**3. Results and discussion**

#### **3.1 Molecular coupling between cellulose and enzyme cellulase**

The molecular model representing five cellulose monomer units was taken as a basis. **Figure 2** shows the theoretical model on which this analysis is based.

From the database of the simulation program AutoDockTools, the cellulase enzyme is extracted into its endo-β-1,4-D-glucanase (rigid substrate), as shown in **Figure 3**.

The results from the simulation were analyzed using the AutoDockTools suite with the option of analyzing the conformational space of the ligand substrate. The initial geometry of the studied complex is shown in **Figure 4**.

**Figure 2.** *Molecular model of cellulose (ligand) of five monomeric units of cellulose.*

**Figure 3.** *Schematic representation of the endo-*β*-1,4-D-glucanase enzyme molecule.*

From the results, the crystallographic coordinates of the crystallized ligand are taken as a reference, grouping the 100 conformations evaluated and obtained in clusters. Each docking is validated after running the same evaluation five times. In the conformational search process, the Lamarckian genetic algorithm (LGA) is

#### *Deinking of Mixed Office Waste (MOW) Paper Using Enzymes DOI: http://dx.doi.org/10.5772/intechopen.99373*

applied. The Intelec parameter is activated, so that the internal electrostatic of the ligand affects the energy of docking (docked energy) that guides the optimization process, but not to the binding energy of the ligand (binding free energy). The algorithm analyzes 100 possible poses of the cellulose in the enzyme, adopting the flexibility property for the cellulose molecule, so as not to rule out this interaction phenomenon between ligand and substrate.

The 100 poses analyzed are grouped into the so-called clusters in such a way that they are ordered from the highest to lowest docking energy (binding free energy). This binding free energy is the ligand-substrate coupling with the highest affinity as it has the most negative or lowest bond free energy. It is worth emphasizing that AUTODOCK returns two energies: the so-called docked energy, used to guide the docking, and the binding free energy, the latter allows us to define the ligand-substrate coupling with the highest affinity, that is to say, with the more negative bond free energy. For the case of endo-β-1,4-D-glucanase and cellulose, the pose with the highest affinity drawn the following parameters: binding energy −0.57 kcal/ mol, inhibition constant 379.8 mM, intermolecular energy −9.52 kcal/mol, internal energy −14 Kcal/mol, and torsional energy of 8.95 Kcal/mol.

**Figure 5** shows the amino acids with the most negative enzyme-cellulose bonds, according to the model: glutamate (GLU, A:207), asparagine (ASN, A:27), tyrosine (TYR, A:118), aspartate (ASP, A:106), tryptophan (TRP, A:29), and phenylalanine (PHE, A:108), whereas **Table 2** shows, according to Morrison and Neilson, the structures of amino acids that interact with cellulose molecules of the endo-β-1,4-D-glucanase.

The experimental results are shown in **Table 3**. A relevant characteristic to determine the efficiency of the deinking process is whiteness, and it is determined through the measurement of the brightness. Brightness ISO is determined using standard sensitivity spectrum centered at a wavelength 457 nm according to ISO 2470 or TAPPI T525 standard and is represented in percentage points. In tests 1 and 2, an ISO whiteness of 85 and 86%, respectively, with 445.2 and 412.5 ppm of black points were obtained. For the L \* tonality, there are values of 93.05 in test 1, and 94.25, in test 2. The a \* and b \* tones manifested with practically equal values, 1.6

**Figure 5.** *Linking amino acids in the best enzyme-cellulose according to the modeling.*

#### **Table 2.**

*Structures of the amino acids that interact with the cellulose molecule [18].*


#### **Table 3.**

*Results of the optical characteristics were measured to the deinked sheets utilizing the use of amino acids.*

#### *Deinking of Mixed Office Waste (MOW) Paper Using Enzymes DOI: http://dx.doi.org/10.5772/intechopen.99373*

and − 3.3 for both cases; the same occurs in reflectance and opacity, with very close values, 41.5 and 42.7% of reflectance, and 88.3 and 89.5%, of opacity.

For glutamate, increasing the dose from 0.1 to 0.2% gives an increase in the whiteness of 2%, that is, from 84.9% it rises to 86.9%. Black spots are a direct reflection of inking quality and flotation effectiveness, and this number also decreases with increasing GLU dosage from 455.3 to 377.1 ppm. The tonality variables L \*, a \*, and b \*, such as opacity, change slightly, and reflectance, on the other hand, is modified to a great extent from 39.57 to 47.15%, as the dose of the amino acid in question increases.

