Biphasic buffer/organic solvent

One method is to run the reaction in an aqueous buffer/organic solvent biphasic system. This makes it possible to work at high substrate and product concentrations and at the pH-optimum of the enzyme. In addition, in water-immiscible solvents the non-enzymahc addition of HCN to the carbonyl group is non-existent or extremely slow. Possible disadvantages are enzyme deactivation at the interface and the presence of organic solvent dissolved in the aqueous phase [15, 17, 18]. 

Hydrolysis of substrates is performed in water, buffered aqueous solutions or biphasic mixtures of water and an organic solvent. Hydrolases tolerate low levels of polar organic solvents such as DMSO, DMF, and acetone in aqueous media. These cosolvents help to dissolve hydrophobic substrates. Although most hydrolases require soluble substrates, lipases display weak activity on soluble compounds in aqueous solutions. Their activity markedly increases when the substrate reaches the critical micellar concentration where it forms a second phase. This interfacial activation at the lipid-water interface has been explained by the presence of a... 

The ability of free or immobilized lipoxygenase to introduce oxygen derived from the air into polyunsaturated fatty acids in media containing organic solvent and aqueous buffer has been investigated [9,56]. The influence of many parameters was tested upon the degree of oxygenation in biphasic systems [36,108]. 

An investigation of different organic solvents, buffer, surfactants, and organorhodium compounds established that the catalytic reduction of tetralin using [ Rh(l,5-hexadiene)Cl 2] proceeds with high efficiency at high substrate-to-catalyst ratios. The reaction occurs at r. t. and 1 atm. pressure in a biphasic mixture of hexane and an aqueous buffer containing a low concentration of a surfactant which stabilizes the catalysts.314... 

As is the case for organic solvents, several workers have investigated two-phase systems in which water-immisdble ionic liquids are mixed vdth aqueous buffers. Such strategies offer all of the advantages of organic/aqueous biphasic mixtures, but without the drawbacks of handling volatile and flammable solvents. Some examples are shown in this section. 

Nikolova and Ward [72,73] studied production of phenylacetyl carbinol from benz-aldehyde and pyruvate by whole-cell yeast biotransformation in two-phase systems. For the biocatalyst preparation fresh pressed commercial baker s yeast (50 g) was suspended in 50 ml 0.05 M sodium citrate buffer (pH 6.0) and lyophilized. Aliquots of 300 mg of lyophilized cells were mixed witii 1 g celite and the mixture was resuspended in 0.05 M sodium citrate buffer (pH 6.0). The suspension was lyophilized again and stored at 4°C. Scanning electron micrographs of the carrier celite and yeast cells lyophilized on celite are given in Fig. 1. Prior to use, organic solvents purchased in anhydrous form were saturated with 0,05 M sodium citrate buffer (pH 6.0). The same buffer was used as an aqueous component of the biphasic systems. 

Operation in biphasic mixtures using water-immiscible solvents introduces a linked equilibrium in the partition of educt and product and possible transport limitations at the interface, which have to be considered. Besides, enzyme deactivation at the interface and possible effects of the residual solvent solubility in aqueous buffers on enzyme stability have to be checked. Table 3 summarizes some data on stability of ADHs dissolved in aqueous buffers in a biphasic mixture with organic solvents [48]. Two different reactor concepts for continuous operation and enzyme catalysis in homogeneous phase have been studied—a bimembrane reactor [13,14] and an emulsion reactor [49]—which are discussed below with regard to reaction engineering. Using water-inuniscible solvents one can make use of the fact that NAD(P)/NAD(P)H are charged molecules and practically insoluble in apolar solvents. The coenzyme introduced in the reaction is therefore confined and physically immobilized with the enzymes in the aqueous phase. This facilitates efficient use of the coenzyme, especially if the volume fraction of the aqueous phase is kept low [13]. 

For this specific task, ionic liquids containing allcylaluminiums proved unsuitable, due to their strong isomerization activity [102]. Since, mechanistically, only the linkage of two 1-butene molecules can give rise to the formation of linear octenes, isomerization activity in the solvent inhibits the formation of the desired product. Therefore, slightly acidic chloroaluminate melts that would enable selective nickel catalysis without the addition of alkylaluminiums were developed [104]. It was found that an acidic chloroaluminate ionic liquid buffered with small amounts of weak organic bases provided a solvent that allowed a selective, biphasic reaction with [(H-COD)Ni(hfacac)]. 

The volumetric ratio of the two liquid phases (j6 = Forg/ Faq) can affect the efficiency of substrate conversion in biphasic media. The biocatalyst stability and the reaction equilibrium shift are dependent on the volume ratio of the two phases [29]. In our previous work [37], we studied the importance of the nonpolar phase in a biphasic system (octane-buffer pH 9) by varying the volume of solvent. The ratio /I = 2/10 has been the most appropriate for an improvement of the yield of the two-enzyme (lipase-lipoxygenase) system. We found that a larger volume of organic phase decreases the total yield of conversion. Nevertheless, Antonini et al. [61] affirmed that changes in the ratios of phases in water-organic two-phase system have little effect upon biotransformation rate. 

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