Enzyme-immobilized microreactors

Another example in which biocatalysis is combined with analysis is the system reported by Honda et al. [436]. A microreaction system, consisting of an enzyme-immobilized microreactor, for optical resolution of racemic amino acids was devel-... 

Smirnova et al. demonstrated the determination of the insecticide, carbaryl, using a two-chip system. The first chip (for the hydrolysis of carbaryl) had a simple Y-shaped channel while the second chip (for the diazo couphng reaction between hydrolyzed products and 2,4,6-trimethylaniline)—the extraction required special channel shapes with a partial surface— modification obtained by using capillary-restricted modification (CARM) (Figure 35.11). " Determination of carbaryl pesticide in water with sufficient sensitivity was carried out with an analysis time of 8 min. In a similar manner, Honda et al. developed a combination of a tube-type enzyme-immobilized microreactor and a microextractor with partial surface modification to produce optically pure amino acids. 

Honda T, Miyazaki M, Nakamura H et al (2006) Facile preparation of an enzyme-immobilized microreactor using a cross-linking enzyme membrane on a microchannel surface. Adv Synth Catal 348 2163-2171... 

Figure 10.21 Schematic representation of enzymatic reaction using enzyme-immobilized microreactors. The substrate was pumped through the enzyme-immobilized microreactors using a syringe pump. The reaction was carried...

Applications of enzyme-immobilized microreactors for processing have also been presented including hydroxylation of macrolide in a microreactor [156]. PikC hydroxylase was immobilized on Ni-NTA agarose beads via in situ attachment. 

Another enzyme that was studied extensively in microreactors to determine kinetic parameters is the model enzyme alkaline phosphatase. Many reports have appeared that differ mainly on the types of enzyme immobilization, such as on glass [413], PDMS [393], beads [414] and in hydrogels [415]. Kerby et al. [414], for example, evaluated the difference between mass-transfer effects and reduced effidendes of the immobilized enzyme in a packed bead glass microreactor. In the absence of mass-transfer resistance, the Michaelis-Menten kinetic parameters were shown to be flow-independent and could be appropriately predicted using low substrate conversion data. 

Most biocatalytic conversions are performed with the enzyme immobilized in the microreactor. Miyazaki et al. [426] developed a simple noncovalent immobilization method for His-tagged enzymes on a microchannel surface. These enzymes contain a polyhistidine-tag motif that consists of at least six histidine residues, often located at the N- or C-terminus. The H is-tag has a strong affinity for nickel and can be reversibly immobilized by a nickel-nitrilotriacetic acid (Ni-NTA) complex (Scheme 4.103), a strategy commonly used in affinity chromatography. 

One solution to the challenges of mixing proteins in microfluidic systems is to immobilize proteins in microreactors [5]. These systems typically consist of chambers of enzymes immobilized on beads, micropillars [6], or porous polymer monoliths [7] (Fig. 2a and b). Such systems have large surface area-to-volume ratios, which minimize diffusion time for reactions with solution-phase reagents. Microreactors can be used either for the conversion of an analyte to another form that is more easily detected or for direct studies of the properties of enzymes and substrates. One of the most common uses is for the digestion of proteins for proteome profiling, but such systems can also be used for the removal of amino acid residues from peptides or proteins or for enzyme kinetic studies. 

Other examples are enzymes immobilized on beads which are trapped in a microreactor by etched weirs [88], enzymes encapsulated in hydrogel patches or sol-gel silica [89] and enzymes attached on the surface of (porous) microstructures (for example, on porous silicon manufactured by anodization of single-crystalline silicon see Figure 1.10 [91]), of mesoporous silica or polymer monoliths or directly... 

To fulfill such requirements, attempts have been made in the past decade by researchers working on peptide mapping and proteomics through development of immobilized microfluidic enzymatic reactors. Microfluidic enzymatic microreactors are an alternative to in-solution method employing immobilization of proteases on microchaimels of chip-based reactors or surfaces of capillaries. The microreactors that enable proteolytic digestion by enzymes immobilized on solid supports are also referred to as immobilized enzyme reactors, IMERs. The great potential of IMERS for proteomic applications comprise rapid and enhance... 

