Enzyme immobilisation in biocatalysis: why, what and how Enzymes are natural sustainable catalysts, biocompatible, biodegradable, and derived from renewable resources. Enzymatic processes are conducted under mild conditions, with high rates and selectivities. Enzymes are used in green and sustainable chemical manufacturing, particularly in the synthesis of pharmaceuticals, fragrances, vitamins, and other fine chemicals. Advances in biotechnology and protein engineering have enabled the production of enzymes at commercially acceptable prices and the manipulation of their properties. However, industrial application of enzymes is often hindered by a lack of long-term operational stability and difficulty in recovery and reuse. Immobilisation of enzymes can overcome these issues, enabling their cost-effective use in continuous operations. Immobilisation also facilitates the efficient recovery and reuse of enzymes, enhancing their stability and reducing allergenicity. Immobilisation methods include adsorption on a carrier, encapsulation in a carrier, and cross-linking. The choice of immobilisation method depends on the reactor configuration and downstream processing. Recent developments include the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs). The terminology of immobilisation includes immobilisation yield, efficiency, activity recovery, and enzyme loading. The physical properties of immobilised enzymes, such as particle size, density, hydrophobicity, and mechanical robustness, are important considerations. Immobilisation on prefabricated supports, such as synthetic organic polymers, natural polymers, and inorganic polymers, has various advantages and disadvantages. The use of smart polymers, such as thermoresponsive polymers, allows for the preparation of 'smart' immobilised enzymes. Entrapment methods include silica sol gels, hydrogels, and other matrices. Carrier-free immobilisation by cross-linking, such as cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs), offers advantages such as high stability and low production costs. CLEAs are particularly useful for industrial applications, as they can be prepared from crude enzyme preparations and exhibit high catalyst productivities and ease of recovery and recycling. CLEAs have been successfully prepared from various enzymes, including lipases, oxidoreductases, and lyases, and have shown improved stability and activity. Combi-CLEAs, which combine two or more enzymes in a single immobilisation, offer further benefits such as enhanced enantioselectivity and improved reaction efficiency. Enzyme-immobilised microchannel reactors offer process intensification, with advantages such as rapid mass and heat transfer and large surface area to volume ratios. These reactors are particularly useful for conducting catalytic reactions in microreactors containing enzyme-immobilised membranes. Overall, enzyme immobilisation is a key enabling technology for the practical and commercial viability of biocatalysis in the production of green and sustainable chemicals.Enzyme immobilisation in biocatalysis: why, what and how Enzymes are natural sustainable catalysts, biocompatible, biodegradable, and derived from renewable resources. Enzymatic processes are conducted under mild conditions, with high rates and selectivities. Enzymes are used in green and sustainable chemical manufacturing, particularly in the synthesis of pharmaceuticals, fragrances, vitamins, and other fine chemicals. Advances in biotechnology and protein engineering have enabled the production of enzymes at commercially acceptable prices and the manipulation of their properties. However, industrial application of enzymes is often hindered by a lack of long-term operational stability and difficulty in recovery and reuse. Immobilisation of enzymes can overcome these issues, enabling their cost-effective use in continuous operations. Immobilisation also facilitates the efficient recovery and reuse of enzymes, enhancing their stability and reducing allergenicity. Immobilisation methods include adsorption on a carrier, encapsulation in a carrier, and cross-linking. The choice of immobilisation method depends on the reactor configuration and downstream processing. Recent developments include the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs). The terminology of immobilisation includes immobilisation yield, efficiency, activity recovery, and enzyme loading. The physical properties of immobilised enzymes, such as particle size, density, hydrophobicity, and mechanical robustness, are important considerations. Immobilisation on prefabricated supports, such as synthetic organic polymers, natural polymers, and inorganic polymers, has various advantages and disadvantages. The use of smart polymers, such as thermoresponsive polymers, allows for the preparation of 'smart' immobilised enzymes. Entrapment methods include silica sol gels, hydrogels, and other matrices. Carrier-free immobilisation by cross-linking, such as cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs), offers advantages such as high stability and low production costs. CLEAs are particularly useful for industrial applications, as they can be prepared from crude enzyme preparations and exhibit high catalyst productivities and ease of recovery and recycling. CLEAs have been successfully prepared from various enzymes, including lipases, oxidoreductases, and lyases, and have shown improved stability and activity. Combi-CLEAs, which combine two or more enzymes in a single immobilisation, offer further benefits such as enhanced enantioselectivity and improved reaction efficiency. Enzyme-immobilised microchannel reactors offer process intensification, with advantages such as rapid mass and heat transfer and large surface area to volume ratios. These reactors are particularly useful for conducting catalytic reactions in microreactors containing enzyme-immobilised membranes. Overall, enzyme immobilisation is a key enabling technology for the practical and commercial viability of biocatalysis in the production of green and sustainable chemicals.