When treating the MOW paper with asparagine 0.1%, important changes are noted concerning the tests with aspartate and glutamate. When 0.1% ASN is dosed, the ISO whiteness obtained is 81.5%, the black points 550.8 ppm, and the L \* of 91.6, these being much lower than those obtained in the rest of the amino acids. For this case, the variables a \*, b \*, reflectance, and opacity are practically similar to what was observed with the rest of the amino acids.

Mixing the three amino acids (test 7), adding 0.1% of each depending on the weight of the dry paper to be inked, a result of whiteness much higher than that obtained in the other tests is obtained. The resulting whiteness was 89.1% and the black points 333.0 ppm. The variable b \* changes to a lesser extent to −4.3. The parameters L \*, a \*, and opacity remain above the average of those obtained in the first tests; however, the reflectance decreases considerably to 35.9%, this being inversely proportional to the whiteness.

In test 8, MOW paper deinked with 0.2% of each amino acid obtained the best quality results, ISO whiteness of 90.8% and black points of 303.4 ppm, values much higher than expected, taking into account that the MOW paper is not being treated with any chemical bleach and is only working with three process stages: pulp, chemical treatment, and flotation. The tonality parameters achieved: L \* = 95.3, a \* = 1.7, and b \* = −4.52, oscillate in the values obtained in the previous tests and fall within the expected, according to the values established by the standard, which are L \*, from 92.5 to 95.5; a \* from 1.3 to 2.3; b \* from −9 to −8, reflectance from 32 to 40% and minimum accepted opacity of 87%.

The variable b \* is controlled with a pigment from aniline, and the negative of its value indicates the color shift toward blue. The addition of such pigment in the manufacture of paper is directly proportional to the negativity of this parameter, that is, the higher the aniline, the more negative the b \*. In deinking, these pigments responsible for the color are detached from the vicinity of the fiber and removed by flotation, so a decrease in this parameter is to be expected after deinking. Opacity is required on all printing papers; it should be sufficient to prevent the printing on the reverse side of the paper from negatively affecting the appearance of a print, so the higher the opacity, the higher the print quality. The result in this test was 89.9%.

The optical properties evaluated in this work are higher than those obtained under similar experimental conditions by using NaOH and the enzyme cellulase *Thricodema* Sp., as defibrillators [19].

#### **3.2 Morphology of the deinked fibers**

Through scanning electronic microscope (SEM), there is possible to appreciate the morphology of the deinked fibers, to appreciate the degree of fibrillation, because its intensity affects the mechanical properties of the fiber. In the micrographs of the pulp deinked with aspartate, glutamate, asparagine (**Figures 6**–**10**), and a mixture of them, it is observed that the depolymerization induced by amino acids does not represent excessive fibrillation that affects the internal morphology of the cellulose fiber and therefore its mechanical properties. No particles of toner

#### **Figure 6.**

*Micrographs of the deinked fibers. Aspartate case. Test 4: (a) 100X, (b) 250X, (c) 500X, (d) 1000X.*

#### **Figure 7.**

*Micrographs of the deinked fibers. Glutamate case. Test 5: (a) 100X, (b) 250X, (c) 500X, (d) 1000X.*

#### **Figure 8.**

*Micrographs of the deinked fibers. Aspargine case. Test 6: (a) 100X, (b) 250X, (c) 500X, (d) 1000X.*

#### **Figure 9.**

*Micrographs of the deinked fibers. 0.1:0.1:0.1 mixture. Test 7: (a) 100X, (b) 250X, (c) 500X, (d) 1000X.*

#### **Figure 10.**

*Micrographs of the deinked fibers. 0.2:0.2:0.2 mixture. Test 8: (a) 100X, (b) 250X, (c) 500X, (d) 1000X.*

or printing ink are seen, only small agglomerations, possibly of starch, precipitated calcium carbonate, and/or residues of other additives typical of the manufacture of paper.

**Figure 10** shows the micrograph of fibers from test 8, which obtained the best results of the optical characteristics, from it can be seen that the morphological structure of the fiber retains its robustness, and some fibers look flatter than others, as a result of forming the sheets of paper; however, there is no fibrillation or excessive fine formation. The fragmentation of inks is another phenomenon that occurs in deinking processes, which is desirable up to a certain point so that the particles formed from these acquire the appropriate size to be floated more efficiently. When this happens, papers with superior brightness and whiteness are obtained, without considerable repercussions on the mechanical properties of the deinked fiber.
