## 3 Results and Discussion
### Enzyme Characterization  
The effect of temperature on \(\beta\)-mannanase activity was evaluated using retentate and Man 58 samples. For both enzyme samples, \(\beta\)-mannanase was more active at 60 \({}^{\circ}\)C with activities of 5.84 and 1.08 IU/mL, respectively. A slow decrease in Man 58 activity was observed at higher temperatures, retaining 96%, 91% and 85% of activity at 65, 70 and 80 \({}^{\circ}\)C, respectively (Figure 4). The pH profile of Man 58 and retentate showed that the enzyme activity remained significant in the acidic and alkaline ranges. Figure 3: SDS-PAGE (12%) of fractions collected from chromatography on Sephacryl S-100 and stained with silver nitrate (**left**). MW (molecular weight) marker: phosphorylase b (97 kDa), albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and \(\alpha\)-lactalbumin (14.4 kDa). Zymogram of chromatography fractions (**right**); the arrows (**left**) indicate protein bands corresponding to \(\beta\)-mannanase activity (**right**) at fraction 58. Figure 2: Elution profile of Man 58 on Sephacryl S-100 chromatography. \(\beta\)-Mannanase activity was seen over a broad pH range (3.0-9.0), with an optimum at pH 4.0. Moreover, 85% and 57% of maximum activity were retained at pH 3.5 and 8.0, respectively. Even at pH 8.5 and 9.0 Man 58 showed retention of 38 and 30% of activity, respectively. \(\beta\)-Mannanase from _Aspergillus niger_ (_A. niger_) was stable at pH range of 3-8 and 55 \({}^{\circ}\)C [23]. Thus, the ability of Man 58 to function at high temperature (60 and 70 \({}^{\circ}\)C) and under acidic and alkaline conditions suggests the potential application of this enzyme in pulp bleaching, which requires a thermostable and alkali-tolerant \(\beta\)-mannanase to cleave the mannan portion in pulps without affecting cellulose structure [24]. The \(\beta\)-mannanase activity from retentate was also more active at pH 4.0. The \(\beta\)-mannanase from _A. niger_ was less stable than Man 58, exhibiting optimum activity at pH 5.5 and 55 \({}^{\circ}\)C.  
An important characteristic of Man 58 and retentate was the high thermostability observed (Figure 5). Under storage conditions at 40 and 50 \({}^{\circ}\)C, no loss of \(\beta\)-mannanase activity was detected after up to eleven days of incubation. It should be noted that both enzymatic samples were stable at 60 \({}^{\circ}\)C for at least 11 days and presented a half-life of 12 days. These data show this enzyme to be far more thermostable than \(\beta\)-mannanase from _A. flavus_, which presented a half-life of 6 h at 60 \({}^{\circ}\)C [25], \(\beta\)-mannanase from _A. niger_, which retained 50% activity after 6 h at 55 \({}^{\circ}\)C or \(\beta\)-mannanase from the bacterium _Pantoea agglomerans_ A021, which was unstable at 60 \({}^{\circ}\)C [24]. Furthermore, Man 58 and retentate showed a half-life of 10 h at 70 \({}^{\circ}\)C. In contrast, both enzyme samples were not stable at 80 \({}^{\circ}\)C with decline of more than 80% of their activities after 1 h of incubation (results not shown). The properties of Man 58 may be advantageous in biotechnological applications, including in the feed and food industries. Thermostable enzymes are capable of maintaining their activities at high temperatures (usually higher than 45 \({}^{\circ}\)C), which favors the reduction in viscosity and solubility of the substrates and products by increasing the rate of reaction. Another important feature is their high resistance to the action of proteases. Additionally, biotechnological processes at elevated temperatures carry a lower risk of microbial contamination. Figure 4: Effect of temperature on Man 58 activity. \(\beta\)-Mannan breakdown is dependent on several factors, including enzyme synergy, interaction with different sub-sites on the heterogeneous substrate, interaction of the subunits within the mannan-degrading enzyme system and the probable presence of binding molecules in addition to the catalytic modules [4].