**4. Ligninolytic enzymes**

Microorganisms that colonize on living and decaying wood are capable of producing oxidative extracellular enzymes which together play a fundamental role in lignin biodegradation. The ligninases, or lignin-degrading enzymes, can oxidize lignin and several related compounds, e.g., environmental pollutants containing polycyclic aromatic hydrocarbons, dyes, and chlorophenols [124].

Lignin-peroxidase (LiP, E.C. 1.11.1.14), manganese-peroxidase (MnP, E.C. 1.11.1.13), and laccase (E.C. 1.10.3.2) are the major lignin-modifying enzyme systems of white-rot fungi and have also been described in actinomycetes and bacteria. These enzymes oxidize phenolic compounds and reduce molecular oxygen to water, generating intermediary radicals as illustrated in **Figure 2** [125, 126].

Accessory enzymes involved in the main reactions of degradation of lignin have also been described and comprise the following: cellobiose-quinoneoxireductase (E.C. 1.1.5.1), aryl alcohol oxidase (E.C. 1.1.3.7), glyoxal oxidase (GO, E.C. 1.2.3.5), manganese-independent peroxidase (E.C. 1.11.1.7), versatile peroxidase (VP, E.C. 1.11.1.16), and cellobiose dehydrogenase (E.C. 1.1.99.18) [127, 128].

Besides ligninolytic enzymes have been used to reduce the lignin content in several feedstock and to degrade recalcitrant aromatic compounds, due to the high chemical similarity of these compounds with lignin [13, 129, 130], the lignin-degrading enzymes have been applied in various industries such as textile dye bleaching, pulp and paper delignification, food, brewery, animal feed, laundry detergents, and xenobiotic compound degradation. Phenol oxidases such as laccases, particularly, have been applied in immunoassay, biosensors, biocatalysts, and oxygen cathode manufacturing [127, 131].

The performance of these enzymes is easily affected by environmental factors including metal ions and other chemical compounds usually found in the aforementioned industries. Ligninases with stronger tolerance to metal ions and organic solvents exhibit high potential for the application in the recalcitrant xenobiotics biodegradation and also improve the effectiveness of biotechnological and industrial enzymatic process [132, 133].

#### **4.1. Laccases (E.C. 1.10.3.2)**

Laccases are multicopper blue oxidases that catalyze the one-electron oxidation of a wide range of substrates with a concomitant four-electron reduction of molecular oxygen to water [126]. The active site of laccase comprises four copper atoms in three groups: T1 (mononuclear

Effect of Metal Ions, Chemical Agents and Organic Compounds on Lignocellulolytic Enzymes Activities http://dx.doi.org/10.5772/65934 149

**Figure 2.** Simplified reactions of lignin peroxidase, manganese peroxidase, and laccase.

copper), T2 (normal copper), and T3 (coupled binuclear copper). The T1 and T2 Cu2+-sites contribute as the primary electron acceptors while T3 is reduced by an intramolecular twoelectron transfer from T1 and T2 Cu2+ sites [126, 134].

#### *4.1.1. Metal ions associate to laccase activity*

a single access route for ligands. The authors classified the inhibitors into two groups: I, single binding inhibitors including cellobiose (4-O-β-D-glucopyranosyl D-glucose), D-glucose, maltose (4-O-a-D-glucopyranosyl-D-glucose), D-xylose, and L-xylose; II, double binding inhibitors including D-arabinose, L-arabinose, D-erythrose, and D-ribose. Both groups have presented

Microorganisms that colonize on living and decaying wood are capable of producing oxidative extracellular enzymes which together play a fundamental role in lignin biodegradation. The ligninases, or lignin-degrading enzymes, can oxidize lignin and several related compounds, e.g., environmental pollutants containing polycyclic aromatic hydrocarbons, dyes,

Lignin-peroxidase (LiP, E.C. 1.11.1.14), manganese-peroxidase (MnP, E.C. 1.11.1.13), and laccase (E.C. 1.10.3.2) are the major lignin-modifying enzyme systems of white-rot fungi and have also been described in actinomycetes and bacteria. These enzymes oxidize phenolic compounds and reduce molecular oxygen to water, generating intermediary radicals as illus-

Accessory enzymes involved in the main reactions of degradation of lignin have also been described and comprise the following: cellobiose-quinoneoxireductase (E.C. 1.1.5.1), aryl alcohol oxidase (E.C. 1.1.3.7), glyoxal oxidase (GO, E.C. 1.2.3.5), manganese-independent peroxidase (E.C. 1.11.1.7), versatile peroxidase (VP, E.C. 1.11.1.16), and cellobiose dehydrogenase