The preparation of an easily replaceable protease microreactor for microchip application was described by Bilkova and colleagues [83]. The most important advantage of microsphere-packed microchip bioreactors is that the enzyme-immobilized microspheres in the channel can be replaced. Magnetic particles coated with... 

An example of enzyme immobilized by adsorption was described by Gao and others, who developed a PDMS microreactor for proteolytic digestion with online ESI-MS identification [89]. Trypsin was adsorbed in a poly(vinylidine fluoride) porous membrane. Peptide identification for Cyt< was reported using as little as 0.04 pmol. 

Immobilization of Enzyme in Capillary Microreactor IMER systems in capillary microreactors are now routinely being used in protein digestion for peptide mapping and proteomics. Similar to microfluidic charmels on chips, enzyme immobilization in capillary can be classified into two categories based on the immobilization location. 

Covalent linking of enzyme on fused silica capillaries was also demonstrated by Stigter [119]. Pepsin was covalently immobilized on dextran-modified capillaries as illustrated in Scheme 10.12. The applicability of pepsin-immobilized microreactor was tested with a number of proteins varying in molecular weight, isoelectric point, and sample composition. This open tubular microreactor demonstrated a flow-dependent digestion as represented by native hemoglobin. Complete digestion of... 

Another category of enzymatic transformations in multiphase systems is enzymes immobilized on the reactor wall as presented in Table 10.4. Enzymes are advantageously used in immobilized form because this strategy allows for increased volumetric productivity and improves stability. Continuous mode of operation is employed in these systems. The approaches commonly used for immobilization in conventional multiphase biocatalysis can also be employed in microreactors such as covalent methods, cross-linked enzyme aggregates (CLEA), and adsorption methods. The experimental setups can either be chip-type reactors with activated charmel surface walls where enzyme binds, or enzyme immobilized monolith reactors, where a support is packed inside a capillary tube. 

Our group has developed a technique that forms an enzyme-immobilizing membrane on the microcharmel surface [154]. This is a modification of CLEA formation, which is used in batchwise organic synthesis [155]. The microreactor has a cylindrical membrane composed of cross-linked polymerized acylase and polylysine matrix product on the internal surface of the microchatmel (Figure 10.28). 

Dried cross-linked (-l-)-Y-lactamase mixed with controlled pore [163] glass in a 1 1 ratio stability of immobilized enzyme for 8h at 80 °C kinetic constants determined in the microreactor Glucose oxidase or choline oxidase were separately immobilized [162] on the surface of PEI coated monolith. Method is simple based on preparation of monolith with controlled porosity low pressure drop, mass transfer limitations avoided enzymes immobilized on PEI-activated surface of monolity through electropositive (PEI and electronegative (enzyme) nature... 

Biocatalytic reactions performed using immobilized enzyme microreactors under continuous flow mode have been found effective for hydrolysis reactions [121,158-161], with the enzyme either trapped in the matrix [159], covalently linked to modified surface wall [160,121], enzymes entrapped in hydrogels [162], or enzymes immobilized on monolith [179]. The experimental setup consists of either chip-type microreactors with activated chaimel walls where enzymes bind, enzymes that bind to beads, enzymes entrapped in the matrix, enzymes adsorbed in nanoporous materials, and most recently, nanosprings as supports for immobilized enzymes in chip-based reactors, or enzyme immobilized monolith reactors, where support is packed inside a capillary tube (Table 10.4). 

The three different enzymes used in combination in this system were FateDH, FaldDH, andADH. FateDH catalyzes the initial reduction of C02 to formate, FaldDH the reduction of formate to formaldehyde, and ADH the reduction of formaldehyde to methanol. Interestingly, the enzymes when immobilized were more active than a combination of the free enzymes, which is claimed to be due to a reduction of spatial interference among the different enzymes. Moreover, due to the immobilization of enzymes within the microreactor, the intermediate species have significantly reduced distances to travel between active sites [21, 22]. 

As mentioned above, in order to extend the potentialities of the luminescence-based optical fibre biosensors to other analytes, auxiliary enzymes can be used. The classical approaches consist either of the coimmobilization of all the necessary enzymes on the same membrane or of the use of microreactors including immobilized auxiliary enzymes and... 

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