3.2.1 Exposure of enzyme extracts to reaction systems
Exposure to reaction systems was performed to evaluate the behavior of keratinases enzymes in various reaction systems. The ultrasonic probe, ultrasonic bath and microwave were evaluated. Two enzyme extracts were exposed to the reaction systems: a) enzymatic extract from FS carried out with swine hair pretreated with ultrasonic probe with macropoint; b) enzymatic extract from FS carried out with swine hair without pretreatment.
3.2.1.1 Ultrasonic Probe
The enzymatic extracts were exposed to an ultrasonic probe using the micropoint. This was studied based on an experimental matrix (Table 2), which presents the results obtained for the independent variables of exposure time, power and pulse, and evaluated relative activity in response.
Table 2
Experimental planning matrix (real and coded values) and relative activity results (%) for the conditions evaluated in the reaction system ultrasonic probe with enzymatic extracts produced from swine hair pretreated by ultrasonic probe and without pretreatment.
Assays | Ultrasonic probe | Relative activity (%) |
Time (min) | Power (%) | Pulse | Pretreatment by ultrasonic probe | Without pretreatment |
1 | -1 (2) | -1 (40) | -1 (1) | 11.3 | 38.4 |
2 | + 1 (10) | -1 (40) | -1 (1) | 0.0 | 1.4 |
3 | -1 (2) | + 1 (80) | -1 (1) | 2.7 | 16.3 |
4 | + 1 (10) | + 1 (80) | -1 (1) | 0.0 | 9.1 |
5 | -1 (2) | -1 (40) | + 1 (3) | 0.0 | 21.1 |
6 | + 1 (10) | -1 (40) | + 1 (3) | 0.0 | 0.0 |
7 | -1 (2) | + 1 (80) | + 1 (3) | 23.3 | 25.2 |
8 | + 1 (10) | + 1 (80) | + 1 (3) | 0.0 | 10.3 |
9 | 0 (6) | 0 (60) | 0 (2) | 0.0 | 0.0 |
10 | 0 (6) | 0 (60) | 0 (2) | 0.0 | 3.0 |
11 | 0 (6) | 0 (60) | 0 (2) | 0.0 | 0.0 |
12 | 0 (6) | 0 (60) | 0 (2) | 0.0 | 0.0 |
Crude enzymatic extract* | 75.0 U mL− 1 | 124.3 U mL− 1 |
* Enzymatic extract obtained after FS without exposure to reaction systems. |
For the enzymatic extract produced from swine hair pretreated using the ultrasonic probe, up to 23.3% were observed in test 7, corresponding to the shortest time evaluated (2 min) (Table 2). When subjected to effects analysis, only the time variable was significant, affecting it negatively: the shorter the exposure time to the ultrasonic probe, the greater the keratinolytic activity. Power and pulse were not significant.
For the enzymatic extracts obtained with swine hair that was not pretreated, more promising increments were observed. Table 2 shows that test 1 showed the largest increase in keratinolytic activity in relation to crude enzyme extract, i.e., 38.4% increase in enzymatic activity. The analysis of the effects of the variables on keratinolytic activity showed that only the time variable was significant in the process, negatively affecting the enzymatic activity: the longer the exposure time to the ultrasonic probe, the lower the keratinolytic activity achieved. As before, power and pulse were not significant in the process. In both cases studied, the statistical model was not validated due to Fcal < Ftab.
The largest increases in activity occurred in the shortest exposure time evaluated (2 min), corroborating the statistical analysis. Ultrasonic systems are responsible for generating waves that propagate through a liquid, which collapse at high temperatures. This phenomenon is called cavitation. The shear force resulting from the explosion of these microbubbles causes rapid heat and mass transfer, promoting increased enzymatic activity (Jin et al. 2015). Thus, the increase in activity may be a result of the shorter exposure time to the ultrasonic probe that favors the decomposition of interfering molecules and the change in enzymatic specificity, making enzymes more easily accessible to the reaction and consequently increasing their activity, providing an ideal environment for the reaction between enzyme and substrate (McClements 1995; Jin et al. 2015). The enzyme also remains more regular and flexible because thermodynamic parameters such as Ea, ΔH and ΔS are reduced with the use of ultrasound, causing an improvement in its activity and improving the operational stability of enzymes (Ma et al. 2011; Wang, Chen and Zhu 2013).
