Non-conventional media

Volume 8 Biocatalysis in Non-Conventional Media (Tramper et al.. Editors)... 

Vofi H,Miethe P (1992) Enzymes entrapped in liquid crystals a novel approach for bio-catalysis in nonaqueous media. In Tramper J, Vermae MH, Beet HH, Stockar UV (eds) Biocatalysis in non-conventional media, progress in biotechnology. Elsevier, London 8 739... 

The scope and limitations of biocatalysis in non-conventional media are described. First, different kinds of non-conventional reaction media, such as organic solvents, supercritical fluids, gaseous media and solvent-free systems, are treated. Second, enzyme preparations suitable for use in these media are described. In several cases the enzyme is present as a solid phase but there are methods to solubilise enzymes in non-conventional media, as well. Third, important reaction parameters for biocatalysis in non-conventional media are discussed. The water content is of large importance in all non-conventional systems. The effects of the reaction medinm on enzyme activity, stabihty and on reaction yield are described. Finally, a few applications are briefly presented. 

Another important advantage with many non-conventional media is that they can solubilise hydrophobic compounds which are poorly soluble in water. Thereby the conversion of these important substrates is facilitated. Further advantages are that the risk of microbial contamination is much lower in non-conventional media, and under optimized conditions the stability of enzymes is often higher than in aqueous solutions. 

Organic solvents constitute the most commonly used non-conventional media for biocatalysis. They are commonly employed to change equilibrium positions of reactions... 

The proportions of water and organic solvent can be varied from pure water to almost pure organic solvent. In order to retain enzymatic activity there seems to be a need for a little water. However, this minimal amount of water is sometimes considerably less than a monolayer of water around the enzyme molecules. The rest of the medium can be an organic solvent. The effects of water on biocatalysis in non-conventional media are treated below. 

Figure 9.2 A schematic presentation of different types of enzyme preparations used in non conventional media, a enzyme powder, b enzyme crystals c enzyme on a porous support d covalently modified enzyme dissolved in the solvent e enzyme solubilised by surfactant ...

The most straightforward way of using sohd enzymes in organic media is to suspend the solid enzyme directly in the solvent. If one wants to get quick results from a bioconversion and does not want to optimise the efficiency of the enzyme, this method is the obvious first choice. There are many example in the hterature where enzymes have been used successfully in synthesis just as powders directly from the enzyme manufacturer. Sometimes there is a need to dissolve the enzyme powder and re-lyophilise it from a buffer with a more suitable composition, see the section 9.6 pH control in non-conventional media . 

In many applications of enzymes in non-conventional media, the enzymes are nsed in the immobilised form on support materials which often are porous. These immobilised preparations usually express considerably higher specific activity (moles snbstrate converted per unit enzyme and time) than enzyme powders. The reason can be facilitation of mass transfer either by the spreading of the enzyme on a large surface area or by better suspension of the catalyst particles (enzyme powders often tend to aggregate). An alternative explanation is that the support might protect the enzyme from inactivation dnring the drying of the preparation. 

Immobilisation methods are treated in detail in chapter 6. Most enzyme immobilisation methods used in coimection with non-conventional media rely on noncovalent interactions between the support and the enzyme. The reason why this works well in many cases is that enzymes normally have a low tendency to dissolve in the reaction media used. Adsorption or deposition on porous supports are often used methods. It is important to remember that other substances (for example salts and other polar substances) are often immobilised on the support because they are present during the immobilisation procedure and not soluble in the reaction medium. Those substances influence the microenviromnent of the enzyme and thereby its catalytic activity. 

General characteristics of the support to be considered are discussed in chapter 6 and those of relevance for the use of enzymes in non-conventional media are listed in Table 9.2. In addition, the specific surface area of the support is of special importance for the applications in non-conventional media. Inactivation can occur if the surface area is too large in relation to the amount of enzyme (Figure 9.3). In order to avoid inactivation at least a monolayer of enzyme molecules should be formed on the accessible surface (Wehtje, Adlercreutz and Mattiasson, 1993). For enzymes of high purity and high activity, the amount of enzyme needed is sometimes quite small. In such cases the enzyme can be mixed with a protecting protein before immobilisation to achieve at least a monolayer of protein and thereby avoid... 

Biocatalyst in non-conventional media 347 even extract it from the support if it is not covalently bound. 

The amount of water in the reaction mixture can be quantified in different ways. The most common way is to nse the water concentration (in mol/1 or % by volume). However, the water concentration does not give much information on the key parameter enzyme hydration. In order to have a parameter which is better correlated with enzyme hydration, researchers have started to nse the water activity to quantify the amount of water in non-conventional reaction media (Hailing, 1984 Bell et al, 1995). For a detailed description of the term activity (thermodynamic activity), please look in a textbook in physical chemistiy. Activities are often very nselul when studying chemical equilibria and chemical reactions of all kinds, but since they are often difficult to measure they are not used as mnch as concentrations. Normally, the water activity is defined so that it is 1.0 in pure water and 0.0 in a completely dry system. Thus, dilute aqueous solutions have water activities close to 1 while non-conventional media are found in the whole range of water activities between 0 and 1. There is a good correlation between the water activity and enzyme hydration and thns enzyme activity. An advantage with the activity parameter is that the activity of a component is the same in all phases at eqnihbrium. The water activity is most conveniently measnred in the gas phase with a special sensor. The water activity in a liqnid phase can thns be measured in the gas phase above the liquid after equilibration. 

Water activity can be nsed to quantify water in all non-conventional media and is used to an increasing extent. However, in microemulsions containing reversed micelles, water is still often quantified as the molar ratio of water to surfactant (abbreviated as Wq or R). This parameter is used because it is correlated with the size of the reversed micelles, which in turn is correlated with enzymatic activity. 

When reactions are carried ont in non-conventional media, water is distributed between the different phases present. Some water is bound to the enzyme and thereby has a large influence on the catalytic activity. Some water is dissolved in the solvent and if supports, polymers or other substances are present these bind water as well. 

As mentioned above, non-conventional media are often used to shift the equilibrium position so that reversed hydrolytic reactions can be carried out. The equilibrium shift can be achieved by different mechanisms. A schematic presentation of two systems for ester synthesis are shown in Figure 9.8. Let us first consider the homogeneous system which... 

Under which conditions in non-conventional media do enzymes normally have the best stability ... 

Hailing (1994). A more thorough description of the fundamental principles of biocatalysis in non-conventional media. 

Hallling, P.J. (1994) Thermodynamic predictions for biocatalysis in non-conventional media theory, tests and recommendations for experimental design and analysis. Enz. Microb. Technol., 16, 178-206. 

After the publication of the first edition of Applied Biocatalysis some five years ago, this field has rapidly been developing. This is evident from the number and types of new applications, but also from the state of the art for some of the important techniqnes, such as protein engineering and the use of non-conventional media, for example. 

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