Enzymes, in SCFs

Bioreactions. The use of supercritical fluids, and in particular C02, as a reaction media for enzymatic catalysis is growing. High diffusivities, low surface tensions, solubility control, low toxicity, and minimal problems with solvent residues all make SCFs attractive. In addition, other advantages for using enzymes in SCFs instead of water include reactions where water is a product, which can be driven to completion increased solubilities of hydrophobic materials increased biomolecular thermostability and the potential to integrate both the reaction and separation bioprocesses into one step (98). There have been a number of biocatalysis reactions in SCFs reported (99—101). The use of lipases shows perhaps the most commercial promise, but there are a number of issues remaining unresolved, such as solvent—enzyme interactions and the influence of the reaction environment. A potential area for increased research is the synthesis of monodisperse biopolymers in supercritical fluids (102). 

Water affects the reaction rate through its effect on reaction kinetics and protein hydration, which is required for optimal enzyme conformation and activity. Enzymes need a small amount of water to maintain their activity however, increasing the water content can decrease the reaction rate as a result of hydrophilic hin-drance/barrier to the hydrophobic substrate, or because of denaturation of the enzyme (189). These opposite effects result in an optimum water content for each enzyme. In SCFs, both the water content of the enzyme support and water solubilized in the supercritical phase determine the enzyme activity. Water content of the enzyme support is, in turn, determined by the distribution/partition of water between the enzyme and solvent, which can be estimated from water adsorption isotherms (141, 152). The solubility of water in the supercritical phase, operating conditions, and composition of the system (i.e., ethanol content) can affect the water distribution and, hence, determine the total amount of water that needs to be introduced into the system to attain the optimum water content of the support. The optimum water content of the enzyme is not affected by the reaction media, as demonstrated by Marty et al. (152), for esterification reaction using immobilized lipase in n-hexane and SCC02- Enzyme activity in different solvents should, thus, be compared at similar water content of the enzyme support. 

Chulalaksananukul et al. measured the residual activity of a lipase from Mucor miehei after one day in SCCO2 at 40-100 °C at various water concentrations. As the temperature rises, the enzyme molecule at first unfolds reversibly and then undergoes one or more of the following reactions formation of incorrect or scrambled structures, cleavage of disulfide bonds, deamination of tryp-sine residues, and hydrolysis of peptide bonds. Each process requires water and is therefore accelerated with increasing water concentration [19]. The role of water on the performance of enzymes in SCFs is described in more detail in Section 4.9.4.3... 

Experiments with enzymes in SCFs revealed very early that scCOa could strip the essential water from the enzyme. Randolph et al. found that damp immobilized enzyme lost its activity when exposed to bone-dry carbon dioxide. The activity was quickly regained when they injected a small amount of water into the system [12]. Dumont also reported results from an immobilized lipase/C02 system where the conversion rate decreased when the enzyme was in contact with a dry COa/substrate flow. Reaction rates were restored completely when they directed the CO2 flow through a water saturator [13]. 

Kus, B., Caldon, C. E., Andorn-Broza, R., and Edwards, A. M. Functional interactions of 13 yeast SCF complexes with a set of yeast F2 enzymes in vitro. Proteins Structure, Function, and Bioinformatics 2004, 54, 455-67. 

Often a cosolvent is used in order to solubilize particularly polar substrates such as sugars and amino acids. Surfactants or additional solvents may also allow adequate solvation of enzymes. In some cases two-phase systems can be used to conduct bioconversion. For example, Reetz and coworkers employed both SCFs and ionic liquids in a semi-continuous... 

After initial findings that some enzymes were active and stable in SCF s [1,2], several other studies on enzyme catalysis in SCF s have been carried out. The majority of systems explored to date have used model reactions [3,4] of which only a few show a possibility for practical and commercial purposes [5],... 

Enzymatic catalysis in SCFs exploits the ability to alter solvent strength with small changes in temperature and pressure in the near-critical region. The versatile solvation characteristics of SCFs, the extreme catalytic specificity of enzymes, and the ability to achieve complete removal of the SCF solvent by depressurization make enzymatic catalysis in SCFs attractive to the pharmaceutical and food industries. 

