**2. Cellulases**

in organic synthesis, and in biological pretreatment of lignocellulosic residues [13] to be used

Many studies have elucidated how cellulases bind to their substrates, as well as their catalytic mechanisms [14–17]. The modes of action of hemicellulases and ligninases have also been explored [18, 19]. The knowledge about these enzymes activators and inhibitors is also relevant, mainly in the context of industrial applications. Metal ions, for example, influence

**Figure 1.** General distribution of activators and inhibitors of lignocellulases. HMF furfural: hydroxymethyl furfural; LPMOs: lytic polysaccharide monooxygenases; XEGIP: xyloglucan-endo-β-glucanase inhibitor proteins; XOS: xylooligosaccharides; SDS: sodium dodecyl sulfate; TAXI: *T*. xylanase inhibitor; XIP: xylanase inhibitor protein; TLXI:

for cellulosic ethanol production.

140 Enzyme Inhibitors and Activators

thaumatin-like xylanase inhibitor.

Cellulases are glycoside hydrolases produced mainly by microorganisms, especially filamentous fungi. Microbial cellulases include endoglucanases, exoglucanases, and β-glucosidases, which synergistically degrade cellulose.

The glycosidic bonds in cellulose molecule are not easily accessible to the active site of cellulases. So, many of these enzymes are modular, consisting of one or more noncatalytic carbohydrate binding modules (CBMs). CBMs associate the enzyme with the insoluble substrate and are connected to the catalytic module by linker peptides varying in length and structure [21, 22].

Endoglucanases (EG, endo-1,4-β-endoglucanases, E.C. 3.2.1.4) hydrolyze the amorphous fraction of cellulose, releasing cellodextrins and cello-oligosaccharides [22] decreasing the substrate polymerization degree. They are classified into 11 families of glycosil-hydrolases: GH 5, 6, 7, 8, 9, 12, 44, 45, 48, 51, and 74 [23]. Some endoglucanases have affinity with others substrates, besides cellulose, such as xyloglucan, xylan, and mannan [24].

Exoglucanases or cellobiohydrolases (CBH, exo-1,4-β-exoglucanases, E.C. 3.2.1.91) degrade the crystalline fraction of cellulose, releasing cellobiose, and are named Type I or II (action in nonreducing or reducing ends, respectively). Exoglucanases are clustered in two families of glycosil-hydrolases: GH 7 (CBH I) and GH 6 (CBH II) [22].

β-Glucosidases or cellobiases (beta-D-glucosideglucohydrolase, BG, E.C. 3.2.1.21) hydrolyze cellobiose to glucose and also remove the nonreducing terminal β-D-glucosyl residue from glycoconjugates [25].

#### **2.1. Metal ions associate to cellulases activities**

Metal ions can be associated to proteins and can also form complexes with other molecules linked to enzymes acting as electron donors or acceptors as Lewis´s acids, or as structural regulators [26]. These ions can either activate or inhibit the enzymatic activity by interacting with amine or carboxylic acid group of the amino acids [27].

Several studies have reported the activation or inactivation of microbial cellulases by metal ions (**Table 1**).

Mono-, di-, and trivalent metal ions such as Na+ , K+ , Ca2+, Mg2+, Mn2+, Fe2+, Co2+, Cu2+, Ni2+, Zn2+, Hg2+, and Fe3+ are commonly studied in the characterization assays of cellulases [46]. Besides


**Table 1.** Effect of metal ions on microbial cellulases activities.

ionic charge, ion radius size has a great influence on the activity and stability of the enzyme. It was demonstrated that larger radius has less influence on catalytic amino acids, while the smaller radius can more intensely attract charged amino acids changing the enzyme's overall conformation with damage on the catalytic site [47, 48].

The studies reported inhibitory effects of Fe2+ and Cu2+ on endoglucanases, exoglucanases, and β-glucosidases activities. However, the effect of other divalent ions on cellulases activities seems to be variable among the enzymes secreted by different microorganisms (e.g., **Table 1**). The effect of divalent ions on cellulases is not well elucidated, and possibly occurs by redox effects on the amino acids, increasing or decreasing their activities [49].

Inhibition of cellulases by Hg2+ is related to the interaction with catalytic amino acid residues containing sulfur, leading to oxidation and irregular formation of disulfide bonds [45, 46, 49]. Fe2+ can complex with D/L-lysine and L-methionine [50], Cu2+ with histidine [51], and Ba2+ with arginine, glutamine, proline, serine, and valine [52].

Sajadi [53] evaluated the interaction of amino acids, such as arginine and glutamine, with metal ions and established the following order of interaction degree: Ca2+ < Mg2+ < Mn2+ < Co2+ < Cu2+ > Zn2+.

#### **2.2. Chemical agents and organic compounds associate to cellulases activities**

Cellulases activities may also be affected by drugs (2,3-dichloride-1,4-nafthoquinone, for example), fungicides (such as phenylmercury acetate and ethylen-bis-dithiocarbamate), antibiotics and disinfectants (Phenylmercury nitrate and 8-hydroxiquinoline, among others), sugars (final product inhibition), protein (such as those secreted by plant as defense mechanism), CBM-binding organic compounds, products from sugar and lignin degradation (such as phenolic compounds) [54], food additives (such as Octyl gallate), plant hormones (auxins, such as indoleacetic acid), and ionic solids (Sodium azide) [55–58].

