Info

A freeze-dried lipase powder (10 mg) was added to 1 ml of an organic solvent containing 0.3 M tributyrin and heptanol; the mixture was shaken at 20° C, and the time-course of the reaction was followed by gas chromatography. Organic solvents contained 0.02% (wt/wt) water, except for the toluene and carbon tetrachloride, in which that water concentration is not attainable and, therefore, it was even lower (our method of measurement does not afford an exact determination in that range). The water content of the lipases was 0.5%, 6.1%, and 4.8% for porcine pancreatic (which was precipitated from pH 8.4), yeast and mold, respectively. The lipases inactivated with diethyl p-nitrophenylphosphate exhibited no enzymatic activity in all organic solvents. From Zaks and Klibanov (1985) with permission of the authors and publisher.

A freeze-dried lipase powder (10 mg) was added to 1 ml of an organic solvent containing 0.3 M tributyrin and heptanol; the mixture was shaken at 20° C, and the time-course of the reaction was followed by gas chromatography. Organic solvents contained 0.02% (wt/wt) water, except for the toluene and carbon tetrachloride, in which that water concentration is not attainable and, therefore, it was even lower (our method of measurement does not afford an exact determination in that range). The water content of the lipases was 0.5%, 6.1%, and 4.8% for porcine pancreatic (which was precipitated from pH 8.4), yeast and mold, respectively. The lipases inactivated with diethyl p-nitrophenylphosphate exhibited no enzymatic activity in all organic solvents. From Zaks and Klibanov (1985) with permission of the authors and publisher.

stable. In principle, protein solvation effects are exerted over a short range, and the stability of an enzyme is rarely influenced by water molecules lying beyond a few layers of solvent molecules from its surface. The properties of three lipases in various organic solvents (Table 7.12) were explored by Zaks and Klibanov (1985), who observed that these lipases retained their catalytic activity in solvents that were immiscible with water, but lost activity when placed in solvents like pyridine, acetone, and formamide. The latter are known to dehydrate the protein surfaces. Zaks and Klibanov (1985) demonstrated that different lipases remain effective catalysts, even in the presence of nearly water-immiscible organic solvents. The catalytic power exhibited by the lipases in organic solvents is comparable to that displayed in water. In addition to transesterification, lipases can catalyze several other processes in organic media including esterification, aminolysis, acyl exchange, and thio-transesterification, such that several actually only proceed to an appreciable extent in nonaqueous solvents. In hexane, porcine pancreatic lipase, for example, still catalyzed various transesterification reactions and, in doing so, obeyed Michaelis-Menten kinetics. The enzyme's pH-dependence in organic media was also bell-shaped, showing maximum coinciding with the pH optimum for enzymatic activity in water.

Zaks and Klibanov (1985) suggested that such behavior could be exploited in enzyme reactors to take advantage of: (a) the high solubility of most organic compounds in nonaqueous media; (b) the potential for carrying out new reactions impossible in water because of kinetic or ther-modynamic restrictions; (c) the enhanced stability of enzymes; (d) the relative facility of product recovery from organic solvents, as compared to water; and (e) the insolubility of enzymes in organic media, thus facilitating enzyme recovery. A fascinating aspect of their findings is that hydrophobic solvents actually result in higher enzymatic activity than their hydrophilic counterparts (Klibanov, 2oo3). This property has been attributed to the presence of a few clusters of water molecules, which are stubbornly bound to charged groups on the surface of freeze-dried enzyme molecules and are required for enzymatic function. Hydrophilic solvents tend to strip some of this essential water off enzyme molecules, thereby lowering the catalytic power. This effect can be prevented, however, with the consequent restoration of enzymatic activity, by adding small quantities of exogenous water to the solvent (Zaks and Klibanov, 1988). Klibanov (2001) reviewed the literature showing that enzymatic selectivity (including substrate selectivity/specificity, stereo-selectivity, and chemo-selec-tivity) can be markedly affected, and even inverted in some cases, by the solvent. These effects were observed at ambient temperature, proving that these properties are not limited to the use as cryosolvents.

While pH profoundly influences enzymatic activity in aqueous solution, pH has no physicochemical meaning in organic solvents (Klibanov, 2oo1). However, when placed in organic media, enzymes and proteins exhibit a "pH memory,'' meaning that their catalytic activity reflects the pH of the last aqueous solution to which they were exposed. This phenomenon appears to be due to the fact that a protein's ionogenic groups retain their last ionization state upon dehydration and subsequent placement in organic solvents. Therefore, the enzymatic activity in such media can be much enhanced, sometimes hundreds of times, if enzymes are lyophilized from aqueous solutions of the pH optimal for catalysis.

