Other Biphasic Systems

Some other systems, which cannot be assigned unambiguously, have also been investigated for HNL catalysis. The driving force for the investigations was the attempt to improve enzyme stability and to suppress the undesired non-enzymatic side reaction. 

Boy et al. [79] used lyotropic liquid crystals (LC) for the immobilization of HfeHNL. The solid LC phase is not used because of the high viscosity. Therefore, the LC is used in a biphasic system consisting of the LC and an organic solvent. Such biphasic liquid crystal systems consist of organic solvent, water, and surfactant, where poorly soluble substrates and products are dissolved in the organic solvent and the liquid crystal matrix, which contains the enzyme, has a protective effect on it. By optimization and by virtue of the immobilization, it is possible to establish an extractive continuous process [79]. 

The immobilization of hyroxynitrile lyase from Sorghum hicolor on a porous membrane incorporated in a standard pump-around reactor was patented by Andruski and Goldberg [80]. A solution of aldehyde and HCN in different organic 

Enzymes can also be entrapped within the pores of a matrix or network. Small molecules can diffuse in and out of the matrix, whereas the macromolecular enzyme is maintained within the network. Precise control of the pore size is not possible, and therefore mass transfer limitations and enzyme leaching always cause problems [59]. 

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Constmction of phase diagrams usually starts with the preparation of series of mixed solutions sufficiently differing in bulk biopolymer concentration. Some of them can be single-phase solutions, others biphasic systems. A true equilibrium between the phases is experimentally obtained by mixing or shaking the water-in-water emulsions under different time-temperature conditions. Separation of the phases by centrifuge provides information about both the number and the volume ratio of the system phases. The closer a system composition is to the critical point, the smaller the difference in density is between the phases and the more difficult their separation is by centrifugation. The amount of each biopolymer in each phase can be quantified by various techniques. Estimation of protein concentration by UV absorbance at 280 nm is widely used because of its simplicity and sensitivity. 

Extractions and separations in two-phase systems require knowledge of the miscibilities and immiscibilities of ILs with other solvents compatible with the process. These are most usually IL/aqueous biphase systems in which the IL is the less polar phase and organic/IL systems in which the IL is used as the polar phase. In these two-phase systems, extraction both to and from the IL phase is important. 

Another interesting recent development is the continuous, Rh-catalyzed hydroformylation of 1-octene in the unconventional biphasic system [BMIM][PF6]/scC02, described by Cole-Hamilton et al. [84]. This specific example is described in more detail, together with other recent work in ionic liquid/scC02 systems, in Section 5.4. 

Other metals can also be used as a catalytic species. For example, Feringa and coworkers <96TET3521> have reported on the epoxidation of unfunctionalized alkenes using dinuclear nickel(II) catalysts (i.e., 16). These slightly distorted square planar complexes show activity in biphasic systems with either sodium hypochlorite or t-butyl hydroperoxide as a terminal oxidant. No enantioselectivity is observed under these conditions, supporting the idea that radical processes are operative. In the case of hypochlorite, Feringa proposed the intermediacy of hypochlorite radical as the active species, which is generated in a catalytic cycle (Scheme 1). 

On the other hand, 3-phenylpropionitrile was synthesized from Z-3-phenyl-propionaldoxime (0.75 M) in a quantitative yield (98gP ) by the use of cells of E. coli JM 109/pOxD-9OF, a transformant harboring a gene for a new enzyme, phenylacetaldoxime dehydratase, from Bacillus sp. strain OxB-1. Other arylalkyl- and alkyl-nitriles were also synthesized in high yields from the corresponding aldoximes. Moreover, 3-phenylpropionitrile was successfully synthesized by the recombinant cells in 70 and 100% yields from 0.1 M unpurified P/Z-3-phenylpropionaldoxime, which is spontaneously formed from 3-phenylpropionaldehyde and hydroxylamine in a butyl acetate/water biphasic system and aqueous phase, respectively. 

