Lipase immobilization is frequently used for altering the catalytic
properties of these industrially used enzymes. Many lipases bind strongly to
hydrophobic surfaces where they undergo interfacial activation. Candida
antarctica lipase B (CalB), one of the most commonly used
biocatalysts, is frequently discussed as an atypical lipase lacking interfacial
activation. Here we show that CalB displays an enhanced catalytic rate for
large, bulky substrates when adsorbed to a hydrophobic interface composed of
densely packed alkyl chains. We attribute this increased activity of more than
7-fold to a conformational change that yields a more open active site. This
hypothesis is supported by molecular dynamics simulations that show a high
mobility for a small ‘lid’ (helix α5) close to the
active site. Molecular docking calculations confirm that a highly open
conformation of this helix is required for binding large, bulky substrates and
that this conformation is favored in a hydrophobic environment. Taken together,
our combined approach provides clear evidence for the interfacial activation of
CalB on highly hydrophobic surfaces. In contrast to other lipases, however, the
conformational change only affects large, bulky substrates, leading to the
conclusion that CalB acts like an esterase for small substrates and as a lipase
for substrates with large alcohol substituents.