Despite the fact that lipases are admittedly stable at water-lipid interfaces, later studies confirmed that water-insoluble organic solvents permitted enzyme activity (Table 7.13). When a lyophilized enzyme (which typically retains ~0.01% water, amounting to a few hundred water molecules per enzyme molecule) is suspended in a suitable anhydrous organic solvent, enzymatic activity is frequently retained. It is important to recognize, however, that the enzyme is suspended (not dissolved) in the organic solvent phase and behaves as a heterogeneous catalyst. To facilitate diffusion of reactants into the enzyme, the freeze-dried enzyme must be mixed vigorously, usually by sonication, to obtain finely dispersed small particles.

Solvent polarity is believed to be the most important factor in balancing enzyme stabilization versus enzyme inactivation by certain organic solvents (Chaplin and Bucke, 1990). Those solvents with low polarity (i.e., greater hydrophobicity) tend to be less disruptive, because they do not strip the enzyme molecules of the tightly bound water molecules that play a major structure-stabilizing role. Perhaps the best measure of polarity is log1oP, the logarithm

TABLE 7.13 Some Benefits of Conducting Enzymatic

Catalysis in Organic Solvents

• Increased solubility of apolar reactants and certain cofactors.

• Suppression of nonenzymatic (spontaneous) reactions and side-reactions.

• Enhanced stability of some enzymes.

• Altered selectivity of an enzymatic toward its substrates.

• Enzymatic properties affected and modulated by the molecular "memory" of the enzyme sample (i.e., persistent retention of pH, and other solutes present prior to addition of the apolar organic solvent).

• Permanent enzyme complexes with ligands insoluble in organic solvents;hence no dissociation of water-trapped solutes, even when enzyme normally exhibits low affinity for its substrate.

• Reversal of hydrolases (Reaction: R-C(=O)-O-R' # R-COOH + R ' -OH) to favor synthesis of apolar esters.

• Altered enantiomeric enrichment, depending on the nature of the solvent.

Based on Klibanov (2003).

of the partition coefficient P = Sn_octanol/Swater, where Sn-octanol and Swater are the respective solubilities for a substance in n-octanol and water (i.e., log P = log{Sn_octanol/ Swater}). Typical logi0 P values are: butanone, 0.3; ethyl acetate, 0.1; butanol, 0.8; diethyl ether, 0.8; methylene chloride (CH2Q2), 1.4; di-isopropyl ether, 2.0; benzene, 2.0; chloroform, 2.2; toluene, 2.1; carbon tetrachloride, 2.8; dibutylether, 2.9; cyclohexane, 3.1; petroleum ether (degree of polymerization = 60-80), 3.5; petroleum ether (degree of polymerization = 80-100), 3.5; heptane, 3.5; petroleum ether (degree of polymerization = 100-120), 4.3; and hex-adecane, 8.1 (Chaplin and Bucke, 1990). A rule-of-thumb is that enzymes are most often inactivated by solvents having logi0 P value less than 2, whereas solvents with logi0 P values exceeding 4 have little effect. An interesting finding is that the presence of deuterium oxide tends to enhance protein stability to a greater degree than H2o.

Chymotrypsin also catalyzes peptide hydrolysis in n-octane (Zaks and Klibanov, 1986), but the substrate specificity is altered. For example, while N-acetyl-L-histi-dine methyl ester is cleaved 200 x more slowly than N-acetyl-L-phenylalanine ethyl ester in aqueous medium, it is cleaved 20 x faster in n-octane.

Enzyme-catalyzed condensation of carboxylic acids with amines and alcohols in the presence of a water-immiscible organic solvent is useful for driving the syntheses of amides and esters that are intrinsically more soluble in organic solvents (Kobayashi and Adachi, 2004). To optimize reaction conditions for maximal product yield, knowledge of the reaction equilibrium and the logi0P value for amide/ester are helpful. In some cases, water-miscible organic solvents may be used, especially when enzyme stability is a consideration. For example, Castillo and Lopez-Munguia (2004) reported that the synthesis of levan, using Bacillus subtilis levansucrase, occurs in the presence of the water-miscible solvents acetone, acetoni-trile and 2-methyl-2-propanol (2M2P). Enzyme activity is only slightly affected by acetone and acetonitrile, but 2-methyl-2-propanol has an activating effect. The enzyme is highly stable in water at 30°C; however, incubation in the presence of 15 and 50% (vol/vol) 2M2P reduced the halflife to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2-methyl-2-propanol, where a half-life of 11.8 days is observed. The presence of methyl-2-propanol in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with i0% (w/v) sucrose in 50/50 water/methyl-2-propanol, sucrose is converted to levan and an aqueous two-phase system (2M2P/ levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high-molecular-weight levan is obtained as a hydrogel and may be easily recovered from the reaction medium.