Ten Brink et al. (2000) have shown how biphasic systems, sometimes with the sparingly soluble alcohols as one phase and an aqueous phase as the other phase, benefit from the strategy for air oxidation to aldehydes/ketones by using water soluble Pd complex of bathophenanthroline disulphonate. This is a nice example of green technology. 

On the other hand, biocatalyst stability can be affected by the presence of organic-aqueous interface. In our previous work [25], we studied the effect of interface with octane on the lipoxygenase stability in an octane-buffer pH 9.6 biphasic system. This loss in activity is more pronounced than that observed in the aqueous system. During lipoxygena-tion, the active enzyme concentration [E] in the aqueous phase of our biphasic bioreactor was ... 

Stability of several enzymes like proteases from thermophilic micro-organisms can be increased in aqueous-organic biphasic systems. Owusu and Cowan [67] observed a strong positive correlation between bacterial growth temperature, the thermostability of free protein extracts, and enzyme stability in aqueous-organic biphasic systems (Table 1). Enzymes, like other cell components (membranes, DNA, (RNA ribosomes), are adapted to withstand the environmental conditions under which the organism demonstrates optimal growth. 

As proven in this review and other papers, organic-aqueous biphasic media have been useful in many areas of biocatalysis applications. We summarize the potential advantages in carrying out biocatalysis in biphasic systems ... 

As will be described in detail below, solute distribution in biphasic systems can be modulated by application of a Galvani potential difference across the interface, thereby leading to the transfer of species from one phase to the other. Therefore, in electrochemical terms, passive transfer simply means the partition across an interface, mediated by a potential-driven process. 

These alternative processes can be divided into two main categories, those that involve insoluble (Chapter 3) or soluble (Chapter 4) supports coupled with continuous flow operation or filtration on the macro - nano scale, and those in which the catalyst is immobilised in a separate phase from the product. These chapters are introduced by a discussion of aqueous biphasic systems (Chapter 5), which have already been commercialised. Other chapters then discuss newer approaches involving fluorous solvents (Chapter 6), ionic liquids (Chapter 7) and supercritical fluids (Chapter 8). 

Other salts of formic acid have been used with good results. For example, sodium and preferably potassium formate salts have been used in a water/organic biphasic system [36, 52], or with the water-soluble catalysts discussed above. The aqueous system makes the pH much easier to control minimal COz is generated during the reaction as it is trapped as bicarbonate, and often better reaction rates are observed. The use of hydrazinium monoformate salts as hydrogen donors with heterogeneous catalysts has also been reported [53]. 

In other cases, organic modification of the sol gel cages markedly protects the entrapped molecular dopant from degradation by external reactants, as shown for instance by the entrapment of the radical 2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). This is a highly active catalyst which in the NaOCl oxidation of alcohols to carbonyls in a CH2CI2-H20 biphasic system becomes highly stabilized upon sol gel entrapment in an ORMOSIL matrix it progressively loses it activity when entrapped at the external surface of commercial silica.25... 

It is essential that all PSs are multiphase. The easiest case to handle is the biphase system consisting of a condensed phase (solid) and a void inside porous particles or between consolidated ensembles of nonporous or porous particles. The void occupies a part of the volume, s, which is referred to as porosity. The other part of a PS volume is equal to ri=(l -e), and is termed density of packing. It is filled with the condensed phase (see Section 9.4). Generally, PSs can include various condensed phases of different structure, including combinations of solid(s) and liquid(s). 

In the simplest binary system comprising two liquids (A and B), adding a small amount of either liquid to the other creates a single phase, as the one liquid dissolves completely in the other. As more of the second liquid is added, in this case B, the first liquid A becomes saturated with B and no more will dissolve. At this point, the system will consist of two phases in equilibrium with each other, one of liquid A saturated with B and the other of liquid B saturated with A. If B is continually added to A, there will come a point at which A becomes the minor component in the system and, ultimately, will dissolve completely in liquid B a single phase will be formed once more. The relative proportions of each liquid that are required to form single or biphasic systems depends both... 

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