Enzyme-polymeric membrane

Another immobilization method was described by Maeda and coworkers [344], They developed a facile and inexpensive preparation method for the formation of an enzyme-polymeric membrane on the inner wall of the microchannel (PTFE) through cross-linking polymerization in a laminar flow. With this approach, a-chymotrypsin was immobilized successfully. The activity of the immobilized enzyme was tested using N-glutaryl-L-phenylalanine p-nitroanilide as substrate, and the reaction products were analyzed offline by HPLC. There was no significant difference in the hydrolysis efficiency compared to solution-phase batchwise reactions using the same enzyme/substrate molar ratio (Scheme 4.87). 

Protein-polymeric membrane in a microchannel is prepared by using a concentric laminar flow (Fig. 43) [267]. Crosslinking condensation of a crosslinked enzyme aggregate (CLEA) [268] with aldehyde groups, which react with amino groups of the enzyme, in a concentric laminar flow results in the formation of a cylindrical enzyme-polymerized membrane on the inner wall of the microtube. The use of this technology for membrane formation in a microchannel can be extended to a broad range of functional proteins. 

Miyazaki and Maeda accomplished immobilization of acylase by the formation of an enzyme-polymeric membrane on the inner wall of the microreactor [172]. The same group used a microreactor system connected to a microextractor, which allowed liquid-liquid microextraction in a flow stream, as shown in Scheme 7.44. Using this microreaction system, optical resolution of racemic acetylphenylalanine was achieved to give D-acelyl phenylalanine with high optical purity [173]. 

Novel chiral. separations using enzymes and chiral surfactants as carriers have been realized using facilitated transport membranes. Japanese workers have reported the synthesis of a novel norbornadiene polymeric membrane with optically active pendent groups that show enantio.selectivity, which has shown promi.se in the. separation of propronalol. 

Generally, a distinction can be made between membrane bioreactors based on cells performing a desired conversion and processes based on enzymes. In ceU-based processes, bacteria, plant and mammalian cells are used for the production of (fine) chemicals, pharmaceuticals and food additives or for the treatment of waste streams. Enzyme-based membrane bioreactors are typically used for the degradation of natural polymeric materials Hke starch, cellulose or proteins or for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industry [50, 51]. In general, only ultrafiltration (UF) or microfiltration (MF)-based processes have been reported and little is known on the application of reverse osmosis (RO) or nanofiltration (NF) in membrane bioreactors. Additionally, membrane contactor systems have been developed, based on micro-porous polyolefin or teflon membranes [52-55]. 

Membrane reactors have been investigated since the 1970s 11). Although membranes can have several functions in a reactor, the most obvious is the separation of reaction components. Initially, the focus has been mainly on polymeric membranes applied in enzymatic reactions, and ultrafiltration of enzymes is commercially applied on a large scale for the synthesis of fine chemicals (e.g., L-methionine) 12). Membrane materials have been improved significantly over those applied initially, and nanofiltration membranes suitable to retain relatively small compounds are now available commercially (e.g., mass cut-off of 400—750 Da). 

Membrane reactors can be considered passive or active according to whether the membrane plays the role of a simple physical barrier that retains the free enzyme molecules solubilized in the aqueous phase, or it acts as an immobilization matrix binding physically or chemically the enzyme molecules. Polymer- and ceramic-based micro- and ultrafiltration membranes are used, and particular attention has to be paid to the chemical compatibility between the solvent and the polymeric membranes. Careful, fine control of the transmembrane pressure during operation is also required in order to avoid phase breakthrough, a task that may sometimes prove difficult to perform, particularly when surface active materials are present or formed during biotransformahon. Sihcone-based dense-phase membranes have also been evaluated in whole-cell processes [55, 56], but... 

There are several methods to selectively open up closed polymeric membrane compartments in order to release entrapped substances (Fig. 37). For uncorking a polymerized vesicle, its membrane has to contain destabilizable areas which could possibly be opened up by variation of pH 70), temperature increase71), photochemical destabilization 72), or enzymatic processes. Such an enzymatic process is the hydrolysis of a natural phospholipid by phospholipase A2 (Fig. 38). This enzyme cleaves the ester bond in position two of a natural phosphoglyceride producing a lysophospholipid and a fatty acid which are both water soluble. This leads to complete destruction of the membrane. 

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. 

Immobilization of a catalyst in (or on) a membrane Immobilization of enzymes or cells on polymeric membranes Immobilization of metals (Pd, Pt) on ceramic membranes... 

