Supercritical fluid enzymatic activity

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]. 

Theoretical predictions are, however, difficult because the activation volumes of reaction steps and the compressibilities of SCFs change with pressure. A further complication is that, by changing pressure, one simultaneously changes the density-dependent physictd parameters of the supercritical fluid. Effects of mass transfer are also always present to some extent. Therefore, only apparent activation volumes have been measured for enzymatic reactions in SCFs. The reaction mechanisms of enzymatically catalyzed reactions are often not known. 

Lipases are able to work in very different media. They work in biphasic systems and in monophasic (in the presence of hydrophilic or hydrophobic solvents) systems where the water content can vary significantly between aqueous and anhydrous media. They have been tested also in ionic liquid media (Lau et al. 2000 Wasserscheid and Keim 2000 Kamal and Chouhan 2004 Ha et al. 2007), in supercritical fluids (Laudani et al. 2007) and in gaseous media (Cameron et al. 2002). The different media for enzymatic catalysis has been outlined before (see section 1.6) and it will not be further discussed here. However, some examples of modulation of activity and selectivity of lipases by medium engineering will be described in this section. 

The application of aqueous / supercritical biphasic media is not restricted to metal complex catalysis but has proven effective also for enzymatic and whole-cell biocatalysis [36]. In general, water plays an important role in coimection with biocatalysis. If water is completely absent, enzymes are often not catalytically active under supercritical conditions [37]. In the literature many examples of biocatalysis with supercritical fluids containing various amounts of water are known and a detailed account of this field is outside the scope of the present discussion. One example to highlight the use of a true biphasic system is the carboxylation of pyrrole... 

Kavcic S, Knez Z, Leitgeb M. Antimicrobial activity of n-butyl lactate obtained via enzymatic esterification of lactic acid with n-butanol in supercritical trifluoromethane. Supercrit Fluids 2014 85 143-50. 

PHA can be synthesised using proper catalysts (i.e., zinc- or aluminium-based catalyst) with water as the cocatalyst. This in vitro system has been shown to be possible using the PHA synthases purified from various sources [141, 179-181]. It is possible to produce homopolymers and copolymers containing 3HB, 3HV, 4-hydroxyvalerate and 3-hydroxydecanoate [90]. Multiple-enzyme systems have been developed that can utilise cheaper substrates as well as recycle expensive cofactors such as coenzyme A. Nevertheless, the in vitro systems are still expensive to use in order to produce PHA for applications as commodity plastics. Furthermore, hazardous organic solvents are generally required to achieve high enzymatic activity. Present studies focus on the replacement of these organic solvents with supercritical fluids [182] and ionic liquids [183]. 

The earliest reported demonstration of enzymatic activity in a supercritical fluid was for the reaction of disodium p-nitrophenyl phosphate to p-nitro-phenol, catalysed by alkaline phosphatase. Randolph et aL [26] detected the product in yields of up to 71% in carbon dioxide at 35°C and 100 atm, in the presence of 0.1% v/v water. Hammond et al. [33] found tyrosinase, a polyphenol oxidase, to be catalytically active for the oxidation of 4-methyl phenol in both supercritical carbon dioxide at (36 2)°C and supercritical trifluoro-methane at (34 2)°C, with oxygen, at a total pressure of 345 bar. Use of a flow reactor permitted isolation of greater quantities of the catecholic product (1,2-dihydroxy, 4-methylbenzene). Oxidative activity for 4-chlorophenol substrate was appreciably lower. 

Enzymatic oxidation in a supercritical fluid medium has also been demonstrated for the formation of cholest-4-ene-3-one from cholesterol in supercritical carbon dioxide by Randolph et aL [32]. Many cholesterol oxidases showed catalytic activity. That isolated from Streptomyces spp. gradually degraded, but gave reaction rates comparable with those sustained in aqueous systems whilst it was yet catalytic. That from Gloecysticum chrysocrea is chemically stable at 35" C and 100 bar, and yielded reaction rates 75 times faster than in water (5 x 10 phosphate, pH 7, and 5% v/v propanol [34]) which were further increased by the addition of aggregating cosolvents. 

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