Greater enantioselectivity was found at 60 bar than at higher pressures. The lipid coating of the enzyme was used to enhance the solubility of the enzyme in nonpolar solvents such as alkanes and SCCHF3 in fact, it was reported to be soluble in the SCF. 

Many chemical reactions carried out in supercritical fluid media were discussed in the first edition, and those developments are included in total here after some recent work is described. In the epilogue (chapter 13) of the first edition we made reference to one of the author s work in enzyme catalyzed reactions in supercritical fluids that was (then) soon to appear in the literature. The paper (Hammond et al., 1985) was published while the first edition was in print, and as it turned out, there was a flurry of other activity in SCF-enzyme catalysis many articles describing work with a variety of enzymes, e.g., alkaline phosphatase, polyphenol oxidase, cholesterolase, lipase, etc., were published starting in mid 1985. Practical motivations were a potentially easier workup and purification of a product if the solvent is a gas (i.e., no liquid solvent residues to contend with), faster reaction rates of compounds because of gas-like transport properties, environmental advantages of carbon dioxide, and the like. 

One of the expected benefits from using enzymes in supercritical fluids (SCFs) is that mass transfer resistance between the reaction mixture and the active sites in the solid enzyme should be greatly reduced if the reactants and products are dissolved in an SCF instead of running the reaction in a liquid phase. It is expected that the high diffusivity and low viscosity of SCFs will accelerate mass-transfer controlled reactions. 

Because enzymes are not soluble in SCFs it should be possible to disperse free enzyme in the SCF and recover the enzyme without the need to immobilize it on a support. 

As SCFs are often hydrophobic, many water-insoluble compounds may be processed in one phase, instead of the two-phase systems involved with using enzymes as homogeneous catalysts in aqueous solutions. For the same reason traditional hydrolysis reactions may be reversed and esterifications may be accomplished in SCFs with hydrolytic enzymes. 

The first reports of enzyme activity in SCFs were published almost simultaneously in 1985 and 1986 [1-3]. Great enthusiasm emerged and since this pioneering work more than 200 papers on enzymatic catalysis in SCFs have appeared. The subject has been reviewed in 1991 [4] and in 1995 [5]. 

This chapter first explains enzyme nomenclature, describes enzymatic, supercritical reactor configurations, and gives a compilation of published experimental results. The- most important topics concerning enzymatic reactions in SCFs are then covered. These are factors affecting enzyme stability, the role of water in enzymatic catalysis, and the effect of pressure on reaction rates. Studies on mass transfer effects are also reviewed as are factors that have an effect on reaction selectivities. Finally, a rough cost calculation for a hypothetical industrial process is given. 

At VTT Chemical Technology, the following procedure has been used for screening enzymes and reaction conditions in SCFs in a batch reactor. Figure 4.9-2 describes the batch reactor conflguration. 

Many enzymes are stable and catalyze reactions in supercritical fluids, just as they do in other non- or microaqueous environments. Many enzymes are more stable in supercritical fluids than in aqueous solutions. However, enzymes are not soluble in SCFs therefore enzymatic catalysis in SCFs is always heterogeneous. 

As one might expect, water has a dramatic effect on enzyme stability in SCFs. Lozano et al. found that the half-life time of a-chymotrypsin decreased exponentially in SCCO2 with increasing water content from 0 to 15 wt% [28]. Kashe et al. found that a-chymotrypsin, trypsin and penicillin amidase partially unfolded during pressure reduction in humid SCCO2. They suggested that... 

Kamat et al. dlso support this hypothesis [18]. They compared lipase-catalyzed transesterification rates in supercritical carbon dioxide, fluoroform, ethylene, ethane, propane, and sulfur hexafluoride as well as in several conventional liquid solvents of different polarities. The reaction rates increased with increasing hydrophobicity of solvent within the SCFs and also within the liquid solvent group. Because the solvent s immiscibility with water and its apolarity, by themselves, are irrelevant to enzymatic activity [8], it appears that the activity loss is the result of the enzyme losing essential water. Although SCCO2 is generally considered to be a hydrophobic solvent, it is more hydrophilic than fluoroform or hexane and capable of stripping essential water from the enzyme in an essentially nonaqueous environment. 

