The biochemical reactions leading to ester synthesis in living organisms are very difficult to use in vitro. However, esters can be obtained through other enzyme-catalyzed reactions. In condensation reactions, or reverse hydrolysis, alcohol and acid molecules react to form the ester. In transesterification, existing esters are modified to produce new esters. The enzymes used for these reactions are various hydrolases: esterases, lipases, and proteases, lipases being the most useful. To minimize the hydrolysis of the produced esters, these reactions are often performed in low water environments, such as organic solvents, using solid or immobilized enzymes. These conditions have an impact on the catalytic activity of enzymes.

Enzyme-catalyzed esterification is very selective and performed in mild conditions, compared to chemical esterification. It can be applied to the modification of oils and fats, the production of chemicals such as nonionic surfactants, flavors, and fragrances, the production of chiral molecules, and the modification of natural compounds.