4.1.4. Adsorption Versus Multipoint Attachment

Adsorption of proteins on surfaces is a straightforward protocol that is susceptible to maintain high enzyme activity, but it may induce enzyme leaching. Covalent attachment fixes the enzyme on the surface, thus preventing enzyme leaching, but must be carefully considered because the mode of attachment can greatly affect the protein structure, hence both stability and activity. Different protocols can be used to attach an enzyme covalently to a support [20]. The most widely used methods are maleimide and carbodiimide coupling, amine aldehyde condensation and various click chemistry reactions. They allow covalent coupling between one available functional group naturally available or engineered on the surface of the enzyme and a complementary function created on the support where the enzyme is immobilized [20]. A spacer can be added to induce some flexibility required for the activity [119]. Multipoint attachment has been widely suggested to be more suitable than one-point attachment for increased stability because of reduced dynamics of the protein [120–122]. On the other hand, a multipoint attachment is challenging, and both the reactive groups on the support and on the enzyme must be carefully chosen to prevent steric hindrance. Furthermore, the decrease in the essential motion of the folded enzyme state required for catalysis often affects the activity. Stabilization factors more than 1000 were for example reported for formate dehydrogenase or alcohol dehydrogenase after immobilization on activated glyoxyl-agarose, while activity decreased by 50 and 10%, respectively [123].
