Enzymatic synthesis biphasic system

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. 

Willeman et al. [26] modeled the enzyme-catalyzed cyanohydrin synthesis in a stirred batch tank reactor. Assumption of a mass transfer limitation (Figure 9.3b) is made, which results in a low concentration of substrate in the aqueous phase, thus suppressing the non-enzymatic reaction. In a well-stirred biphasic system the enzyme concentration was varied, keeping the phase ratio constant A maximum rate of conversion is reached at the concentration where mass transfer of the substrate becomes limiting. Further increase of enzyme concentration does not enhance the reaction rate [27]. The different results achieved by the two groups are explained by the different process strategies. No mass transfer limitation could be detected by Hickel et al. because the stirring rate in the aqueous phase was not varied [26]. 

The asymmetric hydrolysis of (exo,exo)-7-oxabicyclo[2.2.1]heptane-2,3-dimethanol, diacetate ester (37) to the corresponding chiral monoacetate ester (38) (Fig. 12B) has been demonstrated with lipases [61]. Lipase PS-30 from P. cepacia was most effective in asymmetric hydrolysis to obtain the desired enantiomer of monoacetate ester. The reaction yield of 75 M% and e.e. of >99% were obtained when the reaction was conducted in a biphasic system with 10% toluene at 5 g/liter of the substrate. Lipase PS-30 was immobilized on Accurel PP and the immobilized enzyme was reused (5 cycles) without loss of enzyme activity, productivity, or e.e. of product (38). The reaction process was scaled up to 80 liters (400 g of substrate) and monoacetate ester (38) was isolated in 80 M% yield with 99.3% e.e. The product was isolated in 99.5% chemical purity. The chiral monoacetate ester (38) was oxidized to its corresponding aldehyde and subsequently hydrolyzed to give chiral lactol (33) (Fig. 12B). The chiral lactol (33) obtained by this enzymatic process was used in chemoenzymatic synthesis of thromboxane A2 antagonist (35). 

The enzymatic synthesis of polyphenols was carried out not only in the monophasic solvents but in interfacial systems such as micelles, reverse micelles, and biphasic and Langmuir trough systems, p-Phenylphenol was polymerized in an aqueous surfactant solution to give the polymer with a narrower molecular weight distribution in comparison with that obtained in the aqueous 1,4-dioxane.20... 

Many examples are to be found in the chemical or biochemical literature e.g. ultrasonically induced emulsification/mixing has been utilized in the two-phase enzymatic synthesis of dipeptides [11]. For the dipeptide synthesis shown in reaction (1) the source of ultrasound was an ultrasonic bath (38 kHz). The importance of sonication in such a system is that it promotes biphasic reaction in solvent mixtures such as petroleum ether/water which are not effective under conventional conditions (Table 2). 

Hernandez FJ, de los Rios AP, Gomez D, Rubio M, ViUora G. A new recirculating enzymatic membrane reactor for ester synthesis in ionic liquid/supercritical carbon dioxide biphasic systems. Appl Catal B 2006 67 121-126. 

Ionic Liquids/Supercritical Carbon Dioxide as Advantageous Biphasic Systems in Enzymatic Synthesis... 

Fig. 8.1 Experimental set-up of the recirculating enzymatic membrane reactor used for the synthesis of butyl propionate from vinyl propionate and 1-butanol catalysed by Candida antarctica lipase B in supercritical carbon dioxide and supercritical carbon dioxide/ionic liquid biphasic system [17]...

Hemdndez FJ, de los Rfos AP, Gomez D et al (2007) Understanding the chemical reaction and mass-transfer phenomena in a recirculating enzymatic membrane reactor for green ester synthesis in ionic Uquid/supercritical carbon dioxide biphasic systems. J Supercrit Flitids 43 303-309... 

Production of an optically active diltiazem intermediate (2R, 3S)-methoxyophenylglyci-date methyl ester ((-)-MPGM) from racemic MPGM by the action of lipase from Serratia marcescens in a toluene aqueous biphasic system (Tanabe Seiyaku Co., Ltd.). For the continuous production of (-)-MPGM, a hollow fiber bioreactor was set up in collaboration with Sepracor Inc. The introduction of this enzymatic step allowed the shortening of the diltiazem synthesis from nine down to five steps. 

Liquid-liquid-solid reactors are commonly used for biphasic reactions catalyzed by immobilized phase-transfer catalysts (which form the third, solid phase). Certain basic aspects of such reactors were considered in Chapter 19. Three-phase reactions of this type are also encountered in biological reactions, for example, the enzymatic synthesis of amino acid esters in polyphasic media (Vidaluc et al., 1983), and the production of L-phenylalanine utilizing enzymatic resolution in the presence of an organic solvent (Dahod and Empie, 1986). Predictably, the performance of these reactors is influenced by the usual kinetic and mass transfer aspects of heterogeneous systems (see Lilly, 1982 Chen et al., 1982 Woodley et al., 1991a,b). Additionally, performance is also influenced by the complex hydrodynamics associated with the flow of two liquids past a bed of solids (Mitarai and Kawakami, 1994 Huneke and Flaschel, 1998). It is noteworthy, for instance, that phase distribution within the reactor is different from that in the feed and is also a function of position within the reactor and within the voids of each pellet in the bed. More intensive research is needed before these reactors can be rationally designed. 

Hernandez, F. J., A. P. de los Rfos, D. Gomez, M. Rubio, and G. Vfllora. 2006. A New Recirculating Enzymatic Membrane Reactor for Ester Synthesis in Ionic Liquid/ Supercritical Carbon Dioxide Biphasic Systems. Applied Catalysis B Environmental Cl (1-2) 121-126. 

P. Lozano,. M. Bernal, M. Vaultier, Towards continuous sustainable processes for enzymatic synthesis of biodiesel in hydrophobic ionic liquids/supercritical carbon dioxide biphasic systems, Fuel 90 (2011) 3461-3467. 

---