Biphasic biocatalytic systems

The general principles of liquid-liquid reactions were described in Chapter 15. Although these principles are equally applicable to biphasic biocatalytic systems, an additional feature must be considered in these reactions, the special role of surface reaction. In regular liquid-liquid reactions there is only one set of intrinsic reactions, whether at the surface or in the bulk. In biocatalytic systems, however, the intrinsic reaction rate at the surface is elevated which complicates the analysis. First we classify the various types of biocatalytic biphasic reactions. Then, considering the simplest case, we present a brief analysis that simultaneously accounts for the elevated rate at the surface and absorption accompanied by reaction in the aqueous film and/or bulk. In regular nonenzymatic liquid-liquid systems, only the latter effect, as analyzed in Chapters 15 and 16, is operative. 

Reaction in a liquid-liquid biocatalytic system is usually confined to the aqueous phase and occurs by mass transfer of the reactant from the organic phase to the 

Both A and R are predominantly in the aqueous phase, that is, the distribution coefficients wja and wr are close to zero. 

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Scheme 6 Biphasic biocatalytic system in conducting polymer synthesis. (Reprinted with permission from Marcilla et al. [48]. 2009, WUey)...

Lozano et al. [64—67], and Reetz, Leitner and co-workers [68, 69], simultaneously reported SCCO2-IL biphasic, biocatalytic systems for lipase-catalyzed transesterifications using vinyl esters as the transesterification agent [Eq. (13)]. Vinyl butyrate was used because the product vinyl alcohol tautomerizes to acetaldehyde and hence the reaction is irreversible. 

Thanks to their special properties and potential advantages, ionic liquids may be interesting solvents for biocatalytic reactions to solve some of the problems discussed above. After initial trials more than 15 years ago, in which ethylammonium nitrate was used in salt/water mixtures [29], results from the use of ionic liquids as pure solvent, as co-solvent, or for biphasic systems have recently been reported. The reaction systems are summarized in Tables 8.3-1 and 8.3-2, below. Table 8.3-1 compiles all biocatalytic systems except lipases, which are shown separately in 8.3-2. Some of the entries are discussed in more detail below. 

An important problem in emulsified organic-aqueous systems is that of scale-up, which is concerned with the realization of stable emulsions and the separation of phases after the reaction. The use of biphasic membrane systems that contain the enzyme and keep the two phases separated is likely to solve this problem. In the case of 5-naproxen an ee of 92% has been demonstrated without any decay in activity over a period of two weeks of continuous operation. A number of examples of biocatalytic membrane reactors have been provided by Giorno and Drioli (2000) and include the conversion of fumaric acid to L-aspartic acid, L-aspartic acid to L-alanine, and cortexolone to hydrocortisone and prednisolone. 

The use of enzymes as biocatalysts for the synthesis of water-soluble conducting polymers is simple, environmentally benign, and gives yields of over 90% due to the high efficiency of the enzyme catalyst. Since the use of an enzyme solution does not allow the recovery and reuse of the expensive enzyme, well-established strategies of enzyme immobilization onto solid supports have been applied to HRP [22-30]. A recent work reported an alternative method that allows the recycle and reuse of HRP in the biocatalytic synthesis of ICPs. The method is based on the use of a biphasic catalytic system in which the enzyme is encapsulated by simple solubilization into an IL. The main strategy consisted of encapsulating the HRP in room-temperature IPs insoluble in water, and the other components of the reaction... 

Since the beginning of the 20th century, organic solvents have been used in enzymatic reaction media [30]. Biocatalytic reactions in water-organic biphasic media were first carried out by Cremonesi et al. [31] and by Buckland et al. [32] less than 30 years ago. Their work aimed at the conversion of high concentrations of poorly water soluble components, particularly steroids. Later, biphasic systems were used for enzyme-catalyzed synthesis reactions that were unfavored in water, changing the reaction equilibrium towards the higher yield of the product, such as esters or peptides. 

Biphasic systems proved to be advantageous as well in the biocatalytic synthesis of (-)-l-trimethylsilylethanol which was performed by asymmetric reduction of acetyltrimethylsilane with an isolate from Rhodotorula sp. AS2.2241 [144]. Immobilized cells were employed due to the easy separation of the product as well as the improved tolerance against unfavorable factors. In an aqueous/organic solvent biphasic system higher product yield and enantiomeric excess were achieved as compared to an aqueous monophasic system. Several organic solvents were examined, and isooctane was found to be the most suitable organic phase for the reaction. 

Enzyme-catalyzed reactions based on such biphasic systems have been shown to be promising alternatives for developing green chemical processes because of their physical and chemical characteristics [9]. By combining these media with enzymes, the possibilities of carrying out integral green biocatalytic processes has been already demonstrated [10-12]. Such biphasic systems can be used for both the biotransformation and extraction of products simultaneously, even under extremely harsh conditions, because of the different miscibilities of ILs and SC-CO2. 

In this report, the nse of ionic Uquids/supercritical carbon dioxide in biocatalytic reactions has been extensively reviewed. Properties of supercritical carbon dioxide, ionic liquids and ionic liqnids/supercritical carbon dioxide biphasic systems have been analysed. Representative examples of the enzyme catalytic reactions in IL/ scCOj biphasic systems have been included. Finally, the effect of the biphasic systems on activity, selectivity and stability of enzymes has been carefully analysed. 

He, J., Sim, Z Ruan, W., and Xu, Y. (2006) Biocatalytic synthesis of ethyl (S)-4-chloro-3-hydroxy-butanoate in an aqueous-organic solvent biphasic system using Aureobasidium pullulans CGMCC 1244. Process Biochem., 41 (1), 244-249. 

The use of biphasic aqueous-organic systems in biocatalytic reductions is of great interest because the enzyme and its cofactor are dissolved in the aqueous phase, where the reaction takes place, while the hydrophobic substrate and product are mostly located in organic solvent layer and partitioned into the aqueous phase. This distribution reduces the concentrations of toxic substrate and product around the enzyme in aqueous layer and relieves the enzyme from substrate and product inhibition. Other distinctive features of this biphasic system are simple separation, easy regeneration of the enzyme, and easy recovery of the products. However, in this system, the reaction rates are relatively low because of a low rate of mass-transfer across the interface. Although this hindrance can be eliminated by intensive agitation, the increased interface often results in faster denaturation and inactivation of the enzyme. 

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