Enzymatic microreactors

Enzymatic microreactors (7.5 nL) have been fabricated in the microfluidic chip to prepare the tryptic digest of equine (horse) myoglobin (14.2 pmol/p.L)... 

Peterson, D.S., Rohr, T., Svec, F., Frechet, J. M.J., Enzymatic microreactor-on-a-chip Protein mapping using trypsin immobilized on porous polymer monoliths molded in channels of microfluidic devices. Anal. Chem. 2002, 74(16), 4081M088. 

In order to increase the efficiency of biocatalytic transformations conducted under continuous flow conditions, Honda et al. (2006, 2007) reported an integrated microfluidic system, consisting of an immobilized enzymatic microreactor and an in-line liquid-liquid extraction device, capable of achieving the optical resolution of racemic amino acids under continuous flow whilst enabling efficient recycle of the enzyme. As Scheme 42 illustrates, the first step of the optical resolution was an enzyme-catalyzed enantioselective hydrolysis of a racemic mixture of acetyl-D,L-phenylalanine to afford L-phenylalanine 157 (99.2-99.9% ee) and unreacted acetyl-D-phenylalanine 158. Acidification of the reaction products, prior to the addition of EtOAc, enabled efficient continuous extraction of L-phenylalanine 157 into the aqueous stream, whilst acetyl-D-phenylalanine 158 remained in the organic fraction (84—92% efficiency). Employing the optimal reaction conditions of 0.5 gl min 1 for the enzymatic reaction and 2.0 gl min-1 for the liquid-liquid extraction, the authors were able to resolve 240 nmol h-1 of the racemate. 

Table 4.5 Application of enzymatic microreactors in the analysis of proteins.

Recent reviews have provided systematic coverage of the enzymatic microreactors used in chemical analysis [4]. Considering that the focus of this chapter is biocatalytic synthesis, it does not consider the analytical applications and the reader is referred to the cited literature ([4] and references given therein). The use of microreactors for high-throughput kinetic characterization of enzymes is another very interesting application of the technology [8], which, for reasons of limited space, is not discussed herein. 

Fig. 11 Top SU-8 based microfluidic system, which includes an enzymatic microreactor, a chromatographic device, and an integrated ionization emitter tip. Bottom SEM photograph of a section of a monolithic phase prepared from LMA/EDMA. Reproduced from [133]...

In addition to serving as the stationary phases, monolithic materials are also finding numerous other applications in the microfluidic world. Their use in on-chip solid-phase extraction and preconcentration,as supports for immobilization of enzymes to form enzymatic microreactors for protein mapping,static mixers,and valves represent just a few examples of modules in the microfluidic toolbox and further growth is inevitable. 

Fig. 2 (a) An enzymatic microreactor containing micropillars that have been coated with trypsin for protein digestion (Reprinted from [6]). (b) Porous monolith microreactors of various pore sizes. Pore size is determined by the ratio of monomer to porogen (solvent) during polymerization (Reprinted from [7]). (c)... 

Urban PL, Goodall DM, Bruce NC (2006) Enzymatic microreactors in chemical analysis and kinetic studies. Biotechnol Adv 24(l) 42-57... 

Urban, P.L., Goodall, D.M., Bruce, N.C. (2006) Enzymatic Microreactors in Chemical Analysis and Kinetic Studies. Biotechnol. Adv. 24 42-57. 

D. Ogoiiczyk, P. Jankowski, and P. Garstecki, Functionalization of polycarbonate with proteins Open-tubular enzymatic microreactors. Lab Chip, 12,2743-2748,2012. 

Another application of microfluidic enzymatic reactors is in the enormous and diverse challenges of proteomic investigations. Enzymatic microreactors present proteomics with a valuable analytical tool for protein analysis. Most of applications of IMERs are currently directed at protein analysis by protein digestion and peptide mapping. 

An important analytical application of enzymatic microreactors is their use in biocatalysis, in order to transform a difficult-to-measure analyte into an easily measurable form. This could be represented by microreactors designed for digestion of proteins to convert them to more readily measured peptides. Applications of microreactors in continuous-flow chemistry have expanded rapidly over the past two decades, with numerous reports of higher conversions and yields compared to conventional batch equipment. In this chapter, a comprehensive discussion on the most recent trends in the development of enzymatic microreactors and their current applications are covered. 

In the following discussion, applications of enzymatic microreactors will be presented. Comprehensive and up-to-date references are included that will give readers access to detailed information on the arising trends in the development of enzymatic microreactors for bioorganic and biocatalytic reactions and their current applications for applied analytical chemistry and biochemical studies. 

The principal field of application of enzymatic microreactors refers to protein and peptide mapping, an essential process in the identification and sequencing of proteins. Different developments and advances in the analysis of proteins by enzymatic microreactors are highlighted in this chapter. 

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... 

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