Enzymes organic solvents

Albumin is a protein and is therefore susceptible to chemical degradation and denaturation by exposure to extremes of pH, high salt concentrations, heat, enzymes, organic solvents, and other chemical agents. 

Although water is the natural medium for enzymes, organic solvents are widely used to improve the solubility of hydrophobic reactants or products, and to shift... 

In order to improve the extraction of enzymes, organic solvents and surfactants are sometimes used. 

Buschie-Diller, G., Mogahzy, E. Y., Inglesby, M. K., and Zeronian, S. H. (1998). Effect of scouring with enzymes, organic solvents and caustic soda on the properties of hydrogen peroxide bleached cotton yam. Text Res. 

Buschle-DiUer G, El Mogahzy Y, Inglesby M K and Zeronian S H, Effects of scouring enzymes, organic solvents, and caustic soda on the properties of hydrogen jjeroxide bleached cotton yarn . Text. Res. J., 1998, 68(12), 920 9. 

If a catalyst is to work well in solution, it (and tire reactants) must be sufficiently soluble and stable. Most polar catalysts (e.g., acids and bases) are used in water and most organometallic catalysts (compounds of metals witli organic ligands bonded to tliem) are used in organic solvents. Some enzymes function in aqueous biological solutions, witli tlieir solubilities detennined by the polar functional groups (R groups) on tlieir outer surfaces. 

In contrast to the hydrolysis of prochiral esters performed in aqueous solutions, the enzymatic acylation of prochiral diols is usually carried out in an inert organic solvent such as hexane, ether, toluene, or ethyl acetate. In order to increase the reaction rate and the degree of conversion, activated esters such as vinyl carboxylates are often used as acylating agents. The vinyl alcohol formed as a result of transesterification tautomerizes to acetaldehyde, making the reaction practically irreversible. The presence of a bulky substituent in the 2-position helps the enzyme to discriminate between enantiotopic faces as a result the enzymatic acylation of prochiral 2-benzoxy-l,3-propanediol (34) proceeds with excellent selectivity (ee > 96%) (49). In the case of the 2-methyl substituted diol (33) the selectivity is only moderate (50). 

The variety of enzyme-catalyzed kinetic resolutions of enantiomers reported ia recent years is enormous. Similar to asymmetric synthesis, enantioselective resolutions are carried out ia either hydrolytic or esterification—transesterification modes. Both modes have advantages and disadvantages. Hydrolytic resolutions that are carried out ia a predominantiy aqueous medium are usually faster and, as a consequence, require smaller quantities of enzymes. On the other hand, esterifications ia organic solvents are experimentally simpler procedures, aHowiag easy product isolation and reuse of the enzyme without immobilization. 

Alcohol dehydrogenase-catalyzed reduction of ketones is a convenient method for the production of chiral alcohols. HLAD, the most thoroughly studied enzyme, has a broad substrate specificity and accommodates a variety of substrates (Table 11). It efficiendy reduces all simple four- to nine-membered cycHc ketones and also symmetrical and racemic cis- and trans-decalindiones (167). Asymmetric reduction of aUphatic acycHc ketones (C-4—C-10) (103,104) can be efficiendy achieved by alcohol dehydrogenase isolated from Thermoanaerohium hrockii (TBADH) (168). The enzyme is remarkably stable at temperatures up to 85°C and exhibits high tolerance toward organic solvents. Alcohol dehydrogenases from horse Hver and T. hrockii... 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

An amount of enzyme preparation equivalent to 900 mg of wet cells was made up to 25 ml with the above potassium phosphate buffer solution. 150 mg (1.15 mmol) of 5-fluorouracil and 1.0 gram of thymidine (4.12 mmol) were dissolved in 15 ml of the above potassium phosphate buffer solution. The mixture was incubated at 37°C for 18 hours. After this time, enzyme action was stopped by the addition of four volumes of acetone and one volume of peroxide-free diethyl ether. The precipitated solids were removed by filtration, and the filtrate was evaporated under nitrogen at reduced pressure until substantially all volatile organic solvent had been removed. About 20 ml of aqueous solution, essentially free of organic solvent, remained. This solution was diluted to 100 ml with distilled water. 

Biocatalysts in nature tend to be optimized to perform best in aqueous environments, at neutral pH, temperatures below 40 °C, and at low osmotic pressure. These conditions are sometimes in conflict with the need of the chemist or process engineer to optimize a reaction with respect to space-time yield or high product concentration in order to facilitate downstream processing. Furthermore, enzymes and whole cells are often inhibited by products or substrates. This might be overcome by the use of continuously operated stirred tank reactors, fed-batch reactors, or reactors with in situ product removal [14, 15]. The addition of organic solvents to increase the solubility of substrates and/or products is a common practice [16]. 

When either the organic solvent or the ionic liquid is used as pure solvent, proper control over the water content, or rather the water activity, is of crucial importance, as a minimum amount is necessary to maintain the enzyme s activity. For ionic liquids, a reaction can be operated at constant water activity by use of the same methods as established for organic solvents [17]. [BMIM][PFg] or [BMIM][(CF3S02)2N], for example, may be used as pure solvents and in biphasic systems. Water-miscible ionic liquids, such as [BMIM][BF4] or [MMIM][MeS04], can be used in the second case. 

As with organic solvents, proteins are not soluble in most of the ionic liquids when they are used as pure solvent. As a result, the enzyme is either applied in immobilized form, coupled to a support, or as a suspension in its native form. For production processes, the majority of enzymes are used as immobilized catalysts in order to facilitate handling and to improve their operational stability [24—26]. As support, either inorganic materials such as porous glass or different organic polymers are used [27]. These heterogeneous catalyst particles are subject to internal and external... 

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

Entries 7, 8, and 10 describe so-called Idnetically controlled syntheses starting from activated substrates such as ethyl esters or lactose. In two reaction systems it was possible to demonstrate that ionic liquids can also be useful in a thermodynamically controlled synthesis starting with the single components (Entry 11) [39]. In both cases, as with the results presented in entry 6, the ionic liquids were used with addition of less than 1 % water, necessary to maintain the enzyme activity. The yields observed were similar or better than those obtained with conventional organic solvents. 

The report from Sheldon and co-workers was the second publication demonstrating the potential use of enzymes in ionic liquids and the first one for lipases (Entry 13) [43]. They compared the reactivity of Candida antarctica lipase in ionic liquids such as [BMIM][PFg] and [BMIM][BF4] with that in conventional organic solvents. In all cases the reaction rates were similar for all of the reactions investigated alcoholysis, ammoniolysis, and per hydrolysis. 

Many substrates currently produced in the chemical industry are immiscible with water, but are readily miscible with organic solvents. Most enzymes, however, will not operate efficiently, or not operate at all, in non-aqueous media. Some exceptions do exist, such as lipases and esterases, which can operate in non-aqueous environments. Currently, there is considerable interest in extending the range of enzymes that do work in organic solvents. 

The specificity of enzyme reactions can be altered by varying the solvent system. For example, the addition of water-miscible organic co-solvents may improve the selectivity of hydrolase enzymes. Medium engineering is also important for synthetic reactions performed in pure organic solvents. In such cases, the selectivity of the reaction may depend on the organic solvent used. In non-aqueous solvents, hydrolytic enzymes catalyse the reverse reaction, ie the synthesis of esters and amides. The problem here is the low activity (catalytic power) of many hydrolases in organic solvents, and the unpredictable effects of the amount of water and type of solvent on the rate and selectivity. 

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