**3. Hemicellulases**

Since hemicellulose is very heterogeneous, its complete degradation requires the synergic action of several enzymes, mainly endoxylanases and β-xylosidases as well as a variety of accessory enzymes that act in substituted xylans and include α-D-glucuronidases, acetyl xylan esterases, ferulic acid esterases, α-galactosidases, acetyl mannan esterases, and α-Larabinofuranosidases [83].

α-L-Arabinofuranosidases (EC 3.2.1.55.; AFases) are exopolysaccharide hydrolases which remove side chains containing arabinose residues linked by α-1,2, α-1,3, and α-1,5 glycosidic bonds to the main chain of arabinananas or arabinoxylans [84]. AFases are grouped into six families of glycoside hydrolases: GH 3, 10, 43, 51, 54, and 62 [85]. A variety of AFases have been purified from fungi, bacteria, and plants [86–88]. These enzymes' activities can be affected by metal ions, ionic and nonionic detergents, and by chelating and reducing agents [85].

Xylans with acetyl and methyl glucuronic acid (MeGlcA) as substituents groups are named *O*-acetyl-4-*O*-methylglucuronoxylans. On the other hand, when α-4-0-methylglucuronic acid and α-arabinofuranose are the substituent groups, xylans are named as arabino 4-*O*-methylglucuronoxylan [89]. α-glucuronidases (EC 3.2.1.139.) hydrolyze α-1,2-glycosidic bond of MeGlcA in the side chain [90]. Among xylan-degrading enzymes, α-glucuronidases are the less studied and characterized ones. They are grouped into three families of glycosylhydrolases: GH 4, 67, and 115 [91].

Endoxylanases (E.C. 3.2.1.8; endo-β-1,4-xylanases) hydrolyze β-1,4 glycosidic linkages in the backbone of xylans that are composed of xylose residues [92]. According to the similarities of amino acid sequences, the majority of xylanases are grouped into glycoside hydrolases (GH) families 10 and 11 and are also classified into families GH 5, 7, 8, and 43 [93].

β-Xylosidases (E.C. 3.2.1.37; β-1,4-xylosidases) release β-D-xylopyranosyl residues from the nonreducing end of xylobiose and some small 4-β-D-xylooligosaccharides [92]. These enzymes have been classified into 10 families: GH 1, 3, 30, 39, 43, 51, 52, 54, 116, and 120, based on the predicted structural motifs of the enzyme's catalytic domain. β-Xylosidases play a crucial role in endoxylanases activities, since their substrates, such as xylobiose, can inhibit endoxylanases action [94, 95].

### **3.1. Metal ions associate to hemicellulases activities**

Another factor that affects the catalysis by cellulases is the enzymes interaction with lignin, the phenomenon called "nonproductive adsorption" or "nonspecific binding." Cellulases can adsorb lignin through their CBMs [21, 74–77], more specifically by its alanine residues [76]. Some cellulases show higher catalytic activity when CBMs are removed by decreasing non-

Nonproductive adsorption of cellulases on lignin can also be decreased by adding surfactants to the reaction media, which increases the efficiency of enzymatic catalysis [78–81]. Tween 20, 40, 60, 80, and 100, Triton X-100, polyethylene glycol (PEG), among others surfactants, tend to decrease the surface tension of aqueous systems, which may alter the properties of liquids such as detergency, emulsification, greasing, and solubilization. Surfactant properties can decrease the nonproductive adsorption of cellulases on lignin, acting as "activators agents"

Chelating agents such as EDTA (ethylene diamine tetra acetic acid), ethylene glycol (or β-mercaptoethanol), and DPPE (1,2-bis diphenylphosphino-ethylene) may activate some enzymes activities, especially cellulases, by sequestering inhibitors' metal ions from the aqueous system [82]. When chelating agents complex with metals in the reaction media, the active site of enzyme is available to react with the substrate, which represents the positive effect of these compounds on cellulases activities. In contrast, the negative effect of chelating agents on enzymatic activity suggests that enzyme activities depend on the inorganic ion that was

Since hemicellulose is very heterogeneous, its complete degradation requires the synergic action of several enzymes, mainly endoxylanases and β-xylosidases as well as a variety of accessory enzymes that act in substituted xylans and include α-D-glucuronidases, acetyl xylan esterases, ferulic acid esterases, α-galactosidases, acetyl mannan esterases, and α-L-

α-L-Arabinofuranosidases (EC 3.2.1.55.; AFases) are exopolysaccharide hydrolases which remove side chains containing arabinose residues linked by α-1,2, α-1,3, and α-1,5 glycosidic bonds to the main chain of arabinananas or arabinoxylans [84]. AFases are grouped into six families of glycoside hydrolases: GH 3, 10, 43, 51, 54, and 62 [85]. A variety of AFases have been purified from fungi, bacteria, and plants [86–88]. These enzymes' activities can be affected by

Xylans with acetyl and methyl glucuronic acid (MeGlcA) as substituents groups are named *O*-acetyl-4-*O*-methylglucuronoxylans. On the other hand, when α-4-0-methylglucuronic acid and α-arabinofuranose are the substituent groups, xylans are named as arabino 4-*O*-methylglucuronoxylan [89]. α-glucuronidases (EC 3.2.1.139.) hydrolyze α-1,2-glycosidic bond of MeGlcA in the side chain [90]. Among xylan-degrading enzymes, α-glucuronidases are the less studied and characterized ones. They are grouped into three families of glycosyl-

metal ions, ionic and nonionic detergents, and by chelating and reducing agents [85].

productive adsorption on lignin [74].

of these enzymes [78].

