*4.4. Cross-Linked Enzyme Aggregates (CLEAs)*

The addition of salts, water miscible organic solvents, or non-ionic polymers to protein solution induces their precipitation as physical protein aggregates, held together by noncovalent bonding. Enzyme tertiary structure can be maintained in the aggregates. Note this is also the traditional procedure for protein purification (in particular see in Section 3 the discussion about salting-out processes) [166]. Subsequent cross-linking of these physical aggregates renders them permanently insoluble while maintaining their pre-organized structure (Figure 9). Covalent binding or cross-linking of enzyme to enzyme to form aggregates is commonly referred to as CLEAs. CLEAs constitute a simple way of enzyme immobilization which is carrier-free and can be used in any reaction medium [22,167,168]. One more advantage of CLEAs is that they can be formed starting from crude enzyme preparations and do not require highly purified enzymes. Typical cross-linking agents are GA, ethylene glycol diglycidyl ether (EGDGE), and dextran, but also polyethyleneimine (PEI) that can generate covalent bonds between two or more biomolecules in the system through the enzyme amino and/or carboxyl surface groups [169]. FTIR analysis of CLEAs highlighted changes in the secondary structure of enzymes, with an increase in β-sheet and α-helix components and a decrease in β-turns compared to free enzymes, showing the ability of CLEAs to stabilize enzymes [169–171]. β α β

**Figure 9.** (**A**) formation and redissolution of enzyme aggregates, (**B**) stable CLEA preparation.

A wide variety of enzymes, including hydrolases, oxidoreductases, lyases, and transferases have been used as CLEAs especially for organic synthesis [172,173], displaying enhanced stability under various conditions: protection against organic solvents [174], enhanced thermostability [175], reduced substrate inhibition [176,177], enhanced stability in acidic or alkaline conditions [178,179] or under storage [180]. Magnetic CLEAs combined the advantages of CLEAs and covalent immobilization onto Fe3O<sup>4</sup> magnetic particles and may show increased stability to changes of pH and temperature [181,182]. Enzyme cascade can also be combined as CLEAs showing enhanced thermal and pH stability [183,184].

Despite a high enzyme concentration per unit volume, decrease in activity is often observed using CLEAs. Diffusional limitation of mass transfer is one of the main issues. Alteration of the enzyme tertiary structure by one of the components required for CLEA preparation or steric hindrance within the aggregate of high particle size hampering inner enzyme activity can also explain the decrease in activity. Hence, the choice of precipitant, cross-linker, and ratio of cross-linker protein is a critical step for maintaining required flexibility of the structure for high activity [185]. These parameters strongly depend on the enzyme, making the CLEA technology delicate [174].