In assays where the exposure time was longer (from 6 to 10 min), the activity was lower and, in some cases, lower than the activity obtained in the enzyme extract without exposure to the ultrasonic probe, possibly due to the enzymatic denaturation caused by the longer exposure time to the ultrasonic probe (Kapturowska, Stolarzewicz and Krzyczkowska 2012). Denaturation of enzymes can occur due to excess pressure, temperature or even shear force generated during the cavitation phenomenon in the ultrasonic system (Grintsevich et al. 2001; Potapovich, Eremin and Metelitza 2003).
It is important to note that the use of ultrasonic systems is efficient in reducing reaction time, promoting increased enzymatic activity in many cases. On the other hand, depending on the conditions studied, there may be an enzymatic denaturation and consequently a decrease in enzyme activity.
3.2.1.2 Ultrasonic bath
The greatest increase in enzymatic activity obtained for the ultrasonic bath occurred in the enzymatic extract from swine hair pretreated with ultrasonic probe (30.2%), with a temperature of 80 °C during 40 minutes of exposure and 0% power, corresponding to test 6.
Analysis of effects on keratinolytic activity showed that temperature and power were important. The temperature variable had significant positive effects; that is, the higher the temperature, the higher the keratinolytic activity. The power variable affected the enzyme activity negatively; the lower the power, the higher the activity. Time had no influence on enzymatic activity. Because Fcal > Ftab, the statistical model was validated.
For enzymatic extract produced from swine hair without pretreatment, only test 3, corresponding to a temperature of 30 °C during 10 minutes and 100% power, provided an increase of keratinolytic activity in relation to the crude enzyme, which was 5.9%. However, when the results were subjected to effects analysis, no variable was significant. For this enzyme extract studied, the model was not statistically validated due to Fcal < Ftab.
In the enzymatic extracts evaluated, different behaviors of the enzyme regarding temperature were observed. For the pretreated residues, the enzyme extract increased the activity at a temperature of 80 °C and for the untreated residues, the only activity increase was at the temperature of 30 °C, the mildest within the study range. This shows that the enzymes produced during submerged fermentation with pretreated and untreated residues are different in the mode of action compared to the ultrasonic bath reaction system.
The behavior observed for ultrasonic bath may be related to the synergistic effect of ultrasonic waves with temperature. Several studies show increased activity of enzymes exposed to ultrasonic bath (Leaes et al. 2013; Mulinari et al. 2017). According to the literature, temperature considerably influences the increase or decrease of enzymatic activity. With a rise in temperature the activity may be increased, but depending on the structure of the enzyme, if there is a very significant rise in temperature, denaturation may occur (Resa et al. 2009). This behavior was described in the studies by Wang et al. (2011) and Ovsianko et al. (2005), where enzymatic denaturation occurred due to the effect of ultrasonic sonication causing an increase in temperature, also stimulating the effect of cavitation.
Ultrasonic bath alters the behavior of exposed enzymes. Thus, the use of ultrasonic systems can promote increases in enzymatic activity, possibly due to conformational changes in protein structure, in addition to considerably reducing the reaction time (Leaes et al. 2013) and also the decrease in activity by denaturing (Resa et al. 2009).
3.2.1.3 Microwave
For the enzymatic extract produced from swine hair pretreated using the ultrasonic probe, test 2 showed an increase of activity, 15.1% at 80 °C for 5 minutes. Performing statistical analysis, we noted that the temperature variable was significantly positive: the higher the temperature, the higher the keratinolytic activity achieved. Time had no influence on enzymatic activity. The model was statistically validated by Fcal > Ftab.
