In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).

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The successful immobilization and stabilization of a nitrile hydratase in the form of a cross-linked enzyme aggregate (CLEA R ) is described. CLEAs were prepared by using ammonium sulfate as an aggregation agent followed by cross-linking with glutaraldehyde. The effect of different glutaraldehyde concentrations on the recovery of enzyme activity in the CLEA and enzyme leakage from the CLEA matrix was investigated. Although activity recovery was low (21%) the CLEA facilitates easy separation and recycling of the nitrile hydratase. It was also found that the nitrile hydratase CLEA had substantially increased storage stability as well as increased operational stability during exposure to high concentrations of acrylamide and acrylonitrile compared to that of the nitrile hydratase in the crude cell-free extract and whole cell formulation.

A one-pot bienzymatic cascade combining a hydroxynitrile lyase (Manihot esculenta, E.C. 4.1.2.10) and a nitrile hydratase (Nitriliruptor alkaliphilus, E.C. 4.2.1.84) for the synthesis of enantiopure aliphatic a-hydroxycarboxylic amides from aldehydes is described. Both enzymes were immobilised as cross-linked enzyme aggregates (CLEAs). Stability tests show that the nitrile hydratase CLEAs are sensitive to water-immiscible organic solvents as well as to aldehydes and hydrogen cyanide (HCN), but are remarkably stable and show useful activity in acidic aqueous environments of pH 4-5. The cascade reactions are consequently carried out by using a portionwise feed of HCN and moderate concentrations of aldehyde in acidic aqueous buffer to suppress the uncatalysed hydrocyanation background reaction. After optimisation, this method was used to synthesise five different kinds of aliphatic a-hydroxycarboxylic amides from the corresponding aldehydes with good yields and with enantiomeric purities comparable to those obtained for the a-hydroxynitriles in the microaqueous hydrocyanation using hydroxynitrile lyase and an excess of HCN.

The pilot-scale production of the peroxygenase from Agrocybe aegerita (r Aae UPO) is demonstrated. In a fed-batch fermentation of the recombinant Pichia pastoris, the enzyme was secreted into the culture medium to a final concentration of 0.29 g L –1 corresponding to 735 g of the peroxygenase in 2500 L of the fermentation broth after 6 days. Due to nonoptimized downstream processing, only 170 g of the enzyme has been isolated. The preparative usefulness of the so-obtained enzyme preparation has been demonstrated at a semipreparative scale (100 mL) as an example of the stereoselective hydroxylation of ethyl benzene. Using an adjusted H 2 O 2 feed rate, linear product formation was observed for 7 days, producing more than 5 g L –1 ( R )-1-phenyl ethanol. The biocatalyst performed more than 340.000 catalytic turnovers (942 g of the product per gram of r Aae UPO).