When long-chain alcohols are used as organic solvents, workers must entertain the possibility of the alcoholysis of acyl intermediates. For example, long-chain alcohols may replace water as the acyl group acceptor in reactions catalyzed by seine proteases and phosphomonoester hydrolases. Depending on the product quantified in rate experiments, alcoholysis of acyl intermediates may give the appearance of enhanced enzymatic activity.

Castro and Knubovets (2003) have discussed the correlation between activity and structure of the intact enzymes suspended in neat organic solvents, as well as modifications of natural enzymes to favor catalytic active in non-aqueous environment. Bruns and Tiller (2005) also described what they called a "nanophase-separated amphiphilic network,'' consisting of an enzyme entrapped within its hydrophilic domains. A substrate that diffuses into the hydrophobic phase of such a network can access the biocatalyst by way of the extremely large interface. Entrapped horseradish peroxidase and chloroperoxidase exhibited dramatically increased activity and operational stability compared to the native enzymes.

Lyophilized salt-enzyme preparations exhibit activities that are increased by factors as high as 35,000 relative to activities of lyophilized salt-free preparations (Lindsay, Clark and Dordick, 2004). Among the factors associated with reduced enzyme activity in organic solvents are decreased enzyme stability and partial denaturation, reduced enzyme flexibility, incompatibility of solvent and enzyme transition states, over-stabilization of the substrate in its ground-state, and dehydration of the enzyme's water shell. Using NMR spectroscopy, Eppler et al. (2006) determined deuterium spin relaxation rates to assess how salt affects the structure of lyophilized enzymes suspended in organic solvents. Such results suggest that the presence of added salt increases in enzyme-bound water mobility, serving as a molecular lubricant to enhance enzyme flexibility. This increased flexibility may facilitate transitionstate mobility and catalysis in the salt-activated enzyme.

The range of organic solvents that support enzymatic activity has been greatly expanded through the advent of nonaqueous ionic liquids composed solely of a charged organic molecule and a suitable counter-ion. Two examples are i-butyl-3-methylimidazolium cation and i-ethyl-3-methyimidazolium cation:

H2C CH2 H2C CH3

h2c—ch3 Nonaqueous Ionic Liquids

H2C CH2 H2C CH3

h2c—ch3 Nonaqueous Ionic Liquids

When combined with a suitable counter-ion, such as trifluoromethanesulfonate anion above, these imidazolium cations form ionic liquids in which many proteins are "soluble." Irimescu and Kato (2004) reported lipase-cata-lyzed enantioselective reaction of amines with carboxylic acids in ionic liquids of this type. Because ionic solutions bind water tightly, hydrolases may catalyze dehydration when carried out in ionic solutions (e.g., R-COOH + R'-OH # R-C(O)-O-R' + H2O| and R-COOH + R'-NH2 # R-C(O)-NH-R' + H2O|, where the symbol Y indicates depletion of water by combination with an ionic solvent). In this respect, ionic solvents may facilitate peptide bond formation.

Finally, by applying two-dimensional mean-field lattice theory to model enzyme immobilization and stabilization on a hydrophobic surface containing grafted polymers, such as polyethylene glycol, Moscovitz and Srebnik (2005) concluded that: (a) short hydrophilic grafted polymers should protect surface-immobilized enzymes from unwanted adsorption and denaturation upon contact with the hydro-phobic surface; (b) screening is most effective when using a combination of short- and long-chain polymer grafts; and (c) grafted hydrophilic polymers should also protect enzymes when organic solvents are needed to solubilize substrates in the bulk solution phase.

Healthy Chemistry For Optimal Health

Healthy Chemistry For Optimal Health

Thousands Have Used Chemicals To Improve Their Medical Condition. This Book Is one Of The Most Valuable Resources In The World When It Comes To Chemicals. Not All Chemicals Are Harmful For Your Body – Find Out Those That Helps To Maintain Your Health.

Get My Free Ebook


Post a comment