The Use Of Enzymes In Non Aqueous Solvents Biology Essay

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Life exists only when water is present because it is one of the most important molecules of life (Ogino and Ishikawa, 2001). The central roles played by water in enzymatic catalysis and as suitable major component in all biological processes like metabolism, catabolism, biosynthesis, and photosynthesis due to its solvent properties have been long detected (Henderson, 1982).

For many years, it has been known that for enzymatic catalysis to occur, water is totally essential. And aside taking part in all non-covalent interactions-maintaining the active conformation of proteins, it also plays an important role in enzyme dynamics (Zaks and Russell, 1988).

Enzymes are catalysts that are highly specific and basically function within mild reaction conditions (Ogino and Ishikawa, 2001).

For instance, enzymes function under aqueous neutral solutions and room temperature and pressure.

The ability to function under a mild aqueous condition is one of the most important properties of enzymes, but sometimes it posses a disadvantage. This is regrettable because, though water being an ideal solvent for most polar species needed for life (such as amino acids, nucleic acids, carbohydrates, and proteins), it is a poor solvent for some commercially important organic compounds which can sometimes be water-insoluble or water-unstable ( Dordick, 1991).

This brings about the need to carry out biotechnological reactions in organic solvents (non-aqueous solvents) because, the water- unstable compound must be synthesized in organic solvents and it is pleasing to carry out the enzymatic reaction of water-insoluble compounds in the presence of an organic solvent or in an organic solvent (Ogino and Ishikawa, 2001).

In addition, synthetic reactions can be catalysed by hydrolytic enzymes which reverse the reactions of hydrolyses in the presence of non-aqueous solvents. Thus, making benefit of thermodynamic equilibrium that is unfavourable in aqueous solvents (Dordick, 1991; Ogino and Ishikawa, 2001). The equilibrium of a reversible reaction is able to shift between hydrolysis and synthesis to complete synthesis when a molar fraction of water in a reaction mixture is reduced by adding organic solvent.

In aqueous solutions, enzyme molecules in their native state have both hydrophilic regions which has high polarity and in contact with the water and hydrophobic regions which has low polarity and is folded inside the molecule (Butler, 1979; Tawanaka and Kawamoto, 1991).

When the polarity of the solvent is reduced by removing water and adding organic solvent, the hydrophobic regions appropriately tend to disperse, which results in disorganisation, destabilization, and unfolding of the enzyme molecules. Non-polar interaction between the enzyme and the substrate may also be disrupted (Maurel, 1978).

For enzymes to be used as catalysts in organic solvents they must be stable. If they are unstable, this can lead to enzyme inactivation. Hence, the problem of enzyme stability must be addressed by appropriate methods or the enzyme be segregated from the organic solvent to avoid inactivation (Ogino and Ishikawa, 2001).

One significant factor in solvent selection is that the solvent has to be compatible with the continuance of the catalytic activity of the enzyme. It is usually favourable to find a solvent in which the enzyme is thermodynamically stable and also catalytically active (Butler, 1979).

Enzyme stability is greatly subjective to the amount of water in the organic solvent (Zaks and Klibanov, 1984). In as much as the amount of water needed for retaining the enzyme catalytic activity is maintained, the removal of water by adding organic solvents is possible without any loss in active conformation (Garcia- Junceda et al., 2004).

In organic solvents, the catalytic activity of enzymes is lower than in water. However, this challenge of enzyme stability in the presence of organic solvents can be prevented by several physical and chemical methods which includes the following;

IMMOBILIZATION OF ENZYMES

In this technique, the enzymes are combined with insoluble support matrices like fibres, particles, membranes, and entrapping the enzyme in gel matrix (Tanaka and Kawamoto, 1991).

Based on this immobilization, enzymes are stabilised against organic solvents (Harvath, 1974) and also they are stabilised against heat (Klibanov, 1979; Klibanov, 1983). Thus, enzymes are isolated from direct contact with organic solvents and this can also contribute to stabilisation. For instance, some whole cells which are types of immobilised enzymes, with or without support materials have been successfully utilised in organic solvent (Nakashima et al., 1988). These cells are regarded as immobilised insoluble support matrices.

