**1. Introduction**

Enzymes are biological catalysts that are believed to be the cornerstones of life. They assure metabolic needs of cells and assist in a great range of life-sustaining biochemical reactions. The majority of natural enzymes are highly efficient and can increase the rate of biotransformation up to 1017 fold [1]. Enzymes can carry their functions at ambient temperatures and pressures, with a minimum of byproducts and waste, leading to the specific product of interest in a single catalyzed step, whereas synthesis of the same product with the means of organic chemistry may require many steps and produce a mixture of undesired isomeric, epimeric, or rearranged compounds [2]. The field of biotechnology strives to exploit isolated enzymes and whole cell cultures as biocatalysts capable of accelerating and refining complex chemical transformations of organic compounds for industrial and synthetic use [3]. Well known examples of such biocatalysts include microbial

lipases that are used to synthesize cost-effective biopolymers, biodiesel, pharmaceuticals, and agrochemicals from renewable natural sources, *b*-glycosidases employed in industrial plant biomass saccharification [4, 5] and fungal oxidoreductases that have a potential to become biocatalysts in a bio-based (circular) economy by converting biomass into renewable building blocks for manufacturing biodegradable materials [6]. Unfortunately, the scope of natural enzymes is limited, and certain challenges have to be overcome before we can rely on biocatalysts for efficient, low-cost industrial transformations and greener synthetic chemistry. Such challenges include instability of enzymes *in vitro* (denaturation in high temperatures or extreme pH), low selectivity, product and substrate inhibition, and low reaction yield in non-aqueous solvents [2, 7]. Four general approaches exist to address the above mentioned limitations: additive approach, chemical modification, enzyme immobilization, and protein engineering [8]. While protein engineering is concerned with modifying functional properties of the enzyme at the genetic level, the other three approaches are focused on physico-chemical alterations of the media, enzymatic surface residues, or support material for biocatalyst stability [9].

Using **Figure 1** as a guide, we will review both physico-chemical and functional modification strategies for enzyme improvement, starting with the earliest methods to address solvent-dependent limitations and leading to the most recent technologies, like *de novo* and computational enzyme design [10, 11] (**Figure 1**).

#### **Figure 1.**

*Enzyme stabilization methods available for improvement of physico-chemical (yellow) and functional (blue) properties of biocatalysts.*

**29**

due to hydration.

high efficiency [13, 14].

**2.1 Addition of water**

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

proteases subtilisin and *a*-chymotrypsin is reduced 104

**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

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

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

–105

times if the enzymes

*Biophysical Chemistry - Advance Applications*

biocatalyst stability [9].

lipases that are used to synthesize cost-effective biopolymers, biodiesel, pharmaceuticals, and agrochemicals from renewable natural sources, *b*-glycosidases employed in industrial plant biomass saccharification [4, 5] and fungal oxidoreductases that have a potential to become biocatalysts in a bio-based (circular) economy by converting biomass into renewable building blocks for manufacturing biodegradable materials [6]. Unfortunately, the scope of natural enzymes is limited, and certain challenges have to be overcome before we can rely on biocatalysts for efficient, low-cost industrial transformations and greener synthetic chemistry. Such challenges include instability of enzymes *in vitro* (denaturation in high temperatures or extreme pH), low selectivity, product and substrate inhibition, and low reaction yield in non-aqueous solvents [2, 7]. Four general approaches exist to address the above mentioned limitations: additive approach, chemical modification, enzyme immobilization, and protein engineering [8]. While protein engineering is concerned with modifying functional properties of the enzyme at the genetic level, the other three approaches are focused on physico-chemical alterations of the media, enzymatic surface residues, or support material for

Using **Figure 1** as a guide, we will review both physico-chemical and functional modification strategies for enzyme improvement, starting with the earliest methods to address solvent-dependent limitations and leading to the most recent technolo-

*Enzyme stabilization methods available for improvement of physico-chemical (yellow) and functional (blue)* 

gies, like *de novo* and computational enzyme design [10, 11] (**Figure 1**).

**28**

**Figure 1.**

*properties of biocatalysts.*