144 Enzyme Inhibitors and Activators

sequestered [20, 33, 45].

**3. Hemicellulases**

arabinofuranosidases [83].

hydrolases: GH 4, 67, and 115 [91].

The inhibitory effect of Hg2+ on AFases activities has been reported [96–99]. Besides Hg2+, Ag2+, and Pb2+ are mixed inhibitors, which do not bind to the active site, but to another region of the enzyme, and thus do not interfere with substrate binding to the catalytic site. In addition, Hg2+ is known to react with histidine and tryptophan residues, reducing the enzyme availability to metabolic function [100]. Zn2+, Cd2+, and Co2+ have also been described as potential inhibitors of AFases [88, 99, 101].

Most scientific works about α-glucuronidases purification and characterization report that these enzymes do not require metal ions for their activities [102–106]. On the other hand, various metal ions exert inhibitory effects on α-glucuronidases activities, such as Ag2+, Zn2+, Cd2+, Hg2+, Mn2+, Fe2+, and Fe3+ (e.g., **Table 2**).

Some GH 10 family enzymes require metal ions for their stability and activities. For example, *Pseudomonas fluorescens* sub sp. produces a xylanase that is one of the first GH 10 enzymes found to contain a calcium-binding site [93]. On the other hand, there are many GH 43 enzymes with crystal structures that showed tightly bound metal ions such as Ca2+, with structural roles [107]. Besides, many studies have reported the apparent activation of fungal β-xylosidases by Mn2+ and Ca2+, suggesting that these ions activate and protect the active site [95].

The negative effect of heavy metals, such as Hg2+, Fe2+, Co2+, Mn2+, Ag2+, Cu2+, and Pb2+ on xylanases activities have been reported [108]. Inhibition by heavy metal ions (such as Zn2+, Pb2+, and Hg2+) may occur due to the formation of a complex with the reactive groups of the enzyme. Metals from group Ilb exhibit high affinity for SH, CONH<sup>2</sup> , NH2 , COOH, and PO4 [109]. Furthermore, inhibition of xylanase by Hg2+ has been reported as related to the presence of tryptophan residues, which oxidize indole ring, thereby inhibiting the enzyme activity [110]. Xylanase from *Bacillus halodurans* TSEV1 was strongly inhibited by Hg2+, Cu2+, and Pb2+, probably due to the catalysis of the cysteine thiol group autooxidation, which leads to the formation of intra- and intermolecular disulfide bonds or to the formation of sulfenic acid [111].

### **3.2. Chemical agents and organic compounds associate to hemicellulases activities**

Some authors have reported that the addition of chelating agents such as EDTA and reducing agents such as β-mercaptoethanol and DTT (dithiothreitol) does not affect AFases activity [85,


**Table 2.** Metal ions that exert inhibitory effects on α-glucuronidases activities.

99, 112]. Such agents are well known as inhibitors of thiol groups, and these data suggest that sulfhydryl groups are not related to the active site of AFases.

There are few studies reporting the action of ionic detergents in AFases activities. At low concentrations (1–2 mM), ionic detergents such as SDS can stimulate the enzyme activity, whereas in higher concentrations (20 mM) they can cause an inhibitory effect [113]. Since SDS interferes in hydrophobic regions of the enzyme, it alters its three-dimensional structure [114], indicating that these concentrations may be critical and cause enzyme denaturation.

Among the compounds that significantly activate the enzyme activity there are 2-mercaptoethanol, DTT (dithiothreitol), L-cysteine, and NAD<sup>+</sup> indicating that these reducing agents are required for maximal activities of α-glucuronidases [115]. Some of the family 4 enzymes are known to be NAD<sup>+</sup> dependent. The role of NAD<sup>+</sup> for the activity of the hydrolytic GHF4 is not well known. The pyridine nucleotide cofactor could have structural and/ or catalytic function and, in addition, could also be important for the regulation of enzyme activity [116].

Xylanases have received great attention in recent years, mainly due to their potential for the application in the processes of xylooligosaccharides (XOs) production, pulp bleaching, removal of antinutritional factors of animal feeds, bread making (improving the separation of wheat or other cereal gluten from starch), juice extraction from fruits or vegetables, clarification of fruit juices and wines, and extraction of more fermentable sugar from barley to produce beer [111, 117].