Besides ligninolytic enzymes have been used to reduce the lignin content in several feedstock and to degrade recalcitrant aromatic compounds, due to the high chemical similarity of these compounds with lignin [13, 129, 130], the lignin-degrading enzymes have been applied in various industries such as textile dye bleaching, pulp and paper delignification, food, brewery, animal feed, laundry detergents, and xenobiotic compound degradation. Phenol oxidases such as laccases, particularly, have been applied in immunoassay, biosensors, biocatalysts,

The performance of these enzymes is easily affected by environmental factors including metal ions and other chemical compounds usually found in the aforementioned industries. Ligninases with stronger tolerance to metal ions and organic solvents exhibit high potential for the application in the recalcitrant xenobiotics biodegradation and also improve the effec-

Laccases are multicopper blue oxidases that catalyze the one-electron oxidation of a wide range of substrates with a concomitant four-electron reduction of molecular oxygen to water [126]. The active site of laccase comprises four copper atoms in three groups: T1 (mononuclear

tiveness of biotechnological and industrial enzymatic process [132, 133].

competitive or noncompetitive inhibition.

**4. Ligninolytic enzymes**

148 Enzyme Inhibitors and Activators

and chlorophenols [124].

trated in **Figure 2** [125, 126].

(E.C. 1.1.99.18) [127, 128].

**4.1. Laccases (E.C. 1.10.3.2)**

and oxygen cathode manufacturing [127, 131].

Although laccases are efficient on a wide range of substrates without cofactors, in most cases, the addition of Cu2+, Cd2+, Ni2+, Mo2+, and Mn2+ ions increases the activity of laccases, whereas Ag2+, Hg2+, Pb2+, Zn2+, NaN3 , NaCl, and H2 O2 inhibit their activity [126].

Apart from the inhibition problem, the influence of metal ions on the performance of enzymecatalyzed reaction is also important, in addition to the study of effects of single metal ions on the enzyme activity. Lu et al. [135] observed that monovalent and trivalent metal ions inhibited the 4-nitrophenol degradation by laccase-Cu2+, as well as the addition of low concentrations of divalent ions. The suppressive effects of cations on laccase activity comprised Mg2+ > Na+ > Al3+ > K+ > Mn2+ > Hg2+ > Co2+.

#### *4.1.2. Chemical agents and organic compounds associate to laccase activity*

The Michaelis-Menten equation has been suitably used to describe the laccase kinetics and apparent binding constant (*K*m) and maximal reaction rate (*V*max) values. In water-miscible solvents, these kinetic parameters can be affected by the changes in water thermodynamic activity. In the case of laccase from the white-rot fungus *Phlebiaradiata*, e. g., pKI values show the linear dependence on solvent hydrophobicity (log*P*) in a system of 2,6 dimethoxyphenol as substrate in the presence of methanol, ethanol, *n*-propanol, acetonitrile, acetone, and DMSO [136].

Previously, the changes in *V*max by the addition of solvents have been compared to free and immobilized laccases. The activity of laccase from *P. radiata* was rather similar to both forms of the enzyme in the presence of 10% of ethanol, methanol, acetone, DMSO, and dioxane. The immobilized laccase was less vulnerable to Cu-chelatorthioglycolic acid, 2,6-dimethoxy-1,4-benzoquinone [128, 137].

In the conditions of low water content, which is the case of water/organic mixtures, the values of the apparent *K*m tend to grow exponentially with water concentration. The apparent *V*max of immobilized laccase from *Coriolusversicolor* increased two orders of magnitude values with a linear increase in water content [138].

#### **4.2. LiP (E.C. 1.11.1.14)**

Lignin-peroxidases are heme-containing glycoproteins that contain Fe3+ in their active site. LiP catalyzes the H2 O2 -dependent oxidative depolymerization of nonphenolic lignin and lignin-model compounds as well as a variety of phenolic compounds [139].

#### *4.2.1. Metal ions, chemical agents, and organic compounds associate to LiP activity*

The decrease in LiP activity is described as inhibition or denaturation according to the concentration of inhibitor compounds in an aqueous reaction system. The hydrogen bonding and anion stabilization are important characteristics to describe the effect of compounds on the active sites of enzymes, as well as water activity (aw), log*P*, and solvation [140].

The addition of Cu2+, Mn2+, and Fe2+ ions increases the activity of LiP, whereas Ag2+ inhibit their activity [141]. On the other hand, different solvents and organic compounds have been described as LiP potential inhibitors: alcohols, aldehydes, ketones, esters, ethers, amines, acids, amides, acetonitrile, cysteine, DMSO, EDTA, DMF, TEMED, CTAB, sodium azide, and H2 O2 [140–144].