For the enzymatic extract obtained from swine hair without pretreatment, we found that no assay showed an increase of keratinolytic activity. However, performing the statistical analysis, we noted that the time variable was significantly negative; that is, the longer the microwave exposure time, the lower the keratinolytic activity achieved. The temperature had no influence on enzymatic activity.
The increase in activity obtained for enzymatic extract produced from swine hair pretreated with ultrasonic probe was because the microwaves provided an increase in enzymatic activity and an increase in reaction efficiency due to instantaneous overheating. Causing tremendous agitation of molecules that induces an increase in energy collisions, also increasing reaction and conversion rates (Ma et al. 2011; Mazinani, DeLong and Yan 2015).
The mechanism of microwave operation occurs by the interaction of the electromagnetic field with matter. This causes a movement of ions, which in turn cause heat generation by two mechanisms, dipole rotation and ion conduction. In chemical reactions, microwaves cause molecular friction due to the polarization of molecules. This process is responsible for increasing the friction of these molecules, consequently increasing the temperature and the reaction rate (Lopes et al. 2015). In this manner, microwave irradiation can cause conformational changes in the exposed structure, leading to increased enzyme activity or even damage such as enzymatic denaturation (Leonelli and Mason 2010; Lopes et al. 2015; Mazinani, DeLong and Yan 2015).
Some authors point out that the use of microwaves can cause changes in thermodynamic properties, providing increased enzymatic activity (Mazinani, DeLong and Yan 2015; Golunski et al. 2017). These findings suggest that conformational changes in the structure of enzymes occur when exposed to microwaves, possibly due to cleavages that occur during the process and the formation of hot spots by instant heating.
On the other hand, high temperatures and exposure times cause enzymatic denaturation. Possibly this occurred in the assays with enzymatic extract produced from untreated swine hair, where mild temperatures of 30 °C were sufficient to cause decreased enzyme activity.
Temperature is one of the factors that most influence the behavior of enzymes exposed to microwave irradiation. This causes collisions between molecules to increase, and this causes an increase in energy, causing faster reaction rates. However, at very high temperatures, the reaction rate is reduced due to the denaturation of enzymes, caused by heat breakage and also the breakdown of ionic and hydrogen bonds, stabilizing the protein structure (Yadav and Borkar 2009; Khan and Rathod 2018).
3.2.1.4 Larger increments of enzymatic activity
Given the results obtained for the three reaction systems evaluated, the ultrasonic probe showed greater potential for increased enzymatic activity compared to the other reaction systems (Fig. 1).
The largest increase was obtained using the ultrasonic probe (38.4%) with the enzymatic extract produced from swine hair without pretreatment. Ultrasonic systems also showed good results of increased enzymatic activity for the enzymatic extract produced from swine hair pretreated by ultrasonic probe, with the increment values being 23.3% for ultrasonic probe and 30.2% for ultrasonic bath. In addition, these values were reached under mild conditions of time, power and pulse, making the process more viable.
3.2.2 Enzymatic concentration
The enzyme concentration technique was performed to verify the possibility of increasing enzymatic activity from an economically viable and simple operation method. Enzyme extracts produced from swine hair without pretreatment were used and the enzyme concentration technique was performed in the presence of NaCl and acetone.
The homemade extract showed an enzymatic activity of 159.3 U mL− 1 after the concentration technique, increasing the activity value by 53.5% when compared to the crude enzyme extract (103.8 U mL− 1). The concentrated enzymatic extract (homemade) was exposed to reaction systems under the conditions of greatest activity increase in experimental designs, where it presented stability against the ultrasonic probe, ultrasonic bath and microwave, possibly due to the absence of interferents that were separated during enzymatic concentration.
After exposure, the concentrated enzymatic extract (homemade) was applied in the degradation of swine hair and chicken feathers, aiming to evaluate the degradation potential of keratinous residues.