Xylanase proteic inhibitors might hamper their efficacy when used in industrial application. Two distinct types of xylanase inhibitors have been identified in barley, wheat, and rye: XIP (xylanase inhibitor protein), a monomeric and glycosylated protein (XIP-I most widely studied in the XIP class), that can inhibit all GH 10 and GH 11 fungal xylanases, except that from *Aspergillus aculeatus*. The other type of xylanase inhibitor, TAXI (*Triticumaestivum* xylanase inhibitor) is a mixture of two proteins, TAXI I and TAXI II, which differ according to xylanase specificities and p*I*. TAXI inhibitors seem to be specific for GH 11 bacterial and fungal xylanases. More recently, a third class of inhibitor called TLXI (thaumatin-like xylanase inhibitor) also purified from wheat, showed variable activities against most of GH 11 xylanases and does not inhibit GH 10 microbial xylanases [117, 118].

Many other substances, such as EDTA (a chelating reagent), β-mercaptoethanol, and DTT (both disulfide bonds reducing agents) have been extensively investigated regarding their influence on xylanases activities. Xylanase from *Talaromyces thermophile* is inhibited by EDTA and DTT, suggesting that disulfide bonds are essential to maintain the enzyme conformation [119]. On the other hand, the activation of xylanases in the presence of β-mercaptoethanol and DTT was reported and indicates the presence of a reduced thiol group of cysteine in these enzymes [120].

The effect of different modulators on the activity of xylanase from *B. halodurans* TSEV1 has been investigated. These modulators include *N*-bromosuccinimide (N-BS), ethyl-3-(3 dimethyl aminopropyl) carbodiimide (EDAC), iodoacetate (IAA), and Woodward's reagent K (WRK). The inhibition of xylanase activity in the presence of NBS suggests the presence of tryptophan residues in their active site. EDAC and WRK inhibited the enzyme activity, which indicates the importance of carboxylic groups in enzyme catalysis [111].

99, 112]. Such agents are well known as inhibitors of thiol groups, and these data suggest that

There are few studies reporting the action of ionic detergents in AFases activities. At low concentrations (1–2 mM), ionic detergents such as SDS can stimulate the enzyme activity, whereas in higher concentrations (20 mM) they can cause an inhibitory effect [113]. Since SDS interferes in hydrophobic regions of the enzyme, it alters its three-dimensional structure [114], indicating that these concentrations may be critical and cause enzyme denaturation.

Among the compounds that significantly activate the enzyme activity there are 2-mer-

agents are required for maximal activities of α-glucuronidases [115]. Some of the family 4

lytic GHF4 is not well known. The pyridine nucleotide cofactor could have structural and/ or catalytic function and, in addition, could also be important for the regulation of enzyme

Xylanases have received great attention in recent years, mainly due to their potential for the application in the processes of xylooligosaccharides (XOs) production, pulp bleaching,

dependent. The role of NAD<sup>+</sup>

indicating that these reducing

for the activity of the hydro-

sulfhydryl groups are not related to the active site of AFases.

**Table 2.** Metal ions that exert inhibitory effects on α-glucuronidases activities.

**Metal ions Microorganism Referees** Ag2+ *Bacillus stearothermophilus* [105]

146 Enzyme Inhibitors and Activators

Zn2+ *Bacillus stearothermophilus* [105] Cd2+ *Thermotoga maritime* [104] Hg2+ *Thermotoga maritime* [104]

Mn2+ *Thermotoga maritime* [104]

Fe2+ and Fe3+ *Aspergillus niger* [102] Ni2+ *Bacillus stearothermophilus* [105]

Cu2+ *Thermotoga maritima* [104]

K+ *Geobacillus stearothermophilus* [105]

*Saccharophagus degradans* 2-40 [106]

*Bacillus stearothermophilus* [105] *Aspergillus niger* [102] *Helix pomatia* [103] *Saccharophagus degradans* 2-40 [106]

*Bacillus stearothermophilus* [105] *Aspergillus niger* [102]

*Saccharophagus degradans* 2-40 [106]

*Bacillus stearothermophilus* [105]

captoethanol, DTT (dithiothreitol), L-cysteine, and NAD<sup>+</sup>

enzymes are known to be NAD<sup>+</sup>

activity [116].

Treatments for deconstruction of the lignocellulosic structure are frequently employed in the use of biomass as sugar's source for ethanol production and can generate besides soluble sugars, other sources such as furan derivatives, organic acids, and phenolic compounds that can act as xylanases inhibitors, as described for cellulase [121].

Significant inhibition of xylanase activity by vanillic acid, syringic acid, acetosyringone, and syringaldehyde has been observed [121]. Boukari et al. [122] reported that endoxylanase from *Thermobacillus xylanilyticus* was inhibited by phenolic compounds, including cinnamic acid, p-coumaricacid, caffeic acid, ferulic acid, and 3, 4, 5-trimethoxy-cinnamic acid by the noncompetitive multisite inhibition mechanism.

Studies on the inhibitory effect of sugars on xylanases (mainly β-xylosidases) are essential for a better understanding about the decrease in the enzyme activity during biomass conversion. This kind of inhibition was subject of research for a long time, bringing up many different opinions about its mechanism. Jordan et al. [123] studied the active site of the GH 43 β-xylosidase from *Selenomonas ruminantium* and reported that it comprises of two subsites and 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 competitive or noncompetitive inhibition.
