**3. Chemical modifications to stabilize enzymes**

Chemical modification of enzymes is a very common stabilization strategy. In fact, covalent modification by the cross-linking with glutaraldehyde reagent can stabilize almost any enzyme, protecting it from denaturing and other effects of the new solvent [27]. This finding led to the development of carrier-free enzyme immobilization methods (to be discussed in detail in Section 4).

Another popular chemical method involves covalent conjugation with an amphiphilic polymer polyethylene glycol (PEG) and is often referred to as enzyme PEGylation [28]. PEGylating permits binding of specific polymeric functional groups to the free amino groups on the enzyme, creating PEGylated biocatalyst soluble in organic solvents [19]. This method is especially useful for preparation of biopharmaceuticals with high stability and low antigenicity [29]. Additionally, reagent methoxymethyl-PEG (mPEG) bound to the enzyme horseradish peroxidase (HRP) can protect the protein from pH extremes and high temperatures, making HRP particularly useful in industrial and clinical biosensors [30].

Chemical alterations of the enzyme can introduce a new functional group for a covalent attachment or modify one of the reactive side chains. For instance, the treatment of *Candida rugosa* lipase with diethyl *p*-nitrophenyl phosphate modified one of the two reactive functionalities and resulted in a more selective lipase catalyzing a single reaction [31].

Chemical modification makes it possible to introduce and attach a new cofactor, which can in turn induce novel enzyme functions [32]. Lastly, some posttranslational enzyme modifications either *in vivo* or *in vitro* have been linked to an enhanced stability of enzymes [33]. For example, when DNA ligase from *Thermus scotoductus* was chemically adenylated, new irreversible covalent binding of the cofactor resulted in structural changes within the active site and overall protein compaction. As the result of this cofactor-induced conformational change, the enzyme gained increased resistance to thermoinactivation [34, 35].

As strategies for enzyme improvement continue to evolve, chemical modification has been rediscovered to become a robust complimentary approach to both protein engineering and immobilization [36].
