**2. Improving biocatalysis in organic solvents by additive approach**

Enzymes may be remarkable catalysts in biological systems where water is ubiquitous, but they are less suited for biotechnology where organic solvents are largely employed for chemical transformations. For example, the activity of intestinal proteases subtilisin and *a*-chymotrypsin is reduced 104 –105 times if the enzymes are transferred from aqueous to anhydrous octane media [12]. Knowing that water is essential for structural integrity of many biomolecules and seeing vast experimental evidence of decreased catalytic activity in non-aqueous solvents, scientists have been skeptical about using enzymes as industrial biocatalysts or about using water as a solvent for industrial applications due to undesired hydrolytic sidereactions [3]. However, these challenges were proved to be surmountable when new, improved properties of the enzymes in organic and ionic solvents were discovered several decades ago. In many cases, enzymes that had been stripped of their folded structure in a non-aqueous solution not only became more thermostable and easier to store (due to higher melting points), but also became capable of catalyzing new reactions, impossible in aqueous media. For instance, hydrolases, such as subtilisin, routinely accelerate hydrolysis in aqueous conditions, but in anhydrous solvent, they are capable of catalyzing transesterification reactions [12]. The possibility of using novel, industrially favored substrates as well as the possibility of controlling enzymatic activity and selectivity by finely tuned modifications of the solvent lead to the discovery of numerous approaches to stabilize the enzyme in a non-aqueous solvent [3]. Early trial and error experiments with lyophilized (freeze dried) enzyme powders and solvent additives resulted in the development of empirical strategies like addition of water or water-mimicking solvents and addition of salts for stabilizing biocatalysts. The additive approach currently employs the addition of wide variety of lyophilized chemical substances, also known as lyoprotectants, to the media and still appeals to scientists, as it incorporates the simplicity of use and high efficiency [13, 14].

### **2.1 Addition of water**

The lubricating effects of water on biocatalyst flexibility in organic systems were highlighted in multiple studies [12, 15–19]. For instance, in chymotrypsin activity trials, the amount of residual water retained on the enzyme after the addition of organic component correlated with the catalytic activity of the enzyme [12]. It was later determined that the addition of trace amounts of water, even if the enzyme have been unfolded in organic media, can remediate some of the activity loss: in the experiments with subtilisin Carlsberg suspended in organic solvents*,* increase in water content from 0 to 1% resulted in the increase of reaction rate 11-fold in isooctane and 50-fold in THF [18]. Moreover, hydration of organic solvent does not prevent the enzyme from acquiring novel properties valuable for synthetic and industrial applications. For example, adding 1% water to glycerol helped to retain the secondary structure of *a-*chymotrypsin similar to that in aqueous solvent, however, the enzyme stability at high pH was still much greater in 99% organic solvent over that in water [19]. More importantly, while the enzyme suspended in water was fully denatured after 1 min at 100°C, *a*-chymotrypsin in 99% glycerol retained 80% of its catalytic activity after incubation at 100°C for 10 h [19].

In recent years, water addition strategy has benefitted many promiscuous biocatalyzed synthesis reactions, such as Henry reaction, Michael addition, Mannich reaction, asymmetric aldol reactions, and others [14]. You can refer to the excellent review by Liang and Lin for the empirical data on yield increase in these reactions due to hydration.

It is important to note that while too few water molecules may be not enough to activate biocatalyst in organic solvent, too many water molecules may result in reduced substrate solubility or hydrolytic reactions side product [14].

#### **2.2 Addition of water mimics and lyoprotectants**

Just like water that is thought to lubricate the enzyme enhancing protein flexibility with its multiple hydrogen bonds, water-mimicking substances, such as glycerol, formamide, ethylene glycol and formic acid can provide similar hydrogen bonding, while avoiding unintended hydrolysis product [14, 20]. One of the early water mimic studies concluded that adding 0.1% ethylene glycol to the solvents with optimal water content of 0.2% can increase the activity and stereo-selectivity of *Candida cylindracea* lipase [21].

Since enzymes often have to be freeze-dried, it is important to ensure their stability during the long-term storage or temperature changes associated with thawing. The most common lyoprotectant up to date is trehalose sugar that helps to preserve enzyme structure and allows for industrial storage of biocatalysts [18].

Most recent reviews also list organic bases, crown ethers, surfactants, and salts as possible additives used to improve catalytic activity of enzymes in chemical synthesis [14]. For the purpose of this section, we will only cover the addition of salts and the role of ionic interactions in biocatalyst enhancement.

#### **2.3 Addition of salt**

In 1994, Khmelnitsky et al. discovered that lyophilization of an enzyme in a salty matrix prior to its suspension in organic media lead to a dramatic enhancement in the rate of catalyzed reaction [22]. In this study, 3750-fold increase in activity of subtilisin Carlsberg was documented when 98% w/w KCl-containing lyophilized enzyme powder was added to hexane, as opposed to salt-less enzyme preparation [22]. The authors explained this phenomenon by the protective ability of the salt that was able to prevent direct contact between enzyme molecules and the organic solvent; however, more recent findings using electron spin resonance spectroscopy suggest that salt-induced ionization stabilizes the charged transition state and thus, increases the polarity of the active site [23]. It is also known that while adding certain ions to enzyme preparation or sometimes directly into the solvent can improve both the reaction rate and enantioselectivity, other ions improve only the rate of the reaction or have no effect on the catalysis [14]. Empirical evidence suggests that only kosmotropic (increasing viscosity of water) salts can stabilize catalysts due to preferential hydration effect that addition of Ca ions is more activating than the addition of Ba, Sr, or Mg divalent metal salts [24, 25], and that by using aqueous solutions of smaller alkali metals or alkaline earth metals rather than hydrating the enzyme with water alone we can markedly increase enantioselectivity of the reaction [26].

