Biocatalytic with peroxidases

Based on IgG-bearing beads, a chemiluminescent immuno-biochip has been also realized for the model detection of human IgG. Biotin-labeled antihuman IgG were used in a competitive assay, in conjunction with peroxidase labelled streptavidin59. In that case, the planar glassy carbon electrode served only as a support for the sensing layer since the light signal came from the biocatalytic activity of horseradish peroxidase. Free antigen could then be detected with a detection limit of 25 pg (108 molecules) and up to 15 ng. 

The use of hydrogen peroxide as an oxidant is not compatible with the operation of a biocatalytic fuel cell in vivo, because of low levels of peroxide available, and the toxicity associated with this reactive oxygen species. In addition peroxide reduction cannot be used in a membraneless system as it could well be oxidized at the anode. Nevertheless, some elegant approaches to biocatalytic fuel cell electrode configuration have been demonstrated using peroxidases as the biocatalyst and will be briefly reviewed here. 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

In conclusion, it is clear that the immobilization of peroxidases into ordered porous materials with high surface areas aids the biocatalytic operation in terms of stability and activity in conditions that simulate an industrial process such as high temperature, the presence of organic solvent, and high H202 concentrations. 

The selection of the most suitable enzyme for a certain purpose mainly depends on its biocatalytic characteristics. Once a correct choice has been made, it is important to minimize the expenses associated with the enzyme use, as the economic feasibility of enzymatic processes is likely to depend on the cost of the enzyme production. In this context, several authors showed that the performance of various peroxidase processes was independent of enzyme purity [1,2], even suggesting that the crude enzyme was protected from inactivation [3, 4]. Microfiltration and subsequent ultrafiltration stages are sufficient to separate biomass and concentrate the enzyme for an economically viable operation [2, 5]. 

As enantiomericaUy pure sulfoxides are excellent chiral auxUiaries for asymmetric synthesis, different approaches for biocatalytic asymmetric oxidations at the S-atom have been explored [30, 31]. Asymmetric peroxidaseorganic sulfides to sulfoxides in organic solvents opens up attractive opportunities by increased substrate solubility and diminished side reactions [32]. Plant peroxidases located in the cell wall are capable of oxidizing a broad range of structurally different substrates to products with antioxidant, antibacterial, antifungal, antiviral, and antitumor activities [33]. Hydroperoxides and their alcohols have been obtained in excellent e.e. in the biocatalytic kinetic resolution of secondary hydroperoxides with horseradish and Coprinus peroxidase [34]. 

In biocatalytic systems, catalase is mainly used in immobilized state. High activity of immobilized catalase was achieved on its sorption immobilization on cellulose [6], on silica gel modified with fatty acids or phospholipids [7] as well as on activated carbon fibres and brics/tissues/ [8]. Biocatalytic activity of catalase immobilized on cellulose was also studied in nonaqueous solvents [9,10]. In [9] it was found that unlike the enzyme dissolved in water-dimethylformamide medium, on the oxidation of o-dianisidine in the presence of dimethylfonnamide the immobilized catalase does not show any peroxidase activity. It was used [10] for working out an organic-phase amperometric biosensor by immobilizing the enzyme in a polymeric film on a glass-carbon surface. 

The results of investigations of enzymatic activity in the immobilized state. When laccase, peroxidase, and other enzymes are adsorbed on dispersive electroconductive carriers, their enzymatic activity perceptibly declines, thus indicating a denaturing of part of the macromolecules. In this case, however, there is a sufficient quantity of denatured enzyme molecules strongly bonded with the carrier, which display a specific biocatalytic activity in model reactions of the substrates. It is these molecules of the enzyme, as will be seen further, that are responsible also for the electrocatalytic activity of the system. 

This chapter is an overview of architectures adopted for the catalytic/biocatalytic composites used in wide applications like the biomass valorization or fine chemical industry. On this perspective, the chapter updates the reader with the most fresh examples of construction designs and concepts considered for the synthesis of such composites. Their catalytic properties result from the introduction of catalytic functionalities and vary from inorganic metal species e.g., Ru, Ir, Pd, or Rh) to well-organized biochemical structures like enzymes e.g., lipase, peroxidase, (3-galactosidase) or whole cells. Catalytic/biocatalytic procedures for the biomass conversion into platform molecules e.g., glucose, GVL, Me-THF, sorbitol, succinic acid, and glycerol) and their further transformation into value-added products are detailed in order to make understandable the utility of these complex architectures and to associate the composite properties to their performances, versatility, and robustness. 

The electrochemical insulation of the enzyme-active site by its protein or glycoprotein shell usually precludes the possibility of any direct electron-transfer with bulk electrodes [15]. However, under carefully controlled conditions, some enzymes can exhibit direct, nonmediated electrical communication with electrode supports, and biocatalytic transformations can be driven by these processes [16, 17]. For example, the direct electroreduction of O2 and H2O2 biocatalyzed by laccase [18] and horseradish peroxidase (HRP) [19], respectively, have been demonstrated. This unusually facile electronic contacting is believed to be the consequence of incompletely encapsulated redox centers. When these enzymes are properly orientated at the electrode surface, the electrodeactive site distance is short enough for the electron-transfer to proceed relatively unencumbered. Direct electron communication between enzyme-active sites and electrodes may also be facilitated by the nanoscale morphology of the electrode. The modification of electrodes with metal nanoparticles allows the tailoring of surfaces with features that can penetrate close enough to the enzyme active site to make direct electron-transfer possible [20, 21]. 

Abstract The in vitro enzyme-mediated polymerization of vinyl monomers is reviewed with a scope covering enzymatic polymerization of vitamin C functionalized vinyl monomers, styrene, derivatives of styrene, acrylates, and acrylamide in water and water-miscible cosolvents. Vitamin C functionalized polymers were synthesized via a two-step biocatalytic approach where vitamin C was first regioselectively coupled to vinyl monomers and then subsequently polymerized. The analysis of this enzymatic cascade approach to functionalized vinyl polymers showed that the vitamin C in polymeric form retained its antioxidant property. Kinetic and mechanistic studies revealed that a ternary system (horseradish peroxidase, H2O2, initiator fS-diketone) was required for efficient polymerization and that the initiator controls the characteristics of the polymer. The main attributes of enzymatic approaches to vinyl polymerization when compared with more traditional synthetic approaches include facile ambient reaction environments of temperature and pressure, aqueous conditions, and direct control of selectivity to generate functionalized materials as described for the ascorbic acid modified polymers. 

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