In aqueous environments, enzymatic activity is sensitive to the pH of the bulk solution. One may therefore suspect that the SCCO2, which is dissolved in the microwater layer of an enzyme in an essentially nonaqueous system, would change the pH of that layer and affect enzyme activity. Kamat et al. clearly showed that this effect is negligible [5]. Increasing the CO2 pressure by a factor of 100 decreases the pH of bulk water by only one unit in an unbuffered system. Enzymes are normally lyophilized from a buffered solution prior to their use as catalysts in SCFs. In lyophilization, the enzyme is first dissolved in water where the pH is adjusted for maximum enzymatic activity. The enzyme/buffer salt solution is then freeze-dried under vacuum to remove almost all of the water. In a typical phosphate buffer solution, the pH may be 7.8. Kamat et al. calculated that at 100 bar CO2 pressure the new pH of the buffer solution would be 7.75, and at 1010 bar the pH would be 7.66. Lyophilization increases the buffer salt concentration in the residual water in the enzyme considerably. Thus, the effect of CO2 on the pH of the remaining microaqueous layer in enzymes becomes even smaller. 

Reaction Enzyme support optimum water concentration in SCF in solid (wt%) 1 Reference... 

Apart from the direct conformational changes in enzymes, which may occur at very high pressures, pressure affects enzymatic reaction rates in SCFs in two ways. First, the reaction rate constant changes with pressure according to transition stage theory and standard thermodynamics. Theoretically, one can predict the effect of pressure on reaction rate if the reaction mechanism, the activation volumes and the compressibility factors are known. Second, the reaction rates may change with the density of SCFs because physical parameters, such... 

Thus, stereoselectivities from 0% to 100% have been reported for enzymatic reactions in SCFs. These results show that enzymatic stereoselectivity can be altered by adjusting the properties of the SCF. However, only very qualitative rules exist at present with which to predict how reaction selectivities might change with conditions for a new substrate/enzyme/SCF system. 

The experimental data that have accumulated since ca. 1992 have greatly increased the understanding of the interactions between enzymes and SCFs. It is now known that enzymatic reaction rates, substrate selectivity and enantiomeric selectivity can be tailored by altering the type and the density of the SCF. Although CO2 would be the most desirable industrial SCF as reaction medium, it is not suitable for all enzymes. It is an inhibitor for some proteases and lipases [20] but there are other common enzymes, including lipases, which are stable and active in SCCO2. Supercritical hydrocarbons and fluoroform are good and tunable solvents for enzymatic reactions. 

The selectivity of enzymes, especially enantiomeric selectivity, can be fully utilized in SCFs. High selectivity can usually be obtained at slightly elevated temperatures when conditions are optimized and conversion is limited to less than the maximum achievable level. The use of extremely low (down to -90 °C) temperatures, often needed for high selectivity in conventional catalysis, may thus be avoided. 

Reactions involving ester bonds have been extensively explored using lipases as catalysts in SCFs. However, enzymes catalyze a variety of reactions and synthetically much more useful reactions may be found. 

Micromicelles have been used to overcome the solubility limitation of polar compounds in SCFs (see Chapter 2.4). The technique involves using SCF-soluble surfactants. Using enzymes in micromicelles for processing hydrophilic substrates in SCFs has not yet been extensively explored and holds considerable promise for future developments. 

Transesterification has also been used to demonstrate that enzyme catalysis can be carried out in SCF-IL biphasic systems. ILs can stabilize enzymes, thereby enabling their use at higher temperatures [61, 62]. SCCO2, on the other hand, may cause reductions in activity for enzymes either because of changes in pH, as a result of the acidic CO2 dissolving in water, or as a result of conformational changes... 

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