Even though additive approach for biocatalysis improvement has offered many successful results, several disadvantages limit its use and call for exploring other methods of enzyme stabilization. Such disadvantages include the fact that the effect of molecular additives varies widely from case to case, depending on the enzymes used, desired substrates and reactions. Since the majority of successful additive methods were discovered by accident, there are no general protocols developed for this approach, and only few stabilizing additives are researched enough for us to clearly understand the molecular mechanisms behind their role in catalysis [14].

**31**

bents [41, 42].

*Biocatalysis and Strategies for Enzyme Improvement DOI: http://dx.doi.org/10.5772/intechopen.85018*

catalyzing a single reaction [31].

**4. Enzyme immobilization**

**3. Chemical modifications to stabilize enzymes**

immobilization methods (to be discussed in detail in Section 4).

HRP particularly useful in industrial and clinical biosensors [30].

enzyme gained increased resistance to thermoinactivation [34, 35].

protein engineering and immobilization [36].

enzyme systems or whole-cell catalysts in biotechnology.

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

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

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

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

As strategies for enzyme improvement continue to evolve, chemical modification has been rediscovered to become a robust complimentary approach to both

It is clear that while additive or chemical approaches represent a simple and attractive route for a small-scale chemical synthesis, they have to be supplemented or substituted with other enzyme-stabilizing approaches when applied to complex

Much like in a living organism, where enzymes are associated with a membrane or a cell structure that ensures their stability, in industrial setting, it is often necessary to anchor the enzyme to a certain area of a reactor in order to stabilize and reuse the same catalytic device over and over [8]. Enzyme immobilization approach usually achieves this goal by constraining enzymes to a more stable support (a carrier), thus, creating insoluble heterogeneous catalyst of native conformation with reduced flexibility [37]. Not only immobilized catalyst is less likely to be deactivated in organic solvent, is not perturbed by lyophilization [38], is more resistant to sheer stress and high temperatures [39, 40], but it is also less costly, as it is recycled in a continuous fixed-bed process and allows for an efficient enzyme purification and recovery with the help of selective adsor*Biophysical Chemistry - Advance Applications*

*Candida cylindracea* lipase [21].

**2.3 Addition of salt**

**2.2 Addition of water mimics and lyoprotectants**

It is important to note that while too few water molecules may be not enough to activate biocatalyst in organic solvent, too many water molecules may result in

Just like water that is thought to lubricate the enzyme enhancing protein flexibility with its multiple hydrogen bonds, water-mimicking substances, such as glycerol, formamide, ethylene glycol and formic acid can provide similar hydrogen bonding, while avoiding unintended hydrolysis product [14, 20]. One of the early water mimic studies concluded that adding 0.1% ethylene glycol to the solvents with optimal water content of 0.2% can increase the activity and stereo-selectivity of

Since enzymes often have to be freeze-dried, it is important to ensure their stability during the long-term storage or temperature changes associated with thawing. The most common lyoprotectant up to date is trehalose sugar that helps to preserve

Most recent reviews also list organic bases, crown ethers, surfactants, and salts as possible additives used to improve catalytic activity of enzymes in chemical synthesis [14]. For the purpose of this section, we will only cover the addition of

In 1994, Khmelnitsky et al. discovered that lyophilization of an enzyme in a salty matrix prior to its suspension in organic media lead to a dramatic enhancement in the rate of catalyzed reaction [22]. In this study, 3750-fold increase in activity of subtilisin Carlsberg was documented when 98% w/w KCl-containing lyophilized enzyme powder was added to hexane, as opposed to salt-less enzyme preparation [22]. The authors explained this phenomenon by the protective ability of the salt that was able to prevent direct contact between enzyme molecules and the organic solvent; however, more recent findings using electron spin resonance spectroscopy suggest that salt-induced ionization stabilizes the charged transition state and thus, increases the polarity of the active site [23]. It is also known that while adding certain ions to enzyme preparation or sometimes directly into the solvent can improve both the reaction rate and enantioselectivity, other ions improve only the rate of the reaction or have no effect on the catalysis [14]. Empirical evidence suggests that only kosmotropic (increasing viscosity of water) salts can stabilize catalysts due to preferential hydration effect that addition of Ca ions is more activating than the addition of Ba, Sr, or Mg divalent metal salts [24, 25], and that by using aqueous solutions of smaller alkali metals or alkaline earth metals rather than hydrating the enzyme with water alone we can markedly increase enantioselectivity of the

Even though additive approach for biocatalysis improvement has offered many successful results, several disadvantages limit its use and call for exploring other methods of enzyme stabilization. Such disadvantages include the fact that the effect of molecular additives varies widely from case to case, depending on the enzymes used, desired substrates and reactions. Since the majority of successful additive methods were discovered by accident, there are no general protocols developed for this approach, and only few stabilizing additives are researched enough for us to clearly understand the molecular mechanisms behind their role in

reduced substrate solubility or hydrolytic reactions side product [14].

enzyme structure and allows for industrial storage of biocatalysts [18].

salts and the role of ionic interactions in biocatalyst enhancement.

**30**

reaction [26].

catalysis [14].
