Enzymes activity in supercritical

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

Rohm O (1908) Preparation of hides for the manufacture of leather. US Patent 886411 Ru MT, Wu KC, Lindsay JP et al. (2002) Towards more active biocatalysts in organic media increasing the activity of salt-activated enzymes. Biotechnol Bioeng 75(2) 187-196 Russell AJ, Beckman E J (1991) Enzyme activity in supercritical fluids. Appl Biochem Biotechnol 31(2) 197-202... 

AJ Russell, El Beckman, AK Chaudhary. Studying enzyme activity in supercritical fluids. Chemtech 24(3) 33-37, 1994. 

S Kamat, EJ Beckman, AJ Russell. Enzyme activity in supercritical fluids. Grit Rev Biotech-nol 15(1) 41-71, 1995. 

Enzymes can express activity in supercritical and near-supercritical fluids, such as carbon dioxide, freons (CHF3), hydrocarbons (ethane, ethylene, propane) or inorganic compounds (SFe, N2O). The choice of supercritical fluids is often... 

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. 

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. 

SCFs offer a nonaqueous environment which can be desirable for enzymatic catalysis of lipophilic substrates. The lipophilic substance cholesterol is 2 to 3 orders of magnitude more soluble in CX>2-cosolvent blends than in waterQ). In CO2 based blends, it may be oxidized to cholest-4en-3one, a precursor for pharmaceutical production using an immobilized enzyim(22). The enzyme polyphenol oxidase has been found to be catalytically active in supercritical CO2 and fluoroform (22). The purpose of using a SCF is that it is miscible with one of the reactants-oxygen. Lipase may be used to catalyze the hydrolysis and interesterification of triglycerides in supercritical OO2 without severe loss of activity(24). These reactions could be integrated with SCF separations for product recovery. 

Enzymes are an increasingly available and important tool in the arsenal of the synthetic chemist. Enzymatic reductions are often straightforward and highly stereoselective. There are now many enzymatic transformations that are compatible with the use of organic solvents.6l5 Other solvents can be used as well, illustrated by the enzyme alcohol dehydrogenase from Geotrichum candidum, which is active in supercritical carbon dioxide.6i6 Prelog studied the reduction of ketones with several enzymatic systems. Reduction of... 

The increasing number of publications dealing with enzymatic reactions in supercritical carbon dioxide displays a new confidence in economical applications of this technology. The problem of inactivation of enzymes is a great challenge for the economic application of biocatalyzed reactions, and therefore a number of recent publications have investigated the nature and dependencies of enzyme activity in SCCO2. 

H Michor, R Marr, T Gamse. Enzymic catalysis in supercritical carbon dioxide. Effect of water activity. Proc Technol Proc 12 (High Pressure Chem Eng), 115-120, 1996. 

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. 

Since then, the process has been extended to a wide variety of lactones of different size and to several lipases, as recently reviewed [93-96]. Interestingly, large-membered lactones, which are very difficult to polymerize by usual anionic and coordination polymerizations due to the low ring strain, are successfully polymerized by enzymes. Among the different lipases available, that fi om Candida antarctica (lipase CA, CALB or Novozym 435) is the most widely used due to its high activity. An alcohol can purposely be added to the reaction medium to initiate the polymerization instead of water. The polymerization can be carried out in bulk, in organic solvents, in water, and in ionic liquids. Interestingly, Kobayashi and coworkers reported in 2001 the ROP of lactones by lipase CA in supercritical CO2... 

The scope and limitations of biocatalysis in non-conventional media are described. First, different kinds of non-conventional reaction media, such as organic solvents, supercritical fluids, gaseous media and solvent-free systems, are treated. Second, enzyme preparations suitable for use in these media are described. In several cases the enzyme is present as a solid phase but there are methods to solubilise enzymes in non-conventional media, as well. Third, important reaction parameters for biocatalysis in non-conventional media are discussed. The water content is of large importance in all non-conventional systems. The effects of the reaction medinm on enzyme activity, stabihty and on reaction yield are described. Finally, a few applications are briefly presented. 

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. 

If the bonded water is extracted by dry CO2 the enzyme is denaturated and loses its activity. Therefore a certain amount of water is necessary in the supercritical fluid because acting with water-saturated CO2 again causes an inhibition of the enzyme and consequent loss of activity. The optimal water concentration has to be determined for each enzyme separately. Table 9.2-1 shows the residual activity of lipase from Candida cylindracea, esterase from Mucor mihei, and esterase from Porcine liver after a incubation time of 22 hours in supercritical CO2 at 40°C. It is obvious that higher water concentrations cause a strong reduction in the residual activity compared to the activity of the untreated enzyme, which was set as 100 %. Further, the system-pressure has an influence because at higher pressures the activity-loss is lower with a larger amount of water in the system [7,8],... 

The temperature effect is much more significant than the pressure effect. For the enzyme stability, a temperature increase above certain levels, depending on the enzyme, results in deactivation of the enzyme. In Table 9.2-2, the residual activities of various enzymes after one hour incubation time, in supercritical CO2 at 150 bar, are given. It is obvious that temperatures over ca. 75°C reduce enzyme activity dramatically. However, no correlation for the stability with the temperature for different types of enzymes is yet available [8-10],... 

Residual activity of different enzymes after 1 hour treatment in supercritical CO2 at 150 bar (I, lipase from Candida cylindracea If, lipase Amano AY III, lipase from Pseudomonas sp. IV, esterase EP10 from Burkholderia gladioli) ... 

It has been shown that, in supercritical carbon dioxide, increases in water concentration result in increases in enzyme activity. The amount of added water needed for this increase varies and can depend on many factors, such as reaction type, enzyme utilized, and initial water content of the system. This is true until an optimal level is reached. For hydrolysis reactions, activity will either continue to increase or maintain its value. For esterification or transesterification reactions, once the optimal level of hydration has been reached, additional water will promote only side reactions such as hydrolysis. Dumont et al. (1992) suggests that additional water beyond the optimal level needed for enzyme hydration may also act as a barrier between the enzyme and the reaction medium and thereby reduce enzyme activity. Mensah et al. (1998) also observed that water above a concentration of 0.5 mmol/g enzyme led to lower catalytic activity and that the correlation between water content of the enzyme and reaction rate was independent of the substrate concentrations. 

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. 

Several authors have shown the stability and the catalytic activity of enzymes in supercritical carbon dioxide (SC C02) [2 to 25] Some authors [14,17,18] have shown the difference of activity of enzyme in SC C02 and in organic solvent. 

Lipases are the most common enzymes used in non conventional media like organic solvents and supercritical carbon dioxide. Lipases usually hydrolyse fats into fatty acids and glycerol. The special property of lipases is their ability to act at the interface between water and oil. In these experiments lipase (EC 3.1.1.34) from Rhizopus arrhizus (Boehringer Mannheim) was used to investigate the effects of lipase under hydrostatic pressure. The analysed reaction was the hydrolysis of p-Nitrophenyllaureate at different concentrations at 35 °C. The dependance of the kinetic constants between 1 bar and 3000 bar is presented in table 2. Like the thermophilic GDH at 1000 bar lipase is activated under pressure as well. The initial reaction rate increases by a factor of 1.5 at 1000 bar compared to the initial reaction rate at ambient... 

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]. 

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. The active site concentration on enzymes, hence the enzyme activity, is found to be higher in the presence of hydrophobic supercritical fluids (ethane, ethylene) compared to hydrophilic supercritical carbon dioxide. 

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. 

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