Enzyme Engineering for In Situ Immobilization

Abstract

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.

Keywords: biocatalyst; enzyme; immobilization; inclusion bodies; protein particles; recombinant enzyme; self-assembly.

Figure 1

General methods for in vitro immobilization. The crystal structure (1TCC) of lipase B from Candida antarctica is depicted.

Figure 2

In situ enzyme immobilization: (A) Different modes of protein assembly leading enzyme inclusions. Black shapes indicate the target enzyme; (B) Immobilization of target enzyme by genetic fusion of a membrane anchor domain. Production of a phage pore enables release of intracellular content while retaining a membrane vesicle with immobilized enzyme.

Figure 3

Schematic outlining in situ formation of polyhydroxyalkanoate (PHA) inclusions displaying enzymes. PhaA, β-ketothiolase; PhaB, acetoacetyl-CoA reductase; PhaC, PHA synthase; PHB, polyhydroxybutyrate.