Solvent systems enzymatic reactions

The use of ionic liquids (ILs) to replace organic or aqueous solvents in biocatalysis processes has recently gained much attention and great progress has been accomplished in this area lipase-catalyzed reactions in an IL solvent system have now been established and several examples of biotransformation in this novel reaction medium have also been reported. Recent developments in the application of ILs as solvents in enzymatic reactions are reviewed. 

We report the results from a molecular dynamics simulation of the serine protease y-chymotrypsin (y-CT) in hexane. The active site of chymotrypsin contains the "catalytic triad" which consists of Ser-His-Asp. y-CT suspended in nearly anhydrous solvents has been found to be catalytically active. In order for proteins to retain their activity in anhydrous solvents some water molecules are required to be present. These "essential waters" have been suggested to function as a molecular lubricant for the protein. Hexane, having a dielectric constant of 1.89, is a suitable non-aqueous solvent for enzymatic reactions. The low dielectric constant of hexane allows it to not compete with the protein for the essential water and allows enzymes to retain their catalytic activity. y-CT in hexane is thus an ideal system to further explore the effect of non-aqueous solvation on protein structure, function and dynamics. 

The choice of solvents for enzymatic reaction has been widened from organic solvents to various types of solvents such as supercritical fluids, ionic liquids, etc. The enzymatic reaction in organic solvent has been reported already in 1970s, the first biocatalysis in ionic liquids [4] was in 2000, and the first biocatalysis in supercritical fluids [5] was in 1985. Currently four kinds of liquid or fluid solvents, aqueous, organic solvents, ionic liquids, and supercritical fluids, are available for biocatalysis as shown in Figure 3.2. Moreover, biphasic or triphasic solvent systems consisting of two or more kinds of the solvents are also often employed for biocatalysis. Solid phase of immobilized enzymes and/or hydrophobic polymer to adsorb substrate and product may also exist. The performance of a biocatalyst depends significantly on the solvent system. The best medium should provide optimum reaction rates and simplify work-up procedure to make the process both economical and environment friendly. 

Some transport proteins merely provide a path for the transported species, whereas others couple an enzymatic reaction with the transport event. In all cases, transport behavior depends on the interactions of the transport protein not only with solvent water but with the lipid milieu of the membrane as well. The dynamic and asymmetric nature of the membrane and its components (Chapter 9) plays an important part in the function of these transport systems. 

An IL solvent system is applicable to not only lipase but also other enzymes, though examples are still limited for hpase-catalyzed reaction in a pure IL solvent. But several types of enzymatic reaction or microhe-mediated reaction have been reported in a mixed solvent of IL with water. Howarth reported Baker s yeast reduction of a ketone in a mixed solvent of [hmim] [PFg] with water (10 1) (Fig. 16). Enhanced enantioselectivity was obtained compared to the reaction in a buffer solution, while the chemical yield dropped. 

Since the beginning of the 20th century, organic solvents have been used in enzymatic reaction media [30]. Biocatalytic reactions in water-organic biphasic media were first carried out by Cremonesi et al. [31] and by Buckland et al. [32] less than 30 years ago. Their work aimed at the conversion of high concentrations of poorly water soluble components, particularly steroids. Later, biphasic systems were used for enzyme-catalyzed synthesis reactions that were unfavored in water, changing the reaction equilibrium towards the higher yield of the product, such as esters or peptides. 

Often the enzyme stability can be improved by using a suitable water-immiscible solvent instead of a water-miscible one. Two-phase systems are obtained with the enzyme and other hydrophilic substances present in the aqueous phase while hydrophobic substrates and products mainly partition to the organic phase (Figure 9.1). Water immiscible solvents often used for enzymatic reactions are hydrocarbons, ethers and esters further details on solvents are found in the section 9.5 Selection of solvents , below. In order for the bioconversion to occur, the substrates must be transferred to the enzyme in the aqueous phase after the reaction hydrophobic... 

Esters are common components in cosmetics and skin-care products. They can be synthesized from fatty acids and alcohols using either chemical or enzymatic reactions. The chemical reactions are normally catalysed by acid catalysts. Enzymatic synthesis is carried out under milder conditions and therefore it provides products of very high purity. A range of esters such as isopropyl palmitate and isopropyl myristate are now produced industrially using enzymatic synthesis. The reactions are carried out in solvent-free systems using an immobilised lipase as catalyst. In order to get high yields in the reactions, water is removed continuously. 

The amounts of water associated with various components in a typical reaction mixture are shown in Table 1.2. Most of the water is dissolved in the reaction medium, and the amount of water bound to the enzyme is obviously just a minor fraction of the total amount of water. If the solvent was changed to one able to dissolve considerably more water and the same total amount of water was present in the system, the amount of water bound to the enzyme would decrease considerably and thereby its catalytic activity as well. Changing solvent at fixed water activity would just increase the concentration of water in the solvent and not the amount bound to the enzyme. Comparing enzyme activity at fixed enzyme hydration (fixed water activity) is thus the proper way of studying solvent effects on enzymatic reactions. 

Eutectic melting (and also similar systems with added adjuvants/solvents) has been used to prepare homogeneous substrate mixtures with extremely high concentration levels as media for enzymatic reactions [37, 68, 69]. 

The application of SCF as reaction media for enzymatic synthesis has several advantages, such as the higher initial reaction rates, higher conversion, possible separation of products from unreacted substrates, over solvent-free, or solvent systems (where either water or organic solvents are used). Owing to the lower mass-transfer limitations and mild (temperature) reaction conditions, at first the reactions which were performed in non-aqueous systems will be transposed to supercritical media. An additional benefit of using SCFs as... 

The enzymatic reactions are performed in the wells of microtiter plates (96-format) in water (as in lipase-catalyzed hydrolytic reaction of (.S)-13C-4/(i )-4), which is followed by a standard automatic extraction step. Depending on the particular substrate to be assayed and the type of solvent used, it may be necessary to remove the solvent. However, this is often not necessary. For enzymatic reactions in organic medium, solvent extraction is not required. For NMR analysis such solvents as CDC13, Dg-DMSO, or D20 are used. A minimum of about 6 pmol of substrate/product per milliliter of solvent is needed. Although the flow-through cell system does not need too much solvent (about 1L in 24 h), the solvents can be mixed with the undeuterated form in 1 9 ratios to reduce costs. 

Further advantages of biocatalysis over chemical catalysis include shorter synthesis routes and milder reaction conditions. Enzymatic reactions are not confined to in vivo systems - many enzymes are also available as isolated compounds which catalyze reactions in water and even in organic solvents [28]. Despite these advantages, the activity and stability of most wild-type enzymes do not meet the demands of industrial processes. Fortunately, modern protein engineering methods can be used to change enzyme properties and optimize desired characteristics. In Chapter 5 we will outline these optimization methods, including site-directed mutagenesis and directed evolution. 

Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused five times without any loss of activity or productivity in the resolution process to prepare A-(+)-(43). The enzymatic process was scaled up to a 640-liter preparative batch using immobilized lipase PS-30 at 4 g/liter racemic substrate (43) in toluene as a solvent. From the reaction mixture, i -(+)-(43) was isolated in 35 M% overall yield with 98.5% e.e. and 99.5% chemical purity. The undesired, S -(-)-acetatc (46) produced by this process was enzymatically hydrolyzed by lipase PS-30 in a biphasic system to prepare the corresponding S -(-)-alcohol (43). Thus both enantiomers of alcohol (43) were produced by the enzymatic process. 

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