Vazquez-Duhalt et al. [145] chemically modified a LiP from the white-rot fungus *Phanerochaete chrysosporium* by reductive alkylation with benzyl, naphthyl, and anthracyl moieties, thereby increasing its superficial hydrophobicity. These modifications altered the kinetics and increased the yield of oxidation of pyrroles, pyridines, and aromatic amines in 10% acetonitrile.

#### **4.3. MnP (E.C. 1.11.1.13)**

>

of divalent ions. The suppressive effects of cations on laccase activity comprised Mg2+ > Na+

The Michaelis-Menten equation has been suitably used to describe the laccase kinetics and apparent binding constant (*K*m) and maximal reaction rate (*V*max) values. In water-miscible solvents, these kinetic parameters can be affected by the changes in water thermodynamic activity. In the case of laccase from the white-rot fungus *Phlebiaradiata*, e. g., pKI values show the linear dependence on solvent hydrophobicity (log*P*) in a system of 2,6 dimethoxyphenol as substrate in the presence of methanol, ethanol, *n*-propanol, acetonitrile, acetone, and

Previously, the changes in *V*max by the addition of solvents have been compared to free and immobilized laccases. The activity of laccase from *P. radiata* was rather similar to both forms of the enzyme in the presence of 10% of ethanol, methanol, acetone, DMSO, and dioxane. The immobilized laccase was less vulnerable to Cu-chelatorthioglycolic acid, 2,6-dimethoxy-

In the conditions of low water content, which is the case of water/organic mixtures, the values of the apparent *K*m tend to grow exponentially with water concentration. The apparent *V*max of immobilized laccase from *Coriolusversicolor* increased two orders of magnitude values with a

Lignin-peroxidases are heme-containing glycoproteins that contain Fe3+ in their active site.

The decrease in LiP activity is described as inhibition or denaturation according to the concentration of inhibitor compounds in an aqueous reaction system. The hydrogen bonding and anion stabilization are important characteristics to describe the effect of compounds on the

The addition of Cu2+, Mn2+, and Fe2+ ions increases the activity of LiP, whereas Ag2+ inhibit their activity [141]. On the other hand, different solvents and organic compounds have been described as LiP potential inhibitors: alcohols, aldehydes, ketones, esters, ethers, amines, acids, amides, acetonitrile, cysteine, DMSO, EDTA, DMF, TEMED, CTAB, sodium azide, and

Vazquez-Duhalt et al. [145] chemically modified a LiP from the white-rot fungus *Phanerochaete chrysosporium* by reductive alkylation with benzyl, naphthyl, and anthracyl moieties, thereby increasing its superficial hydrophobicity. These modifications altered the kinetics and increased the yield of oxidation of pyrroles, pyridines, and aromatic amines in 10% acetonitrile.

nin-model compounds as well as a variety of phenolic compounds [139].

*4.2.1. Metal ions, chemical agents, and organic compounds associate to LiP activity*

active sites of enzymes, as well as water activity (aw), log*P*, and solvation [140].


*4.1.2. Chemical agents and organic compounds associate to laccase activity*

Al3+ > K+

150 Enzyme Inhibitors and Activators

DMSO [136].

1,4-benzoquinone [128, 137].

**4.2. LiP (E.C. 1.11.1.14)**

LiP catalyzes the H2

H2

O2 [140–144].

linear increase in water content [138].

O2

> Mn2+ > Hg2+ > Co2+.

Manganese-peroxidases catalyze the H2 O2 -dependent oxidation of Mn2+ into Mn3+, which is stabilized by fungal chelators such as oxalic acid or different organic acids. Then, the oxidation of various phenolic substrates (e.g., amines, dyes, lignin related compounds) occurs under the action of chelated Mn3+ ions that comprise a diffusible charge-transfer mediator in these reactions [141, 146].

#### *4.3.1. Metal ions associate to MnP activity*

MnP activity is completely inhibited by Hg2+, Pb2+, Ag<sup>+</sup> , lactate, NaN3 , CaCl2 , TEMED, ascorbic acid, β-mercaptoethanol, and dithreitol [147, 148]. Partial inhibition of MnP activity was observed with EDTA, a metal chelating compound that complexes with inorganic cofactors and prosthetic groups of enzymes. High concentrations of Cu2+ and Fe2+ (~4 mM) could enhance MnP activities [148]. Youngs et al. [149] related that Cd2+ is a reversible competitive inhibitor of Mn2+ to MnP activity. The inhibition was not observed in reaction systems containing 2,6-dimethoxyphenol or guaiacol in the absence of Mn2+.