Butterfield DA, Colvin J, Liu J, Wang J, Bachas L, Bhattacharrya D (2002) Anal Chim Acta 470 29 Electron paramagnetic resonance spin label titration a novel method to investigate random and site-specific immobilization of enzymes onto polymeric membranes with different properties... 

Approaches aiming at creating biocompatible environments consist in modifying the surface of polymeric membranes by attaching functional groups like sugars, polypeptides and then to adsorb the enzymes. 

Figure 2 Mode of action of the prototypical lantibiotic nisin. (a) The peptidoglycan precursor lipid II is composed of an N-acetylglucosamine-p-1,4-N-acetylmuramic acid disaccharide (GIcNAc-MurNAc) that is attached to a membrane anchor of 11 isoprene units via a pyrophosphate moiety. A pentapeptide is linked to the muramic acid. Transglycosylase and transpeptidase enzymes polymerize multiple lipid II molecules and crosslink their pentapeptide groups, respectively, to generate the peptidoglycan. (b) The NMR solution structure of the 1 1 complex of nisin and a lipid II derivative in DMSO (6). (c) The amino-terminus of nisin binds the pyrophosphate of lipid II, whereas the carboxy-terminus inserts into the bacterial membrane. Four lipid II and eight nisin molecules compose a stable pore, although the arrangement of the molecules within each pore is unknown (5).

Surfactant aggregates (microemulsions, micelles, monolayers, vesicles, and liquid crystals) are recently the subject of extensive basic and applied research, because of their inherently interesting chemistry, as well as their diverse technical applications in such fields as petroleum, agriculture, pharmaceuticals, and detergents. Some of the important systems which these aggregates may model are enzyme catalysis, membrane transport, and drug delivery. More practical uses for them are enhanced tertiary oil recovery, emulsion polymerization, and solubilization and detoxification of pesticides and other toxic organic chemicals. 

Figure 4 14 Potentiometric enzyme electrode for determination of biood urea, based on urease enzyme immobilized on the surface of an ammonium ion-seiective polymeric membrane electrode.

The separation of proteins and enzymes is performed with ultrafiltration membranes. Branger et al. [93] use Carbosep Ml and M4 (40,000 and 20,000 Dalton respectively) for the separation of enzyme hydrolysates. The fluxes with these membranes compare favourably with polymeric membranes 37-102 l/m hvs. 7-41 l/m h. 

The aim of the present review is to present development of new biosensors for mycotoxines determination in foods, utilizing new polymeric membranes with immobilized enzymes and antibodies. 

Enzymes are covalently immobilized primarily onto the surface of the membrane exposed to the feed solution, known as the "active side" of the asymmetric membrane. In general, it is not clear whether reaction between enzymes and polymeric membranes via coupling agents simply results in enzyme attachment to the membrane, or if it leads to an enzyme-carrier network inside the polymer matrix. For the sake of simplicity let us assume that asymmetric membranes are used, that suitable active groups are available on the polymeric surface and that the membrane molecular weight cut-off is such that the active layer is enzyme-impermeable. In this way, even though their activity is often drastically reduced, surface bound enzymes are in close proximity to the substrate solution-thus reducing the mass transfer resistance to that associated with the boundary layer. When enzymes are covalently immobilized in the... 

HUal N, NigmatuUin R, Alpatova A (2004) Immobilization of cross-linked lipase aggregates within microporous polymeric membranes. J Membrane Sci 238(1-2) 131-141 Hiol A, Jonzo MD, Rugani N et al. (2000) Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit. Enzyme Microb Technol 26 421 30... 

Molecular separation along with simultaneous chemical transformation has been made possible with membrane reactors [17]. The selective removal of reaction products increases conversion of product-inhibited or thermodynamically unfavourable reactions for example, in the production of ethanol from com [31]. Enzyme-based membrane reactors were first conceived 25 years ago by UF pioneer Alan Michaels [49]. Membrane biocatalytic reactors are used for hydrolytic conversion of natural polymeric materials such as starch, cellulose, proteins and for the resolution of optically active components in the pharmaceutical, agrochemical, food and chemical industries. Membrane bioreactors for water treatment were introduced earher in this chapter and are discussed in detail in Chapters 2 and 3. 

In order to automate the analysis, these methods frequently combine immobilized enzymes with flow or sequential injection techniques. These methods may include a separation step such as solid-phase extraction, gas diffusion, or pervaporation. The latter is a nonchromatographic separation technique, which selectively separates a liquid mixture by partial vaporization through a nonporous polymeric membrane. Separation is not based on relative volatilities as in distillation, but rather on the relative rates of permeation through the membrane. 

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