Enzymes in supercritical fluids

The use of enzymes in supercritical fluids presents the problem that the number of parameters influencing the stability of the enzymes increases dramatically. This is the reason why, up to now, no prediction can be made of whether an enzyme is stable under supercritical conditions and if the equivalent of even higher activity and selectivity is available compared with reactions in organic solvents. In the following chapters the influence of parameters on the enzyme stability will be given. Although much research has been done [2-5] it is very difficult to compare these results because non-standard methods have been applied. 

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. 

T. Hartmann, E. Schwabe, T. Scheper, Enzyme catalysis in supercritical fluids in R. Patel, Stereoselective Biocatalysis, Marcel Dekker, 2000, 799. 

Fontes tt al. [224,225 addressed the acid—base effects of the zeolites on enzymes in nonaqueous media by looking at how these materials affected the catalytic activity of cross-linked subtilisin microcrystals in supercritical fluids (C02, ethane) and in polar and nonpolar organic solvents (acetonitrile, hexane) at controlled water activity (aw). They were interested in how immobilization of subtilisin on zeolite could affected its ionization state and hence their catalytic performances. Transesterification activity of substilisin supported on NaA zeolite is improved up to 10-fold and 100-fold when performed under low aw values in supercritical-C02 and supercritical-ethane respectively. The increase is also observed when increasing the amount of zeolite due not only to a dehydrating effect but also to a cation exchange process between the surface proton of the enzyme and the sodium ions of the zeolite. The resulting basic form of the enzyme enhances the catalytic activity. In organic solvent the activity was even more enhanced than in sc-hexane, 10-fold and 20-fold for acetonitrile and hexane, respectively, probably due to a difference in the solubility of the acid byproduct. 

High-pressure FT-IR spectroscopy has been used to clarify (1) the rotational isomerism of molecules, (2) characteristics of water and the water-head group, and (3) RSO3 Na4- interactions in reverse micellar aggregates in supercritical ethane. This work demonstrates interesting pressure, temperature, and salt effects on an enzyme-catalyzed esterification and/or maintenance of a one-phase microemulsion in supercritical fluids from practical and theoretical points of view (Ikushima, 1997). 

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). 

Over the past decade, much progress in supercritical fluid technology has occurred. For example, supercritical fluids have found widespread use in extractions (2-5), chromatography (6-9), chemical reaction processes (10,11), and oil recovery (12). Most recently, they have even been used as a solvent for carrying out enzyme-based reactions (14). Unfortunately, although supercritical fluids are used effectively in a myriad of areas, there is still a lack of a detailed understanding of fundamental processes that govern these peculiar solvents. 

Many enzymes are stable and catalyze reactions in supercritical fluids, just as they do in other non- or microaqueous environments (7). Enzyme stability and activity may depend on the enzyme species, supercritical fluid, water content of the enzyme/support/reaction mixture, decompression rates, exposure times, and pressure and temperature of the reaction system. 

A series of enzyme-catalyzed reactions recently conducted in both conventional and supercritical fluid medium has shown that while no loss of enzyme activity was experimentally observed for the conventional medium, the same was no longer valid for supercritical C02 systems (1,4,10,11). For instance, Steinberger and Marr (12) have pointed out that the stability of an enzyme in supercritical C02 depends onboth its tertiary structure and several parameters during exposure to high-pressure fluid. They argued that high temperatures, the water content in C02 and pressurization/de-pressurization steps might cause enzyme inactivation. 

Supercriticality in an environment does not, in itself, prohibit life. Some terran enzymes are known to be active in supercritical fluids.30-32 Subsequent reviews can be found in Aaltonen and Rantakyla,33 Kamat et al.,34 and Aaltonen.35 Although most of that work concerns supercritical carbon dioxide as the solvent, fluorinated hydrocarbons (HCF3) and simple alkanes (e.g., ethane, propane) have also been reported,36 providing a formal demonstration that terran-derived proteins can function in these media. Any enzyme adapted to the supercritical media would undoubtedly be different from those used in the studies cited. 

The prospect of using enzymes as heterogeneous catalysts in scC02 media has created significant interest. Their low viscosity and high diffusion rates offer the possibility of increasing the rate of mass-transfer controlled reactions. Also, because enzymes are not soluble in supercritical fluids, dispersion of the free enzymes potentially allows simple separations without the need for immobilization. 

Solubilization of Cytochrome C in propane/AOT/water solutions is particularly convincing evidence for micelle formation in supercritical fluids because it excludes the possibility of a simple ion-pair mechanism of solubilization. It seems likely that this large, water soluble enzyme is solvated by the highly hydrophobic fluids only if the polar functional groups on the surface of the protein are shielded from the fluid by surfactant molecules. 

A number of other important potential applications of a micellar phase in supercritical fluids may utilize the unique properties of the supercritical fluid phase. For instance, polar catalyst or enzymes could be molecularly dispersed in a nonpolar gas phase via micelles, opening a new class of gas phase reactions. Because diffusivities of reactants or products are high in the supercritical fluid continuous phase, high transport rates to and from active sites in the catalyst-containing micelle may increase reaction rates for those reactions which are diffusion limited. 

In addition to chemicals, biological catalysts such as enzymes can be used to catalyze reactions in SC CO2. Since the first attempt to operate reactions in supercritical fluids published by Randolph et al. [34], various type of enzymes were studied lipase, oxidase, decarboxylase, dehydrogenase, proteinase, etc. [33,35-37]. The effect of different parameters was extensively reported by Ballesteros et al. [35]. Enzyme activity and stability in supercritical conditions as well as the benefits of using supercritical fluids for enzymatic reactions (improved reaction rates, control of selectivity, etc.) have been demonstrated [36]. 

Perrut, M. Enzymic reactions and cell behavior in supercritical fluids. Chem. Biochem. Eng. Q. 1994, 5 (1), 25-30. 

Nakamura, K. Enzymic synthesis in supercritical fluids. In Supercritical Fluid Technology in Oil and Lipid Chemistry, AOCS Press Champaign, 1996 306-320. 

Kamat SV, Beckman EJ, Russell AJ. Enzyme activity in supercritical fluids. Crit Rev Biotechnol 1995 15 41-71. 

Kamat SV, Iwaskewycz B, Beckman EJ, Russell AJ. Biocatalytic S5mthesis of acrylates in supercritical fluids tuning enzyme activity by changing pressure. Proc Nat Acad Sci USA 1993 90 2940-2944. 

Enzyme reactions have been successfully operated in a variety of organic solvents (Table 8.4) as well as in supercritical fluids (e.g., carbon dioxide and fluoroform) and gases. - The latter two categories offer some intriguing possibilities and potential advantages relative to solvents, including enhanced substrate diffusivity, tunable solvent phase properties (via temperature and pressure), reduced solvent... 

Chaudhary, A. K., Beckman, E. J., and Russell, A. J., Rational control of polymer molecular weight and dispersity during enzyme-catalyzed polyester synthesis in supercritical fluids, J. Am. Chem. Soc., 117, 3728-3733, 1995. 

Fontes, N., Almeida, M.C., and Barreiros, S. (2001) Biotransformations in supercritical fluids. Methods Biotechnol., 15, (Enzymes in Nonaqueous Solvents), 565-573. 

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. 

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