3.1. Production of xylanases from Penicillium sp. by SSF
The pH is a variable that is very difficult to control in solid-state cultivation due to the absence of free water. However, it is essential to use microorganisms capable of growing over a wide pH range. Most microorganisms grow best in environments with pH variations close to neutrality, between pH 6.5 and 7.5. However, filamentous fungi and yeasts thrive in pH variations between 5 and 6 [30, 31].
Figure 1 displays the pH profiles during cultivation via FES. The pH values ranged from 6.1 to 7.6 in the cultures with only wheat bran as the substrate, while the range expanded from 5.6 to 7.6 in the culture using both wheat bran and sisal fiber blend. The slight decrease in pH observed can be attributed to sisal, which played a significant role in modifying the environmental conditions. Overall, the resulting conditions became more favorable for the expression of the enzymes in question.
Figure 1.
Figures 2 and 3 depict the kinetics of xylanase production by Penicillium sp. FSDE15 using wheat bran and wheat bran/sisal fiber blend. Enzymatic activity values increased significantly over time in both cultures. The enzymatic activity in the wheat bran culture increased from 119.32 ± 7.90 U/g on 72h to 261.14 ± 5.19 U/g on the seventh day 168h, representing a 119% increase. In the culture with the wheat bran/sisal fiber blend, xylanase activity exceeded 100 U/g after 72h of the experiment.
These results highlight the strain's potential as a xylanase producer. Penicillium sp. FSDE15 performance is comparable to recognized xylanase producers [32, 33]. Maximum xylanase activity was recorded at the most extended experiment times (168 h). At this cultivation time, xylanase activities reached 201.67 U/g in the culture with the wheat bran/sisal fiber blend and 261.13 U/g in the wheat bran culture.
In both cultures, productivity increased in the initial days of the process and reached a plateau after 72 h (1.68 ± 0.11 U/g.h for wheat bran and 1.50 ± 0.24 U/g.h for the blend). Indeed, other conditions even achieved higher average productivity values, such as in the wheat bran culture after 120 h (1.89 U/g.h) or in the blend culture after 96 h (1.27 U/g.h). However, no statistical differences were observed in the means at 95% confidence in the wheat bran and blend cultures at 72 and 96 h, respectively. The culture with wheat bran achieved 1.68 ± 0.11 U/g.h. Regarding the SSF medium sisal fiber/wheat bran, the best productivity was at 96 h with 1.66 ± 0.09 U/g.h and 72 h with 1.50 ± 0.24 U/g.h. It is worth noting that productivity was used as the criterion for selecting the cultivation time for crude extract enzymatic production used in the following stages of the study.
Figure 2.
Figure 3.
Sisal fiber generally has a lower hemicellulose content than wheat bran (11% vs. 51%) [34, 35]. This evidence aligns with the findings of Da Silva et al. [36], who pointed out that the production of lignocellulosic enzymes via SSF is directly associated with the availability of the inducer.
The use of wheat bran has already been investigated [37]. They used four low-cost agro-industrial residues, wheat bran, sorghum straw, corn cob, and soybean meal, the xylanase activity resulting from 72 h FES was 1137 ± 104, 257 ± 35, 380 ± 25 and 365 ± 20 U/g, respectively. The maximum xylanase production was obtained with wheat bran compared to the other three agro-industrial residues.
Productivity played a crucial role in the decision-making process. The choice was based on criteria involving the maximization of enzymatic activity, minimization of processing time, and overall productivity enhancement. Therefore, after analysis, a cultivation period of 72 h using pure wheat bran as the substrate was chosen.
3.3 Lyophilized Enzyme Characterization
After lyophilization, xylanase activity was stabilized at 102.34 U/mL. The freeze-drying process is recommended to preserve enzymatic activity, with xylanase activity showing notable concentration. The activity of other enzymes was also analyzed. The concentrations of cellulases and beta-glucosidases activities reached 4.13 and 45.31 U/mL in the lyophilized extract, respectively. Notably, the total protein concentration was also maintained at higher values (2.17 mg/mL), indicating efficient enzyme recovery during the lyophilization process.
The electrophoretic profile of xylanase revealed the presence of a predominant visible protein band with an approximate molecular mass of 66 kDa (Fig. 4), however, other lighter bands were observed, which may indicate cellulase and β-glucosidase. This result is consistent with typical characteristics of xylanases produced by microorganisms, as reported in the literature, where the molecular masses of these enzymes generally fall within a weight range of 8 to 145 kDa [38]. The electrophoretic profile reinforces the identification of xylanase as the primary enzyme of interest in this context, confirming its presence and integrity after lyophilization [38].
Figure 4.
