations govern the construction of functional hybrid PKSs. Fusion sites should be chosen either in the surface-accessible linker regions between enzymatic domains, as previously revealed, or just inside the conserved margins of domains. The interaction of an ACP domain with the adjacent KS domain, whether on the same polyketide or not, is of particular importance, both through conservation of appropriate protein-protein interactions, and through optimising molecular recognition of the altered polyketide chain in the key transfer of the acyl chain from the ACP of one module to the KS of the downstream module.

Water is the liquid of life. Nature has therefore evolved countless enzymes that catalyse the addition of water to C=C bonds, isolated or conjugated. These reactions are regio- and enantioselective, they are part of primary metabolism as well as the secondary metabolism. The enzymes that catalyse these reactions (hydratases or hydro-lyases) are applied industrially in selected cases. However, they are not generally used in the laboratory although they outperform all currently available catalytic chemical methodologies. This feature article highlights the potential that hydratases have for chemistry compared to the acid catalysed addition of water.

Multiple versions of the DEBS 1-TE gene, which encodes a truncated bimodular polyketide synthase (PKS) derived from the erythromycin-producing PKS, were created by replacing the DNA encoding the ketoreductase (KR) domain in the second extension module by either of two synthetic oligonucleotide linkers. This made available a total of nine unique restriction sites for engineering. The DNA for donor "reductive loops," which are sets of contiguous domains comprising either KR or KR and dehydratase (DH), or KR, DH and enoylreductase (ER) domains, was cloned from selected modules of five natural PKS multienzymes and spliced into module 2 of DEBS 1-TE using alternative polylinker sites. The resulting hybrid PKSs were tested for triketide production in vivo. Most of the hybrid multienzymes were active, vindicating the treatment of the reductive loop as a single structural unit, but yields were dependent on the restriction sites used. Further, different donor reductive loops worked optimally with different splice sites. For those reductive loops comprising DH, ER and KR domains, premature TE-catalysed release of partially reduced intermediates was sometimes seen, which provided further insight into the overall stereochemistry of reduction in those modules. Analysis of loops containing KR only, which should generate stereocentres at both C-2 and C-3, revealed that the 3-hydroxy configuration (but not the 2-methyl configuration) could be altered by appropriate choice of a donor loop. The successful swapping of reductive loops provides an interesting parallel to a recently suggested pathway for the natural evolution of modular PKSs by recombination.