3.2.1. Chemical Modification of Enzymes

Chemical protein modification can be achieved either by modification of one sort of amino acid on the protein surface or by polyfunctional modification using reticulating agents or by conjugation to water-soluble polymers. One widely used method consists in reticulation through cross linking via glutaraldehyde (GA) that increases the protein rigidity and mimics disulfide physiological bonds or salt bridges [25]. Activity is however very often affected. Stabilization of proteins through cross-linked-enzyme-aggregates (CLEAs) will be further discussed below in Section 4 [67,68]. Conjugation through watersoluble polymers such as PEG, chitosan, alginate, dextran, etc. has also been widely reported [38,69,70]. Those polymers present either one functionality or are bifunctional to enable reaction with N- or C-terminal or with one individual amino-acid residue on the protein. Increase in the half-life of proteins is frequently observed [71,72]. Other polymers responding to temperature stimuli can tailor the temperature dependence of enzyme stability [73]. Two different methods can be carried out to synthesize the bioconjugate. In

the "grafting to" strategy, the polymer is first synthetized; then, an end-group functionality is attached to one amino-acid on the protein surface. As an example, a mono-PEGylated arginase was constructed by linkage of PEG-maleimide to a cysteine residue on the enzyme surface [74]. The protein was protected against proteases thanks to the shielding effect of PEG, allowing the modified arginase to operate as a promising anticancer drug candidate. In the "grafting from" method, radical polymerization occurs at sites attached to the protein. A recent work describes the ligation of Atom Transfer Radical Polymerization (ATRP) initiators to lysine residues on the surface of a laccase [75]. The polymer-enzyme hybrid obtained by this "grafting-from" procedure showed enhanced solvent and thermal stability, as well as a clear enhancement of activity in a much wider pH range than the free enzyme. Combination of PEGylation and chemical modification by GA can also be efficient [76]. GOx was first PEGylated to provide steric protection; then, it was crosslinked with GA which stabilized the tertiary and quaternary structures. Intermolecular crosslinking-induced aggregation was prevented by the PEG shield. The GA-modified PEGylated GOx retained 73% of activity after 4 weeks at 37 ◦C against 8.2% for the control (Figure 4).

**Figure 4.** PEGylation of GOx (**B**) followed by chemical modification (**C**) enhances enzyme stability, while preventing intermolecular crosslinking contrary to direct cross-linking (**A**). Reprinted with authorization from [76].

#### 3.2.2. Enzyme Engineering

Protein engineering is another relevant strategy to enhance stability [4,77,78] (Figure 5). Rational design takes benefit of structures and sequences of already known stable proteins. Through identification of amino acids which are assumed to participate in (de)stabilization, new variants of proteins of interest are created through site-directed mutagenesis [79]. As an example, asparagine is a thermolabile residue prone to deamination which can be mutated into threonine or isoleucine, with similar geometry but more thermostable. Replacement of lysines (or histidines) by arginine residues increases thermostability by increasing intramolecular or inter-subunit salt bridges. Otherwise, we will discuss further in this review the interest in the screening of biodiversity to search for more stable enzymes such as in extremophilic organisms. However, because the production of extremophile enzymes may be delicate, genes from hyperthermophiles can be implemented into suitable mesophilic hosts, coupling advantages of high productivity and high thermostability [22,80]. Use of directed evolution to design more stable enzymes is now common. With this method, mutant libraries are created by random changes, and the most promising variants are subjected to further rounds of evolution [81–85]. Rational approach based on molecular

dynamic (MD) and QM/MM simulations may help in identifying and redesigning variants with increased stability [86].

**Figure 5.** Protein engineering methods to improve enzyme stability. Reprinted with permission from [87].