In this study, the protein appeared at a specific location on the gel, which shows that this represents the molecular mass of the xylanases produced by Penicillium sp. FSDE15. This molecular weight value supports previous findings from other investigations, which show the production of xylanases by other microorganisms with molecular weight values similar to those found in this work. Thiagarajan et al. [39]evaluated xylanase production by Aspergillus fumigatus MKU1 in SSF using wheat bran as a substrate. They observed a single xylanase band on SDS-PAGE gel electrophoresis, indicating high-purity xylanase with an approximate molecular mass of 66 kDa. In Liu et al. [40], xylanase produced by Escherichia coli was investigated, and four bands corresponding to molecular masses of 110, 84, 72, and 66 kDa were observed.
3.4 Influence of pH and Temperature on Xylanase Activity by Penicillium sp. FSDE15
The highest xylanase activity values were recorded in acidic environments, while an increase in pH appeared to compromise the enzyme's action. Figure 5A shows the xylanase activity in the pH range from 4 to 9. It can be observed that the highest xylanase activity was 92.89 ± 1.74 U/g at pH 4.8 and 76.83 ± 6.32 U/g at pH 5. However, it was evident that at neutral and alkaline pH, the enzymatic activity decreased considerably, reaching 8.60 ± 1.80 U/g at pH 9, indicating low stability at alkaline pH levels. Regarding the effect of temperature on xylanase production shown in Fig. 12B, it was revealed that the ideal temperature is between 50–60°C, with an activity of 102.35 ± 1.94 U/mL, followed by 102.60 ± 4.50 U/mL at 55°C and 102.85 ± 5.19 U/mL at 60°C. Xylanase activity sharply declined at temperatures above 65°C.
Figure 5.
The pH effect on xylanase activity has been reported. It is widely described that slightly acidic environments can favor xylanase action. It was shown that [41], the activity of xylanases produced by Penicillium citrinum and Aspergillus fumigatus in SSF using wheat straw and sugarcane bagasse at different pH levels and temperatures displayed an optimal pH of around 5, and the optimal temperature was around 50°C [33].
In Poornima et al. [42], the maximum production of xylanase by Streptomyces geysiriensis occurred at an optimal incubation temperature of 40°C at pH 8 [34, 35] found that the optimal temperature for xylanase produced by Aspergillus niger at pH 5 was 25°C. These studies demonstrate that the pH and temperature range for optimal xylanase activity depends on the species.
The xylanase produced by Penicillium sp. FSDE15 in SSF, using wheat bran as a substrate, was evaluated at different temperatures and pH levels. Therefore, the enzyme's activity at 50°C and pH 4.8 was considered optimal, as observed in Fig. 5. Thus, the enzyme's acid pH stability has significant potential for application in biotechnological processes conducted under low pH conditions.
3.5 Thermal and pH Stability of Xylanase
The ability to tolerate pH and temperature variations mitigates the makeup of enzymes in the process. In the present study, the stability of enzymes produced by Penicillium sp. FSDE15 in SSF is evaluated based on the relative activity observed after incubating the supernatant at different temperatures and pH levels. Figure 6 presents the profile of relative xylanase activity at different temperatures at pH 4.8.
Figure 6.
The relative activity of xylanase produced by Penicillium sp. FSDE15 significantly decreased with increasing temperature due to enzyme denaturation. A decline in the relative xylanase activity can be observed within the first 15 min at all studied temperatures (Fig. 6). The increase in temperature leads to the breaking of weak bonds and can cause alterations in enzyme conformation [43]. This effect was intensified in trials at higher temperatures (60 and 70°C), with nearly an 80% loss in residual activity.
The effect of pH on the relative xylanase activity was assessed at pH values ranging from 4 to 9 over a 120 min incubation period, as these are two important external factors influencing enzymatic activity, as shown in Fig. 7. Xylanase stability was highest at pH 4.0, with residual activity of 44% at 30 min. However, its activity decreased to 22% at 120 min. When the extract was incubated at pH 9.0, the residual activity reached 24% at 30 min and dropped to 8% at 120 min. It can be noted that enzyme stability decreased at alkaline pH levels, given that most fungal xylanases function efficiently at pH values ranging from 4.5 to 6.5 [44, 45].
Exposure to alkaline pH represents a challenging condition for xylanases, as demonstrated in previous studies. A paper conducted by Gowdhaman and collaborators [46] investigated xylanases from Bacillus aerophilus and revealed that xylanase production significantly decreased as the medium's pH exceeded 5.0, with the maximum xylanase production occurring at a lower, more acidic pH of 4.0. These findings highlight the sensitivity of xylanases to alkalinity in the environment, underscoring the critical importance of pH as a determinant factor in regulating enzymatic activity. Furthermore, several studies on xylanase production have reported that an initial medium pH of around 4.5 favors efficient xylanase production by different fungi during fermentation. These findings suggest that most fungi prefer an acidic environment for their growth and xylanase production [47–49]. However, it is important to note a discrepancy in our study, which emphasizes the disappointment resulting from the enzyme's low stability at a pH of 4.0, considering that the optimal pH for this xylanase was established at 4.8.