The major benefit of immobilised enzymes is that they can be utilised continuously and repeatedly (Ogini and Ishikawa, 2001).

An example of immobilised enzyme utilised in organic solvent is the immobilised protease (Rao et al., 1998) which is used for the production of non-calorific artificial sweetener Aspartame.

CHEMICAL MODIFICATION OF ENZYMES WITH AMPHIPATIC COMPOUNDS

As a means of improving stabilization under conditions of the presence of organic solvents, enzymes can be chemically modified by amphipatic compounds, making them become soluble in the organic solvent (Inada et al., 1986). For example, according to (Takahashi et al., 1984), the modification of enzymes by using an amphipatic polymer, polyethylene glycol (PEG), makes the enzyme soluble in organic solvents like benzene.

This is possible because PEG modified enzymes are able to maintain the catalytic activity of the enzymes even in organic solvents by creating an aqueous shell about the enzyme molecules and by so doing, prevent the denaturation of the enzyme in organic solvents.

PHISICAL MODIFICATION OF ENZYME WITH LIPIDS

The process of freeze-drying or dehydration by reducing the surrounding pressure and adding enough heat in order to allow the frozen water to sublime can be used to coat enzymes with lipids via lyophilisation.

These lyophilised enzymes are soluble and have high activity in organic solvents (Okazaki et al., 1997).

The nature of the lipid used determines the performance of the lipid coated-enzymes. The lipid-coated enzymes are said to be similar to the enzymes chemically modified by amphipatic compounds but the structural configuration of lipid-coated enzymes are not well understood (Ogino and Ishikawa, 2001).

The catalytic activity yield of lipid-coated enzymes is higher when compared with enzymes that are chemically modified with amphipatic compounds. However, lipid-coated enzyme applications may be limited because these enzymes contain large amounts of lipids which may block the isolation and recovery of the reaction mixture products.

ENTRAPMENT OF ENZYMES IN THE REVERSED MICELLES

When surfactants like detergents, phospholipids, etc are dissolved in non-polar organic solvent, reversed micelles are formed (Martinek et al., 1986).The outer shell is formed by the hydrophobic tails of surfactant molecules while the inner core comprises polar heads of these molecules.

Polar compounds are solubilised in reversed micelles and they tend to form a micro-droplet that is isolated against organic solvents through a surfactant layer (Luisi and Magid, 1986).

At the molecular level, the reversed micellar system is said to be a 2-phase system in where the properties of the micellar-water is absolutely different from those of bulk water based on polarity, viscosity, and nucleophilicity. Hence, these enzymes are trapped and they often exhibit a high level of catalytic activity (super activity) in comparison with other native native enzymes in the bulk aqueous phase (Martinek, 1989).

SOLID ENZYMES

With the use of crude commercial powder enzymes as catalysts in organic solvents and in the suspension form, solid enzymes can be prepared by crystallization, lyophilisation, and precipitation with acetone from aqueous solutions (Klibanov, 1989).

These solid enzymes have the ability to preserve their catalytic activity in organic solvents via forming a layer of denatured protein on their surface and shielding the inner layer from adverse contact with the organic solvent.

The enzyme molecules inside the solid enzyme particles are tightly squeezed from all sides by neighbouring molecules which results in a high level of conformational stability (Zaks and Klibanov, 1985). This process is known as the ''cage effect'', which can also be used to demonstrate enzyme stability.

Due to the very small specific surface area of the enzyme, solid enzymes posses a specific activity when suspended in organic solvents. Thus, a considerable amount of enzyme is needed to achieve the desired catalytic activity (Zaks and Klibanov, 1985).

Advantages of the utilization of enzymes in non-aqueous solvents in comparison with water are very many (Ogino and Ishikawa, 2001). They include, the increased level of solubility in non-polar substrates, the shifting of thermodynamic equilibrium favouring synthesis over hydrolysis, and the elimination of microbial contamination in the reaction mixture (Dordick, 1991; Gupta, 1992).

