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Aalto University School of Chemical Technology CHEM-E3140 Bioprocess Technology II Summary: Strategies for Stabilization of Enzymes in Organic Solvents Group Members: Evgen Multia Jimi Leivo Karl Mihhels 1 1 Introduction Enzymes are used in the industrial biocatalysis in wide variety of applications. These vary from synthesis of pharmaceuticals to large-scale production of biofuels. Most important biocatalysts are hydrolases, particularly lipases, since they are able to convert wide variety of substances. They have many favourable properties; stability at extreme temperatures, high chemo-, regio-, and enantioselectivity, and no need for cofactors. Biocatalysts as such have evolved to work in cellular environments, which makes them intolerant to harsh industrial conditions. The solvent in cellular environment is water, which is not the best solvent for most synthetic reactions. This raises a problem, even though organic solvents make synthesis more favourable, it affects the enzyme activity, and majority of enzymes display lower catalytic efficiency under organic solvents. This can be solved by either optimizing the process conditions for existing enzymes, or prepare new biocatalysts that can work under optimal process conditions. The latter is the strategy most often employed strategy today. With help of modern technology biocatalysts have been engineered to work even in neat organic solvents, which makes enzyme stability engineering both of commercial and scientific interest. 2 Biocatalyst stability The main factor that affects biocatalysis in nonconventional media is water content. Biocatalysts that work efficiently in neat organic solvents are lipases, proteases and many others, but even these biocatalysts have few water molecules bound to protein molecule. This leads to a general belief that waterless proteins are inactive. While water promotes the conformational mobility required for optimal catalysis, organic solvents lead to stronger intermolecular interactions. Thus water content of an organic solvent promotes conformational mobility which leads to tendency of an enzyme to denature. This results in decrease of enzyme activity when watermiscible cosolvent is added to water. For most enzymes the limit of organic cosolvent 2 concentration is 60-70 % (v/v), after which they become inactive. Hydrophilic solvents have a tendency to remove protein-bound water, which is essential for proteins structure and function. Hydrophobic organic solvents are thus much better for maintaining the protein structure. Because enzymes are not soluble to hydrophobic organic solvents, they are added to hydrophobic solvents as powders prepared by lyophilization. Lyophilization dehydrates the proteins, thus altering the structure, which makes the enzyme activity lower also in hydrophobic solvents. The denaturation of lyophilized protein is reversible upon addition of some water to the protein, however, not unproblematic when protein is in hydrophobic solvent, since it reduces the structural mobility of the protein. 3 Assessing biocatalyst stability Protein stability can be defined as energetics of unfolding reactions, which occur via reversible and irreversible unfolding. Reversible unfolding is characterized by an equilibrium between native and unfolded states, and is called thermodynamic stability or conformational protein stability. It reflects protein’s stability to refold after different extreme conditions. Irreversible unfolding can be due to protein aggregation, misfolding, chemical modification, or a lack of chaperons. Irreversible unfolding is kinetically driven, it is thus termed the kinetic stability. Thermodynamic stability alone doesn’t guarantee that protein will remain active during the process, since typical conditions in bioprocesses often result in irreversible denaturation. This makes kinetic control of operational stability important for successful biocatalytic process. 4 Assessing biocatalyst stability Three major strategies for obtaining stable biocatalysts in organic solvents can be employed: (i) isolation of new enzymes that can work under extreme conditions, 3 (ii) modification of enzyme structure to make it more resistant to organic solvents, (iii) modification of solvent environment so that it has less denaturing effect on the enzyme. Figure 1: Strategies for stabilization of enzymes toward organic solvents. [1] 4.1 (i) Isolation of stable biocatalysts Biodiversity prospecting can be utilized to isolate the enzymes that are staying functional under harsh conditions from living organisms. These so-called extremozymes are collected from microorganisms that can grow under extreme conditions. In these cases nature employs many different structural strategies to obtain highly stable enzymes. The most important characteristic of these extremozymes is probably their improved hydration capabilities, since loss of water molecules under extreme conditions is a main factor affecting the functionality of an enzyme. This makes it possible to use extremozymes in nonaqueous environments. Important sources of these extremozymes are for example various strains of Pseudomonas aeruginosa, also some halophilic and alkaliphilic organisms. Since there is certain difficulties related to isolation of extremophilic organisms, it is more convenient to clone genes of these organisms into suitable mesophilic hosts. 