This contradiction between the optimal pH for activity and enzyme stability at a specific pH underscores the complexity of enzymatic regulation and the need to consider not only the optimal pH but also stability under different pH conditions when designing biotechnological processes involving xylanases. This discrepancy may be attributed to structural or microenvironmental peculiarities of the xylanase, emphasizing the importance of detailed studies to understand the nuances of enzyme activity and stability under different pH conditions[50].
Figure 7.
3.6 Enzymatic Saccharification
The production of glucose and xylose after 48 h of saccharification with different enzymes can be observed in Table 2.
Table 2.
Table 2
Production of glucose and xylose from Na₂CO₃ pretreated sugarcane straw hydrolysis after 48 h with application of enzymes produced by Penicillium sp. FSDE15, commercial Celluclast®, and enzyme cocktail (Celluclast® + NS50012) under optimal ph and temperature conditions with 5% solids loading.
Residue | Enzymes | Glucose (g/L) | Xylose (g/L) |
Untreated Straw | Penicillium sp. FSDE15 | 3.26 ± 0.13 | 4.37 ± 0.19 |
Celluclast® | 3.04 ± 0.04 | 0.55 ± 0.43 |
Pretreated straw | Penicillium sp. FSDE15 | 4.16 ± 0.43 | 2.73 ± 0.21 |
Celluclast® | 8.21 ± 0.39 | 4.14 ± 0.12 |
The saccharification of untreated material with the enzyme cocktail from Penicillium sp. FSDE15 and Celluclast exhibited similar performance in terms of glucose (3.26 g/L versus 3.04 g/L, respectively), but the release of xylose was higher using the enzymes from this study. Conversely, the enzymes from Penicillium sp. FSDE15 were less effective in the saccharification of pretreated material. The glucose release with pretreated material was 4.16 g/L, approximately 51% less glucose than the Celluclast cocktail. These results indicate that the hemicellulose in the pretreated straw was less susceptible to hydrolysis than cellulose. This behavior is attributed to the accumulation of cellulose in the pretreated straw, likely reducing the accessibility of xylanases to hemicellulose [51].
Alkaline pretreatment removes lignin and alters the molecular configuration in the cell wall, eventually increasing the surface area for enhanced receptivity to the cellulase cocktail [52]. There is no specific combination of parameters that ensures high sugar yield. Enzymatic hydrolysis depends on various process parameters, such as the type of pretreatment, enzyme loading, biomass concentration, temperature, agitation, pH, and hydrolysis time [53]. Indeed, the commercial enzyme cocktail exhibited superiority when considering the context of second-generation ethanol, where pretreatments are common. Although this outcome may not immediately attract attention, it is pertinent to emphasize the unique effectiveness of the enzyme produced when dealing with untreated straw, a substrate with higher recalcitrance due to its high lignin and extractive content.
3.7 Xylose oligosaccharides (XOS)
In this present study, XOS production was investigated under different loads of xylanases from Penicillium sp. FSDE 15, and the results are shown in Table 3.
Table 3.
Table 3
Xylooligosaccharide (XOS) Production from corn cob xylan using different loads of Penicillium sp. FSDE 15 xylanase at 30°c and 150 rpm for 24 h.
Xylanases (U/mL) | XOS (g/L) | X2 (g/L) | Xylose (g/L) |
1.00 | 2.07 ± 0.04 | 0.43 ± 0.02 | 0.14 ± 0.00 |
2.00 | 1.94 ± 0.01 | 0.54 ± 0.03 | 0.26 ± 0.00 |
5.00 | 1.80 ± 0.08 | 0.43 ± 0.01 | 0.16 ± 0.01 |
XOS reached a production of 2.07 g/L when exposed to a concentration of 1 U/mL. Subsequently, this production decreased to 1.94 g/L when the xylanase activity was increased to 2 U/mL and, finally, to 1.80 g/L when the xylanase activity was raised to 5 U/mL. Regarding X2 (xylobiose), the highest yields were obtained at 0.54 g/L when the xylanase activity was 2 U/mL, while at concentrations of 1 and 5 U/mL, the production was 0.43 g/L.
In a study conducted by Boonchuay and collaborators [54], corn cob was employed as a substrate for xylose oligosaccharides (XOS) production using the xylanase_1948 enzyme, which underwent a 12 h purification process. After a 12 h reaction period, the results indicated the remarkable content of XOS in the reaction medium, totaling 30.6 g/L. Furthermore, the presence of X2 was highlighted, with a specific concentration of 12.82 g/L, and xylose occurred at a concentration of 7.16 g/L. These findings underscore the effectiveness of the approach adopted in converting corn cob into XOS, demonstrating the potential of this methodology for industrial applications, with a particular focus on X2 and xylose production.
It is noteworthy that the xylanase from Penicillium sp. FSDE15 demonstrated remarkable efficiency, as it allowed XOS to be produced even without a prior purification process. This aspect highlights the versatility and catalytic capacity of xylanases, indicating that it can play a significant role in hydrolysis, resulting in XOS formation. This discovery may have promising implications for biotechnological processes involving the production of XOS from natural raw materials.