Versatile peroxidase , production

Besides addition of heme, the influence of culture temperature on heterologous production of peroxidases has also been reported. For example, lowering the culture temperature from 28 to 19°C enhanced the level of active versatile peroxidase of P. eryngii 5.8-fold and reduced the effective proteolytic activity of the A. nidulans host strain by 2-fold. In this way, a maximum peroxidase activity of 466 U/L was reached [42]. Efficient heterologous production of peroxidases is not always dependent on the availability of heme. The heterologous production of Arthromyces ramosus peroxidase (ARP) has been analyzed in A. awamori under the control of the inducible endoxylanase promoter. Secretion of active ARP was achieved at up to 800 mg/L in shake flask cultures without addition of hemin [43]. This represents a 1,600-fold increase in production compared to ARP production in S. cerevisiae and 38-fold increase compared to ARP production in P. pastoris (see Sect. 12.2). These observations support that several filamentous fungi are more effective secretors of proteins than yeast strains like S. cerevisiae and P. pastoris. Also for... 

This catalytic asymmetric oxidation yielded J -methylphenylsulfoxide with a productivity of30g/l/day andane.e. >98% [35]. Chloroperoxidase is the most versatile peroxidase with better stability compared to other peroxidases, because spontaneous oxidation can be suppressed in the presence of ascorbic acid or dihydroxyfu-maric acid, and with better enantioselectivity because substrate access to the heme iron and ferryl oxygen favors stereoselective oxygen transfer [36]. Chloroperoxidase has been used for catalyzing the oxidation of cis-cydopropylmethanols with much higher enantioselectivity than trans-isomers [37]. 

Similarly, chloroperoxidase has proven to be a versatile peroxidase enzyme for synthetic applications (Dai and Klibanov, 1999 Loughlan and Hawkes, 2000 Spreti et al., 2004). The industrial usage of chloroperoxidases under aqueous conditions have been hampered by poor stability (inactivation via peroxide) of the enzyme and poor solubility of the substrate (Spreti et al., 2004). However, suspension of the peroxidase in PEG 200 increased product yields (Dai and Klibanov, 1999 Spreti et al., 2004). Other researchers have determined the effect of DMSO, DMF, MeOH and acetonitrile on the chlorination activity of the enzyme (pH 2.8), where Log P < 0 was preferred (Loughlan and Hawkes, 2000). 

MnP is the most commonly widespread of the class II peroxidases [72, 73], It catalyzes a PLC -dependent oxidation of Mn2+ to Mn3+. The catalytic cycle is initiated by binding of H2O2 or an organic peroxide to the native ferric enzyme and formation of an iron-peroxide complex the Mn3+ ions finally produced after subsequent electron transfers are stabilized via chelation with organic acids like oxalate, malonate, malate, tartrate or lactate [74], The chelates of Mn3+ with carboxylic acids cause one-electron oxidation of various substrates thus, chelates and carboxylic acids can react with each other to form alkyl radicals, which after several reactions result in the production of other radicals. These final radicals are the source of autocataly tic ally produced peroxides and are used by MnP in the absence of H2O2. The versatile oxidative capacity of MnP is apparently due to the chelated Mn3+ ions, which act as diffusible redox-mediator and attacking, non-specifically, phenolic compounds such as biopolymers, milled wood, humic substances and several xenobiotics [72, 75, 76]. 

The use of europium chelates, with their unusually long fluorescence decay times, as labels for proteins and antibodies has provided techniques that are referred to as time-resolved fluoroimmunoassays (TRFIA). Fluorophores as labels for biomolecules will be the topic of Sect. 3. Nevertheless, TRFIAs always have to compete with ELISA (enzyme-linked immunosorbent assays) techniques, which are characterized by their great versatility and sensitivity through an enzyme-driven signal amplification. Numerous studies have been published over the past two decades which compare both analytical methods, e.g., with respect to the detection of influenza viruses or HIV-1 specific IgA antibodies [117,118]. Lanthanide luminescence detection is another new development, and Tb(III) complexes have been applied, for instance, as indicators for peroxidase-catalyzed dimerization products in ELISAs [119]. 

In spite of their catalytic versatility and their capacity to transform a variety of pollutant compounds, peroxidases are not applied at large scale yet. The challenges that should be solved to use peroxidases for environmental purposes have been recently reviewed [146], Three main protein engineering challenges have been identified (a) the enhancement of operational stability, specifically hydrogen peroxide stability (see Chap. 11) (b) the increase of the enzyme redox potential in order to widen the substrate range (see Chap. 4) (c) the development of heterologous expression and industrial production (see Chap. 12). 

Plant peroxidases versatile catalysts in the synthesis of bioactive natural products. Studies in Natural Products Chemistry, 27, 735-791. 

This review will focus on the general properties of class III secretory plant peroxidases, and emphasis will be put on their broad metabolic plasticity, which is what makes them versatile catalysts in the synthesis of bioactive plant products. Examples of the role of peroxidase in the synthesis of such compounds are reviewed. 

So far, we have summarized strategies to exploit the chemical versatility of polymer brushes to either immobilize biomolecules by covalent attachment or for significantly decreasing protein adsorption. However, the extended interface created by the brush in a good solvent also provides a swellable, soft layer that can promote the nonspecific immobilization of enzymes and provide an environment that supports their activity. We have tested the functionality of enzymes physisorbed from solution [11]. Because this type of binding is weak, the conformation and activity of the proteins is expected to remain largely intact. To assess the influence of polymer brush chemistry, wettability, and swellability on the physisorption of proteins, model enzymes were chosen. Alkaline phosphatase (ALP) and horseradish peroxidase (HRP) were selected because they both catalyze the transformation of a colorless substrate to a colored product, and the enzymatic activity can therefore be easily monitored with colorimetry. The substrate of choice for ALP is /lara-nitrophenyl phosphate (pNPP), which is hydrolyzed to yield yellow /lara-nitrophenol (pNP) (Figure 4.14). 

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. 

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