Enzyme-catalyzed reactions in supercritical

Chrisochoou, A. Schaber, K. Bolz, U. Phase Equilibria for Enzyme-Catalyzed Reactions in Supercritical Carbon Dioxide. Fluid Phase Equilib. 1995,108, 1-14. 

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. 

Table 4.9-2 A compilation of enzyme-catalyzed reactions in supercritical fluids.

Although not specifically fitting in the category of enzyme catalyzed reactions in supercritical fluids, there have been other studies that have investigated the effects of supercritical fluids on enzyme stability. In some cases there was a loss of activity when the enzyme was exposed to supercritical fluids, and the loss of activity was a desirable feature. In other studies, there was no loss of activity, and the retention of activity was advantageous. 

A Ghrisochoou, K Schaber, U Bolz. Phase equilibria for enzyme-catalyzed reactions in supercritical carbon dioxide. Fluid Phase Equilib 108(1-2) 1-14, 1995. 

Knez, Z. Habulin, M. Lipase Catalyzed Esterification in Supercritical Carbon Dioxide. In Biocatalysis in Non-Conventional Media Tramper, J., Vermiie, M. H., Beeftink, H. H., Eds. Elsevier Science Amsterdam, 1992, pp. 401-406. Knez, Z. Habulin, M. Krmelj, V. Enzyme Catalyzed Reactions in Dense Gases. J. Supercrit. Fluids 1998, 14, 17-29. 

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. 

The first study of enzyme catalyzed reactions in a supercritical fluid was reported by Randolph (44) who... 

Water is known to be essential for the enzyme activity. Small amounts of water enhance enzyme activity, however excess water hinders the rate of some enzyme catalyzed reactions. Also, supercritical water cannot be used as the reaction medium either, because its critical temperature and pressure are too high for the enzymes used in biotransformations. The active site concentration on enzymes, hence the enzyme activity is found to be higher in the presence of hydrophobic supercritical fluids (ethane, ethylene) as compared to hydrophilic supercritical carbon dioxide. 

U Bolz, K Stephan, P Stylos, A Riek, M Rizzi, M Reuss. Comparison of enzymic catalyzed reactions in organic solvents and in supercritical fluids. Biochem Eng —Stuttgart, [Proc. Int. Symp.], 2nd Meeting Date 1990, 82-5. Edited by Reuss, Matthias. Fischer Stuttgart, Fed. Rep. Ger., 1991. 

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. 

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]. 

In many instances, reaction rates are limited by diffusion in the liquid phase. The rate of these reactions can be increased if the reaction is carried out in the supercritical phase. Typical examples are enzyme-catalyzed reactions as well as very fast reactions such as some free radical reactions. Selectivity considerations usually dominate in complex reactions. If some steps of the complex reaction are controlled by diffusion, changing the diffusivity changes relative rates of the reaction steps and affects the selectivity. 

Because of their tunable properties, supercritical solvents provide a useful medium for enzyme-catalyzed reactions.f The mechanism of enzyme-catalyzed reactions is similar to the mechanism described for solid-catalyzed reactions. External as well as internal transport effects may limit the reaction rate. Utilizing supercritical fluids enhances external transport rate due to increase in the diffusivity and therefore mass transfer coefficient. Internal transport rate depends on the fluid medium as well as the morphology of the enzyme. Supercritical fluids can alter both. 

The effect of pressure on enzyme-catalyzed reactions can be explained in terms of the transition theory. Supercritical fluids that exhibit very high negative activation volumes for certain reactions are expected to improve the rate of the reaction. 

Although, supercritical carbon dioxide has the advantage of being nontoxic and abundant, it is practically immiscible with water. Therefore, supercritical fluids used as the reaction medium in enzyme-catalyzed reactions include fluoroform, sulfur hexafluoride, and ethane, while lipases are the enzymes utilized in such reactions. ... 

Randolph s tests with alkaline phosphatase were carried out in a stirred autoclave. An amount of the disodium salt and some water, which is required for the enzyme catalyzed hydrolysis, were placed in the autoclave along with a sealed glass ampule containing the enzyme. In this case water is necessary not just to render the enzyme active, as Klibanov found, but also to serve as a reactant in the hydrolysis. Carbon dioxide was admitted, the temperature and pressure adjusted to the level desired, and the sealed ampule shattered to expose the enzyme and to mark the zero point of the reaction sequence. In their studies they investigated the effects of changing the relative amount of enzyme on the rate of conversion of the disodium salt of p-nitrophenyl phosphoric acid to p-nitrophenol. They measured the amount of conversion by UV analysis of the solution removed from the autoclave at the end of a reaction test. The results are shown in Figure 11.1 based upon these results and other experimental results, the authors concluded that the rate-determining step of the enzyme-catalyzed reaction was the dissolution of disodium p-nitrophenyl phosphate in supercritical carbon dioxide. 

Biocatalysis in supercritical fluids, fluorous solvents, and under solvent-free conditions was recently reviewed (80). In this book, de Geus et al (17), Villarroya (41) and Bruns et al (32) all provide important examples of how supercritical CO2 can be used for enzyme-catalyzed reactions. Furthermore, Srienc et al (38) used ionic liquid media for enzyme-catalyzed polymerizations of p-butyrolactone in order to prepare poly(hydroxyalkanoic acids), PHA. The role of ionic liquids was to both maintain enzyme activity and propagating chain solubility so that high molecular weight products could be obtained in monophasic media. 

Turner C, Persson M, Mathiasson L, Adlercreutz P, King JW. Lipase-catalyzed reactions in organic and supercritical solvents application to fat-soluble vitamin determination in milk powder and infant formula. Enzyme Microb Tech 2001 29(2-3) 111-21. 

Enzyme-catalyzed transesterification under supercritical CO2 medium Improves diffusion and reaction rate, salvation ability can be engineered, can be used in extraction of lipids as well, easy separation from products Expensive technique, requires sophisticated instrumentation... 

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