Enzymes encapsulation

Erythrocyte Entrapment of Enzymes. Erythrocytes have been used as carriers for therapeutic enzymes in the treatment of inborn errors (249). Exogenous enzymes encapsulated in erythrocytes may be useful both for dehvery of a given enzyme to the site of its intended function and for the degradation of pathologically elevated, diffusible substances in the plasma. In the use of this approach, it is important to determine that the enzyme is completely internalized without adsorption to the erythrocyte membrane. Since exposed protein on the erythrocyte surface may ehcit an immune response following repeated sensitization with enzyme loaded erythrocytes, an immunologic assessment of each potential system in animal models is required prior to human trials (250). 

However, it has to be realized that biological templates remain inserted in the final nanoparticles and this is not acceptable for many applications. Nevertheless, some recent examples indicate that such biomimetic materials may be suitable for the design of biotechnological and medical devices [32]. For instance, it was shown that silica gels formed in the presence of p-R5 were excellent host matrices for enzyme encapsulation [33]. In parallel, biopolymer/silica hybrid macro-, micro- and nanocapsules were recently obtained via biomimetic routes and these exhibit promising properties for the design of drug delivery materials (see Section 3.1.1) [34,35],... 

Caruso F, Trau D, Mohwald H, Renneberg R. Enzyme encapsulation in layer-by-layer engineered polymer multilayer capsules. Langmuir 2000 16 1485-1488. 

Betancor L, Luckaiift HR (2008) Bioinspired enzyme encapsulation for biocatalysis. Trends Biotechnol 26(10) 566-572... 

Erythrocyte Entrapment of Enzymes. Erythrocytes have been used as carriers for therapeutic enzymes in the treatment of inborn errors. Exogenous enzymes encapsulated in erythrocytes may be useful both for delivery of a given enzyme to Ihe she of its intended function and for file degradalinn of pathologically elevuied. diffusible substances in the plasma. 

Membranes can be used as well as a supporting material for immobilisation (e.g. polycarbonate membranes with created amino groups on the surface that allow covalent binding with glutaraldehyde). Entrapment of enzymes on electrode surfaces can be carried out with polymeric membranes such as polyacrylamide and gelatine, or by electropolymerisation of small monomers (o-phenylenediamine). Enzyme encapsulation within a sol-gel matrix has also been reported. 

Palmitoyl-l-ASPT a chemically modified derivative of the enzyme encapsulated in liposomes, has also been studied in animals [44] and shown to exhibit prolonged half-life (about 10-fold), no acute toxicity, but preservation of in vivo antitumor activity. 

The hyperbolic architecture of a variety of enzymes has been pointed out in Chapter 6. It seems natural then that enzymes encapsulated in a hyperbolic (lipid) environment (of suitable dimensions) are able to adopt their favoured architecture, and thus perform their biochemical task efficiently. In this context, it is wortii noting that remarkable increases in enzymatic efficiency of biochemical s3mthesis have been achieved by containment of enzymes in cubosomes, rather than conventional (flat) liposomes. 

Up to now there is still not much research on the mechanism of how the MPS enhance the catalysis of proteins and how the direct electron transfer occur while the proteins encapsulated in the MPS pore. The mechanism is not clear. Besides, as this chapter referred above, many scientists found that the protein/enzymes encapsulated on normal MPS surface and in the pore were not stable and lots of endeavor was devoted to solve this problem [34-38], Abundant of work is currently carrying out on the study of the electrochemical... 

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... 

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. 

Encapsulation of enzymes in LMs offers further improvements for immobilization of complex enzyme systems, as the enzymes / cofactors, etc. are situated in aqueous droplets surrounded by a stable liquid hydrocarbon film (Figure 1). Instead of the physical pores present in microcapsules, the HC barrier, which has a diffusion thickness of about 0.1-1.0 p, effectively blocks all molecules except those which are oil-soluble or transportable by the selected carriers. Encapsulation of enzymes in LMs is accomplished simply by emulsifying aqueous enzyme solutions. Hence, LMs offer many advantages over other systems used for separation and eirzyme immobilization they are inexpensive and easy to prepare they promote rapid mass transport they are selective for various chemical species they can be disrupted (demulsified) for recovery of internal aqueous solutions gradients of pH and concentration (even of small molecules) can be maintained across the HC barrier multiple enzyme / cofactor systems can be coencapsulated and enzymatic reaction and separation can be combined. Some of the potential disadvantages of LMs for enzyme encapsulation have been discussed earlier. 

Significant research on LM enzyme encapsulation systems has also been conducted at the University of Hannover, West Germany. Scheper et al. (19) proposed the use of LMs to resolve racemic D,L-phenylalanine methyl ester with encapsulated chymotrypsin. This enzyme cleaves the ester bond of the L-isomer only. The process employed Adogen 464 (TOMAC) as an anion carrier, but the pHs used were such that any L-phenylalanine formed would be zwitterionic LM transport of zwitterions would be expected to be poor. Further work has included development of an LM enzyme reactor for detoxification of blood (33), reductive amination of a-ketoisocaproate by L-leucine dehydrogenase with a coencapsulated... 

FTIR analysis was also used to study the efficiency of the sol-gel enzyme encapsulation method that uses TEOS as precursor. The silica matrix gelation procedure produces a tridimensional reticulate formed by interacting polymeric inorganic chains that form a holding net around the enzyme, and FTIR can be used to follow this encapsulating process. The FTIR spectrograms obtained for the pure silica matrix and the encapsulated CGTase are shown at Fig. 6. 

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... 

As enzymes encapsulate multiple functionalities within their catalytic cavity, they have also served as a major source of inspiration for the fields of biomimetic chemistry and supramolecular catalysis. Early mechanistic theories about how enzymes work have prompted scientists from various fields to explore similar approaches for synthetic systems. One of these approaches is host-guest catalysis, where one or more substrates are bound in a cavity next to the catalytically active site. 

Enzyme-Encapsulated Layer-by-Layer Assemblies Current Status and Challenges Toward Ultimate Nanodevices... 

As one can easily imagine from the above-mentioned examples, it is high time to develop biological nanodevices based on LbL techniques. We here summarize recent developments on enzyme-encapsulated LbL devices and related functions where encapsulated does not always entail entrapment within spherical structures but generally includes immobilization of enzymes within LbL structures. 

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