Non aqueous solvents may disrupt enzyme molecules or in some cases become competitive inhibitors by specifically hindering the interaction of the enzyme which may result in alteration of the reaction kinetics. However, these changes can be prevented (Ogino and Ishikawa, 2001).

Over the past 20 years, the applications of enzymes in non-aqueous solvents have increased with more developments and discoveries (Zaks and Russell, 1988). The hydrolases like lipases, proteases, and esterases are the most important classes of enzymes utilised.

LIPASE-CATALSED REACTIONS

One of the first important areas of applications of lipases in organic solvents is their utilisation in the processing of fats and oils producing triglycerides having desirable physical properties (Yokozeki et al., 1982).

The major benefit of using these enzymes is because of their specificity; thus, allowing esterification to be limited to specific positions based on the enzyme used and not randomly. For instance, it has been reported that immobilised lipase (Rhizopus delemar) can successfully catalyse the inter-esterification of oils with stearic acid in hexane (Yokozeki et al., 1982).

In synthetic organic chemistry, the production of optically pure compounds are of immerse importance. Asymmetric hydrolysis of corresponding esters involves the utilization of lipases in racemic alcohols and carboxylic acids. And the possibility of enzymatic resolution of these racemic mixtures using esterification, aminolysis, etc is further enhanced (Kirshner et al, 1985).

This transesterification reaction is impossible in water because of the hydrolsis reaction which dominates the reaction, but in organic solvents, optically pure esters can be produced on a gram scale because lipases are continuously active in organic solvents and hydrolysis is insignificant (Kirshner, 1985).

Presently, there is demand on the availability of various peptide structures and the organic synthesis and biological production are difficult and costly processes but enzymatic preparation of these peptides is an easy option or alternative.

The synthesis of peptides, catalysed by proteases can be achieved from amino acids. However, the drawback here is that even as the process of synthesis is achieved, these proteases also catalyse the hydrolysis of the growing chain.

A well developede alternative to this problem is the utilization of non-proteolytic enzymes like lipases in organic solvents (West and Wong, 1986; Margolin and Klibanov, 1987).

OXIDOREDUCTASE-CATALYSED REAACTIONS

Alcohol dehydrogenases and its co-factors have a unique property of stereo-specificity and this can be exploited in the oxidoreduction of several organic compounds (Eliel and Otsuka, 1982). The exploitation of alcohol dehydrogenases in organic solvents erases the problem of instability in aqueous surroundings.

The co-factor (NAD/NADH) - which is actually insoluble in organic solvents has been found to be efficiently regenerated when in combination with alcohol dehydrogenases and deposited on a surface of glass beeds, then suspended in water immiscible solvent containing substrates (Grunwald et al., 1986).

Polyphenol oxidase which is a highly region-specific enzyme can be utilized in the hydroxylation of phenols. Unfortunately, polyphenol oxidase is unstable in water and this is a major drawback in the use of this enzyme. However, this drawback can be prevented by making use of the enzyme in organic solvents (Kazandjian and Klibanov, 1985).

The horseradish peroxidise is another enzyme that catalyses reactions vigorously in various organic solvents (Kazandjian et al., 1986). For instance, hydrogen peroxide has been found to bring about the oxidation of p-anisidine in several solvents like toluene, ethyl acetate and ether. (Kazandjian et al., 1986).

DETECTION DEVICE

Enzymes in non-aqueous solvents can be utilised in the production of enzymatic sensors for numerous thermolabile products (Boeriu, 1986).

Since there is no hindering in the level of diffusion in the solution state, horseradish peroxidise has also been found to cause the oxidation of p-anisidine in liquid hexadecane a million times quicker than in the solid hydrocarbon (xxx).

Therefore, when there is solidification of horseradish peroxidise, p-anisidine, hydrogen peroxide, and hexadecane by freezing and allowed to stay at 4oC for about 3 weeks, there is no reaction observed ( though this eventually leads to a brown colouration).

However, when allowed to stay at room temperature for about an hour, reaction was observed by a distinctive reddish-brown colour being produced (xxx). Enzymatic detection devices can be made by using this principle.

Gupta, hf. N.: Enzyme function in organic solvents. Eur. J.

Biochem., 203, 25-32 (1992).

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