Laccase, testing

Dimeric aldehydes analogous to (II) and (VI) have in fact been isolated from incubates of coniferyl alcohol with laccase (58). There are only 3% such aldehydic groups in lignin 4) [cf. Unit 10 in Fig. 9] but these suffice to give a intense red color with phloroglucinol and concentrated hydrochloric acid, the conventional Wiesner test for lignin. 

Lignin peroxidase activity, (i.e., peroxide-dependent oxidation of veratryl alcohol at pH 3) was not detected over the 30 days tested, while laccase appeared at day 7. Culture medium from day 7 onwards could also oxidize veratryl alcohol to aldehyde with concomitant conversion of oxygen to hydrogen peroxide. This activity, which was optimal at pH 5.0, was named veratryl alcohol oxidase (VAO). The extracellular oxidative enzyme activities (laccase and veratryl alcohol oxidase) could be separated by ion-exchange chromatography (Figure 2). Further chromatography of the coincident laccase and veratryl alcohol oxidase (peak 2), as described elsewhere (25) resulted in the separation of two veratryl alcohol oxidases from the laccase. 

These problems were overcome by immobilizing the enzyme. Since the usual methods for immobilization of laccase did not work, we adopted a new method, details of which will be described elsewhere (29). Reasonable measurements were possible with this technique. Typical patterns of laccase activity could be monitored via the changes of absorbance of 2,6 DMP and syringaldazine. When the reaction took place in organic solvents, the absorption spectra of the products were similar to those obtained for the same reaction in buffer. Furthermore, the catalytic action of the T. versicolor laccase followed Michaelis-Menten-kinetics in most of the organic solvents which were tested (see Table II for specific examples). 

Expression of the catalytic capacity of the immobilized laccase was also observed in more than a dozen different solvents, provided that they were either saturated with water or, in the case of solvents miscible with water, small amounts of water had been added (Table III). No enzymatic reaction was observed when the solvents tested were free of water. No correlation was found between the activity of the immobilized laccase and the hydrophobicity of the solvent in which the reaction took place. The rate of laccase reaction in ethylacetate was only twice that in toluene, despite the fact that water-saturated ethylacetate contains 50 times more water than... 

Catecholases and laccases may be differentiated by the use of substrate specificity tests and selective specific inhibitors (Walker and McCallion, 1980 Ferrar and Walker, 1996 Table C4.1.1). Salicylhydroxamic acid (SHAM), PVP, and/or cinnamic acids (cinnamic, p-coumaric, or ferulic) are probably the best choice for catecholase inhibitors, whereas cetyltrimethylammonium bromide (CETAB) has been found to inhibit most laccases. 

Another approach to curing SSL by oxidative polymerization employs the use of enzymes (54 ) In that study, SSL was treated with the white-rot fungus Fomes annosus and a laccase-inducing phenoloxidase until the solution reached a honeylike consistency. This viscous liquid was then subsequently used to bond wood particles together at a pressure of 0.03 kg cm-2. It now needs to be established whether the mechanical properties of these boards survive accelerated aging tests. 

A wide range of oxidoreductases have been tested in ionic liquid media including peroxidases, chloroperoxidases, laccases and dehydrogenases [28-34],... 

Mediators essential for laccase bleaching of pulp have been the subject of coupled experimental and computational research by Sealy et al. [55]. This study examined the effect of substitution on N-hydroxybenzotriazole, and the activity of phthalim-ides. N-hydroxybenzotriazole itself was more effective in lowering kappa numbers than any of the derivatives that were tested. Computationally, the bond dissociation energy was generally lower for N-hydroxybenzotriazole than the derivatives. Similarly, the most active of the phthalimide mediators had the lowest bond dissociation energy. 

Bark of P. taeda obtained from a pulp mill in Mississippi was milled, extracted, activated, and reacted with bisaminopropylethylenediamine (BAPED). The modification was made to increase the capacity of the bark to adsorb anions. Modified bark (0.5 to 0.7 g) in 5 mL of 40 mM tartarate buffer pH 5.0 and 10 pL laccase was mixed for 30 min at room temperature. Phloroglucinol (1 mL of 0.3 M in ethanol) or catechol (1 mL of 0.3 M) were added and incubated for 3 h at room temperature, followed by 18 h at 5°C. The particles were rinsed and recovered by filtration and freeze dried. Controls included laccase addition without substrate and untreated modified bark. The bark was tested for the ability to remove phosphate from solution. 

Substrates were tested in 20 mM pH 5.0 tartarate buffer and 0.2 units laccase using a YSI oxygen meter with a temperature-controlled stir cell (2 mL). Substrates are listed in order of increasing reactivity with laccase. 

Laccase creates free radicals that can fiirther react with odier compounds. Ideally, conditions for each substrate would be optimized for the grafting reaction, which would require investigation of concentration, pH, affinity of the enzyme, and other reactants in the pulp. Because optimizing the conditions for each substrate is beyond the scope of a screening test, the concentration of substrate (3.75 mM), enzyme (5 units), and fiber were held constant. We incubated 12 bar loblolly pine pulp in the presence of laccase with and without various laccase substrates, as described in Methods. These pulps were treated with laccase, washed, and fi-eeze dried. High affinity laccase substrates were. oxidized once before 1 h had elapsed. The enzyme reaction was continued for several hours to allow for lower affinity substrates to react. 

Laccase has also been proposed, and utilized, as a method for quantifying the extent to which mold Botrytis cinerea) deterioration has occurred on fruit (Dubourdieu et al., 1984). This colorimetric method relies on laccase-catalyzed oxidation of syringaldazine to its corresponding colored quinone. This test has an additional advantage in that the analysis has been reduced to kit form for field assay. Details of these procedures are described by Zoecklein et al. (1995). 

That pyrogallol is oxidized by blood, saliva, gum arable, and malt extract to give orange colored products was first noticed by Struve (332) in 1872. A study of the structure of these products was made by de Clermont and Chautard (109), and the first specific enzyme test using pyrogallol, namely for laccase, was made by Bertrand (60). 

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