Lipase-Catalyzed Reactions with Supercritical Fluids

5 LIPASE-CATALYZED REACTIONS WITH SUPERCRITICAL FLUIDS 

In biodiesel production, methanol and lipid reaction products are immiscible and form two phases at room conditions. This results in low reaction effidency and lipase deactivation. Hydrophobic solvents can minimize this effect, however, they are toxic and require a separation unit, which further increases the overall production cost Supercritical carbon dioxide (SC-COj) has frequently been used to replace organic solvents in various chemical processes. Due to its properties— including the easy and complete removal of the solvent, an ability to manipulate the physical properties of the solvent by simply changing the pressure or tanperature, nontoxidty, nonflammability, and enhancement of substrate mass transfer properties— it was suggested as a green solvent in biocatalyst reactions. A chemical feature of SC-COj is its low critical temperature (below the denaturation temperature of lipase). This feature combines the good solubility of nonpolar compounds, such as lipids, and makes SC-CO2 the perfect medium for biodiesel production. 

Although high pressures are involved in such processes, pressures up to 300 bar were found to have a minimal effect on enzyme activity and stability in esterification reactions (Novak et al, 2003). Giessauf and Gamse (2000) reported an increase in pancreatic lipase activity when exposed to SC-CO2 at 150 bar for 24 h. No loss in activity was reported when stored in this condition for 6 months. 

The focus on lipase-catalyzed reaction in SC-CO2 is due to enhanced reaction rates (Lee et al, 2013). In biodiesel production systems, SC-CO2 offers easy product separation from the reaction mixture by selectively dissolving the biodiesel, due to its high solubility compared to the glycerol by-product (Rodrigues et al, 2011). In a continuous system, the product is continuously removed from the reaction system, which can then be easily separated from SC-CO2 by simple depressurization. 

FIGURE6.2 (See color insert.) Mixture of microalgae lipids and methanol in 10 ml view cells 

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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. 

Reacting lipophilic substrates with hydrophilic compounds, as in the case of most transesteriflcation reactions, is one of the major difficulties in lipase-catalyzed reactions. Several parameters need to be considered to overcome this immiscibility problem. One commonly proposed strategy is the use of a nonaqueous medium. In this chapter, the advantages of using nonaqueous media in biochemical synthesis reactions, over aqueous and solvent-free systems, are discussed. The use of hydrophobic solvents is also discussed, followed by a presentation of the alternatives that can overcome the limitations of solvents. The focus of this chapter is mainly on the use of supercritical fluids (SCFs) as a green alternative reaction medium. The chapter also discusses ionic liquids (ILs) as another alternative. These solvents and the factors affecting their physical properties and their effect on the activity and stability of lipase are also discussed. 

Although, supercritical carbon dioxide has the advantage of being non-toxic and abundant, it is practically immiscible with water. Also, side reactions such as carbamate formation and carbonic acid formation can occur when carbon dioxide is employed as the supercritical fluid in enzymatic reactions. 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." However, ethane and propane have the disadvantages of being expensive and flammable. 

Erickson et al. studied the effects of pressure in a lipase-catalyzed transesterification using the same mol per volume substrate concentration at all fluid densities. Because they obtained a similar pressure effect in CO2 and in ethane, they concluded that the changes in reaction rate are due to changes in the supercritical fluid phase and not in the immobilized enzyme phase, such as pH changes. They found that the decrease of rate with increasing pressure could be modeled accurately by using the mole fractions of the substrates instead of mol/volume concentrations in the rate law [51]. Saito et al. made the same observation the reaction rate decreased with the decrease of substrate mole fraction due to increasing pressure and density of CO2 [52]. 

In the past decade or so, lipase-catalyzed esterifications and transesterifications in anhydrous media (e.g., organic solvents and supercritical fluids) have been an area of intensive research. In particular, the use of organic solvents, which normally allow a higher stability of enzymes than in water (Bock, Jimoh, Wozny, 1997), has been demonstrated. Reviews of the applications have been made by Hail Krishna and Karanth (2002) and Gandhi et al. (2000), dealing with fundamental and practical aspects of lipase catalysis. In particular, they concentrated on various immobilization strategies and factors (e.g., temperature, reaction medium, water activity) as weU as the methods of preparation (which affect and influence the stability of the lipases). 

In the aqueous phase enzymatic hydrolysis of racemic IPGA leads to IGP (mainly one enantiomer), which can be extracted into the supercritical fluid. Buffering of the pH in the aqueous solution is necessary to provide optimal conditions for the enzyme. In the following lipase-catalyzed esterification IPG reacts with vinyl but) to IPG-butyrate (lipase was from P. cepacia). In this sequential resolution of racemates no processing of intermediates is necessary both reactions take place in different phases (Fig. 19). 

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