Liquid membrane encapsulated enzymes

Mohan, R. R. Li, N. N. "Reduction and Separation of Nitrate and Nitrite by Liquid Membrane-Encapsulated Enzymes" Biotech, and Bioeng. 1974,16, pp 513-523. 

Spherical liquid membranes consist, in simplest terms, of an emulsion suspended in a liquid that does not destroy the emulsion. In a typical application, small droplets of aqueous solution are encapsulated in a thin-film oil this emulsion is then suspended in another aqueous solution. Alternatively, small droplets of oil can be emulsified with water and the emulsion suspended in oil. In the first case, the oil phase is the liquid membrane in the second case, the water is the Uquid membrane. A typical droplet might be about 100 p,m in diameter. These spherical liquid membrane systems have many potential medical applications in the emergency treatment of drug overdoses and for oxygenating the blood system. Spherical liquid membranes may be applied in resource recovery and water purification, as encapsulated cells as well as liquid membrane encapsulated enzymes [331). 

In the future, novel developments of liquid membranes for biochemical processes should arise. There are several opportunities in the area of fermentation or cell culture, for the in situ recovery of inhibitory products, for example. Another exciting research direction is the use of liquid membrane for enzyme encapsulation so that enzymatic reaction and separation can be combined in a single step. Chapter 6 by Simmons ial- (49) is devoted to this technique. The elucidation of fundamental mechanisms behind the liquid membrane stability is essential, and models should be developed for the leakage rate in various flow conditions. Such models will be useful to address the effect of parameters such as flow regime, agitation rate, and microdroplet volume... 

Miyako, E., Maruyama, T., Kamiya, N., Goto, M. (2004). Highly enantioselective separation using a supported liquid membrane encapsulating surfactant-enzyme complex. J. Am. Chem. Soc., 126, 8622-3. 

A liquid membrane bioreactor was developed as a means of encapsulation for a multi-enzyme system incorporating an oxidation and carbohydrate cleavage, demonstrated using a-glucosidase and glucose oxidase in the conversion of maltose to gluconic acid ... 

The rising need for new separation processes for the biotechnology industry and the increasing attention towards development of new industrial eruyme processes demonstrate a potential for the use of liquid membranes (LMs). This technique is particularly appropriate for multiple enzyme / cofactor systems since any number of enzymes as well as other molecules can be coencapsulated. This paper focuses on the application of LMs for enzyme encapsulation. The formulation and properties of LMs are first introduced for those unfamiliar with the technique. Special attention is paid to carrier-facilitated transport of amino acids in LMs, since this is a central feature involved in the operation of many LM encapsulated enzyme bioreactor systems. Current work in this laboratory with a tyrosinase/ ascorbate system for isolation of reactive intermediate oxidation products related to L-DOPA is discussed. A brief review of previous LM enzyme systems and reactor configurations is included for reference. 

Liquid membranes are double emulsions formed when a water-in-oil emulsion (w/o) is gently dispersed in a second aqueous phase, the external aqueous phase. The internal (emulsified) and external aqueous phases are kept separate by a layer of hydrocarbon, forming the liquid membrane. Since the two aqueous phases are not in contact, LM systems can be useful for separation processes as well as for enzyme immobilization separation is accomplished by selective transport of solutes across the hydrocarbon "membrane," and enzyme immobilization is accomplished by encapsulating enzyme(s) via emulsification of an aqueous enzyme solution. It is in fact possible to combine enzymatic reaction(s) with separations in a single LM system. Figure 1 depicts an LM-enzyme system. 

Liquid membranes as reactors ate especially promising for biological systems,11 For example, sensitive enzymes may be encapsulated to protect tbem from deactivating substances while meintaining free uccess to the substrate ( immobilization ). The substrate may be encapsulated with the enzyme or il may diffuse from the external phese into the internal phase where the reaction lakes place. In either case, it is... 

Emulsion liquid membranes (ELM) are double emulsions formed by mixing two immiscible phases and then dispersing the resulting emulsion in another continuous phase under agitation. Proposed applications for emulsion liquid membranes have included selective recovery of metal ions (1-12), separation of hydrocarbons (13 16), removal of trace organic contaminants (17-27), and encapsulation of reactive enzymes or whole cells (28-36). 

A recent study with biotechnology applications relates to amino acid extraction. Schugerl and co-workers (71 ) used a quaternary ammonium carrier in an emulsion liquid membrane system for enzyme catalyzed preparation of L-amino acids. Frankenfield et al. (72) discuss a wide variety of biomedical ELM applications including enzyme encapsulation, blood oxygenation, and treatment of chronic uremia. 

Boyadzhlev et al. (78) discussed phenol extraction using a combined ELM and film pertractlon scheme. Volkel et al. (79) discuss an interesting application. They used an enzyme encapsulated in an ELM to remove phenol from blood. Kitagawa et al. ( ) discussed applications of the liquid membrane technique to the removal of ammonia and various metal ions from Industrial waste water. For ammonia removal, the formulation used was similar to that for phenol separation except that the trapping agent was an acid. Various commercial... 

Figure 8.50 Liquid membrane capsule. The diameter of the capsule is 150 to 1000 /im. The diameter of encapsulated micro droplets is 1 to 5 /im. The encapsulated active phase can contain a catalyst, such as an enzyme the LMC then function as reactors . The reactants diffuse into the catalyst-active phase, and the products diffuse out. The encapsulated phase can also be a reagent. LMC encapsulating a reagent-active phase can be formulated to function as traps. Here the species to be removed diffuses from the solution being treated through encapsulating phase to the reagent-active phase. The reagent converts the material to a non-permeable species which cannot diffuse back through the encapsulating phase and becomes trapped [262].

Enzyme micro-encapsulation is another alternative for sensor development, although in most cases preparation of the microcapsules may require extremely well-controlled conditions. Two procedures have usually been applied to microcapsule preparation, namely interfacial polymerization and liquid drying [80]. Polyamide, collodion (cellulose nitrate), ethylcellulose, cellulose acetate butyrate or silicone polymers have been employed for preparation of permanent micro capsules. One advantage of this method is the double specificity attributed to the presence of both the enzyme and the semipermeable membrane. It also allows the simultaneous immobilization of many enzymes in a single step, and the contact area between the substrate and the catalyst is large. However, the need for high protein concentration and the restriction to low molecular weight substrates are the important limitations to this approach. 

LEM systems have also been shown to be successful in separating commodity-type biochemicals such as propionic acid (10) and acetic acid (10,22) and have been used for the preparation of L-amino acids from racemic D,L mixtures by means of enzymatic hydrolysis of amino acid esters (23). In addition to biochemical separations, the work of Mohan and Li showed that enzymes could be encapsulated in liquid emulsion membranes with no deleterious effect on enzyme action (24). Later work by these authors indicated that encapsulated live cells could remain viable and function in the LEM interior phase for period as long as five days (25). 

Nanocapsules, also known as hollow nanoparticles or nanospheres, are submicrometric colloidal objects composed of a liquid core (an internal phase) surrounded by a thin silicone shell (a membrane) (see Figure 4.2B). Hollow nanoparticles are potentially useful for encapsulation of various chemical molecules, Hving cells, or enzymes [26]. The following properties of nano capsules made their application very attractive ... 

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