Cellulose degradation products such as cello-oligosaccharides and cellobiose can inhibit endo- and exoglucanase activities, respectively. Endoglucanases that act on xyloglucan and xylan can be inhibited by the xylooligomers released [59]. The addition of xylanase to the reaction media is an alternative to remove these products [60]. The inhibition of β-glycosidases activities by glucose is frequently observed [6, 61]. Disaccharides such as cellobiose and xylobiose, and monosaccharides such as mannose and galactose can inhibit some exoglucanases activities [22, 59, 62].

Gluconolactone, resulting from cellulose oxidation by lytic polysaccharide monooxygenases (LPMOs) activities, can act as β-glycosidases inhibitor. Cellobiose and also other substrates of β-glycosidases compete with gluconolactone and other LPMO-degrading products [63–65]. On the other hand, β-glycosidases can be activated by soforose and lactose [66, 67].

It is relevant to consider that sugars released by enzymatic hydrolysis of lignocellulose can be degraded and converted into inhibitory compounds. Under acidic conditions, glucose, mannose, and galactose can be converted into furan aldehydes such as hydroxymethylfurfurals (HMF). HMF, in turn, can be converted into levulinic and formic acids [68].

Lignin degradation during the hydrolysis of some lignocellulosic materials such as alkali or acid pretreatment, or else during enzymatic hydrolysis (by laccases action) can release phenolic compounds [68] such as vanillin, syringaldehyde, trans-cinnamic acid, and hydroxybenzoic acid. These compounds are potential inhibitors of endo/exoglucanases and β-glycosidases activities due to the presence of hydroxyl, carbonyl, and methoxyl groups [69, 70].

ionic charge, ion radius size has a great influence on the activity and stability of the enzyme. It was demonstrated that larger radius has less influence on catalytic amino acids, while the smaller radius can more intensely attract charged amino acids changing the enzyme's overall

**Enzyme Microorganism Activator metals Inactivating metals Reference**

Endoglucanase *Aspergillus niger* Ca2+ and Mn2+ Co2+, Fe2+, Cu2+ [30]

*Aspergillus niger NRRL 567* Zn2+, Ca2+, Mn2+, Co2+ Mg2+, Fe2+, Hg2+ [32]

, Ca2+, Mg2+,

, K+

Zn2+

Cellobiohydrolase *Trichoderma reesei* Mn2+, Ba2+, Ca2+ Hg2+ [35] β-Glucosidase *Aspergillus niger*322 – Pb2+, Hg2+, Mn2+, Fe2+ [36]

Endoglucanase *Mucor circinelloides* Ca2+, Mg2+, Co2+, Cu2+ Mn2+ [38] β-Glucosidase *Penicillium citrinum* YS40-5 Na+ Zn2+, Cu2+ [39]

Cu2+

β-Glucosidase *Monascus sanguineus* – Ca2+, K+ [41] Exoglucanase *Aspergillus fumigatus* Ca2+, Mg2+, Zn2+ – [42]

Cellobiohydrolase *Agaricus arvencis* Ca2+, Cu2+, Mg2+ Zn2+ [44] Endoglucanase *Aspergillus terreus* Cu2+, Mg2+, Ca2+, Na+ Fe2+, Mn2+, Zn2+, K+ [45]

Endoglucanase *Aspergillusniger*ANL301 Mn2+, Fe2+, Mg2+, Ca2+, Cu2+, Zn2+,

, Mn2+, Na+

Mg2+ and Sn2+ Cu2+, Co2+, Li2+, Fe2+, Mn2+ [29]

Hg2+

Ca2+, Co2+ Hg2+, Cu2+, Fe2+ [33]

– Fe2+, Hg2+ [43]

Fe3+, Pb2+, Ni2+, Cd2+, Hg2+

Cu2+ [34]

, Cu2+, Hg2+, Fe2+, Pb2+, Ni2+, Mn2+, Cd2+

Hg2+ [40]

, Cu2+, Fe2+,

[28]

[31]

[37]

Endoglucanase *Aspergillus fumigatus* Co2+ and Mg2+ K+

Endoglucanase – Cu2+

Endoglucanase *Penicillium pinophilim* MS20 Co2+, Zn2+, Mg2+ Na+

β-Glucosidase *Fusarium oxysporum* Mn2+, Fe2+, Ca2+, Mg2+,

*purpurogenum*KJS506

**Table 1.** Effect of metal ions on microbial cellulases activities.

Endoglucanase *Penicillium simplicissimum* H-11

Exoglucanase Cu2+

(Ehrenb.:Fr.)

β-Glucosidase *Melanocarpus* sp. Na+

Endoglucanase *Daldiniaeschscholzii*

Cellobiohydrolase *Penicillium* 

Endoglucanase exoglucanase

142 Enzyme Inhibitors and Activators

The studies reported inhibitory effects of Fe2+ and Cu2+ on endoglucanases, exoglucanases, and β-glucosidases activities. However, the effect of other divalent ions on cellulases activities seems to be variable among the enzymes secreted by different microorganisms (e.g., **Table 1**). The effect of divalent ions on cellulases is not well elucidated, and possibly occurs by redox

conformation with damage on the catalytic site [47, 48].

effects on the amino acids, increasing or decreasing their activities [49].

As mentioned above, another class of cellulolytic inhibitors has a proteic origin. Specific xyloglucan endo-β-glucanase inhibitor proteins (XEGIPs) are presented in the cell walls of some vegetables such as tomatoes, tobacco, and wheat and inhibit endoglucanases that act on xyloglucan [71–73]. These proteins are part of the plant protecting mechanism against pathogens and act by forming high-affinity complexes with the enzyme [73].

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 nonproductive adsorption on lignin [74].

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" of these enzymes [78].

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 sequestered [20, 33, 45].