4 4.2 (ii) Modification of biocatalysts Modification of biocatalysts can be divided into five parts: enzyme immobilization, chemical modification, propanol-rinsed enzyme preparation, ionic liquid coating and genetic modification. The most effective method to enhance enzyme stability is replacing free enzymes to immobilized forms. This immobilization creates stiffer structure of biocatalyst preventing them to and unfold enzymes and form malformations of its active sites. Furthermore, the method increases the recovery of enzymes and lowers the industrial costs by improving usability in continuous processes. Basically, immobilization can be occurred onto or within enzymes or form water-insoluble particles. The most effective way for increasing stability is physical adsorption of an enzyme. Chemical modification of amino acid residues was one of the most common way to improve chemical stability before broader understanding of molecular biology. Even though the use of other methods have been increased, chemically modifying has kept its popularity as a decent alternative. Usually polyethylene glycol (PEG) and its derivatives have been used to process the properties of organic solvents. Surface modification of proteins and covalent modification of subtilisin Carlsberg are examples PEG can be used. Other methods for modifying enzymes have not yet been able to achieve same popularity than previously mentioned methods. The basics of propanol-rinsed enzyme preparation (PREP) are based on reactions with dry n-propanol. The goal is to achieve low-water media by rinsing enzymes with n-propanol. Apart from immobilized ones, studies has shown that it is also possible to run PREP with free form enzymes. Another less-studied method is ionic liquid coating which utilizes ionic liquids (IL) as cosolvents and enzyme-coating agents. IL are organic salts in a liquid form in room temperature. They consist of only different cations and anions, and their properties bring new innovative ways to process enzymes e.g. polar substrates. Some of those properties are low volatility and melting point (usually under 100 C), non- 5 flammability and thermal stability. IL have been examined in an organic solvent by coating Burkholderia cepacia enzyme in [PPMIM][PF6]. In spite of increased stability, the activity of enzyme wasn’t improved, however compared to that, activation improved during lyophilization. Genetic modification manipulates directly the genome of organisms using biotechnology. It is technology to improve the genetic characterization of different cells. This kind of modification has three different categories: directed molecular evolution, rational design and semirational design. The difference between methods are how mutations are generated (randomly, predicted, with degenerate oligonucleotide, etc.). 4.3 (iii) Modification of solvent environment Solvent environment can be modified with additives or surfactants. Modification is relatively easy to perform, as additives, such as inorganic salts, polyols and sugars, can be added straight to the reaction solution or before lyophilization. Currently, the most of the market enzymes have been produced using additives. The method needs still more studying, since often estimations are based on presumptions and empirical values. Several studies have been shown that simple salts can affect to enzymes by activating them in organic media by lyophilization. Furthermore, other proofs about the salt activation have been obtained using spectroscopic methods. Instead when adding polyols or sugars in aqueous solution, they have been reported to strengthen the hydrophobic interactions among nonpolar amino acid residues. Thus, proteins stiffen. Polyols and sugars also affect water activity and microbial contamination by lowering it. When stabilizing and activating proteins in nonaqueous conditions, crown ether is one of the alternatives to consider. Surfactants lower the surface tensions between two liquids or liquid and solid. When adding into organic solvents, enzymes can form reverse micelles or water-in-oil microemulsions. Especially microemulsions have several benefits what comes to stabilization. They are clear, thermodynamically stable and isotropic liquid mixtures of 6 water, surfactants and organic compounds which are in different phase than water. The enzyme stands in the aqueous phase while reacting hydrophobic substrate can be dissolved in the organic phase. The encapsulation of enzymes to microdroplets are the main reason for chemically activation and stabilization. New studies have given new promising results, when conventional organic solvent has been replaced by ionic liquid. 5 Conclusions Enzymes play a significant role in today’s chemical technology and wide range of reactions. Water can be used as a solvent for many different reactions, however, it is relatively ineffective in most synthesis where organic solvents are needed. Using organic solvents at the same time with the enzymes require modification and optimizing. One way is to produce new biocatalysts. Lipases and proteases are good and effective examples of enzymes that are stable in organic liquids. The main factor for the biocatalysts in nonconventional media is water content. Stabilization is needed to improve reaction economy. For organic solvents, there are three main methods to produce stable biocatalysts: Isolation of stable catalysts, modification of biocatalysts and modification of solvent environment. Isolation is done by separation of biocatalysts from extremozymes. Modification of biocatalysts can be performed in several ways such as enzyme immobilization, chemical and genetic modification, or with the presence of ionic liquids. Solvent environment is basically modified with additives or surfactants, of which additives are predominant. 6 References [1] V Stepankova, S Bidmanova, T Koudelakova. . . - Acs . . . , 2013 - ACS Publications