Pure fungal lineages associated with cassava leaves (LV) and stems (ST) were successfully isolated. Table 3 shows the identification of eighteen fungi from the stems and five fungi from the leaves. Notably, six fungi derived from the stems (ST1 – Microdochium lycopodinum, ST6 - Phyllosticta elongata, ST9 - Diaporthe endophytica, ST12 - Diaporthe caatingaensis, ST13 - Stenocarpella maydis, and ST15 - Sordariomycetes sp) and one fungus from the leaves (LV2 - Xylaria sp.) displayed significant laccase activity.
Fungi ST1, ST15, and LV2 exhibited the highest potential for laccase production. These findings align with previous studies by Fillat et al. (2016) and Hassan et al. (2019), which reported the ability of the Ascomycotas species, particularly those belonging to the Xylariales, Sordariomycetes, and Dothideomycetes orders, to produce oxidoreductase enzymes like laccases [28–29]. Additionally, Castaño et al. (2015) and Liers et al. (2007) highlighted the laccase expression by Xylaria sp., emphasizing the high potential of this species for laccase production [30–31]. Furthermore, Bankole and collaborators (2018) demonstrated laccase production in Xylaria polymorpha. These studies provide a rationale for the observed robust laccase activity during the selection of laccase-producing fungi [32].
Table 3
Identification of fungi associated with cassava leaves and stems.
Code | Identified Species | GenBank accession numbers (ITS5 + ITS4) | Phylum | Order | Specific activity (U.mg− 1.min− 1) |
ST1 | Microdochium lycopodinum | NR_145223 | Ascomycota | Xylariales | 0.010 |
ST2 | Colletotrichum sp. | MF076603.1 | Ascomycota | Glomerellales | 0.001 |
ST3 | Cladosporium sp. | MH047202.1 | Ascomycota | Capnodiales | 0.000 |
ST4 | Colletotrichum brevisporum | KU319458.1 | Ascomycota | Glomerellales | 0.000 |
ST5 | Alternaria sp. | EF432274.1 | Ascomycota | Pleosporales | 0.000 |
ST6 | Phyllosticta elongata | EU167584.1 | Ascomycota | Botryosphaeriales | 0.005 |
ST7 | Phomopsis sp. | KM979805.1 | Ascomycota | Diaporthales | 0.001 |
ST8 | Diaporthe phaseolorum | AY577815.1 | Ascomycota | Diaporthales | 0.000 |
ST9 | Diaporthe endophytica | AB899789.1 | Ascomycota | Diaporthales | 0.003 |
ST10 | Vouchered mycorrhizae | AB449173.1 | Basidiomycota | Unclassified | 0.000 |
ST11 | Phanerochaete australis | o KP135075.1 | Basidiomycota | Polyporales | 0.000 |
ST12 | Diaporthe caatingaensis | KY085926.1 | Ascomycota | Diaporthales | 0.003 |
ST13 | Stenocarpella maydis | MF076624.1 | Ascomycota | Diaporthales | 0.003 |
ST14 | Annulohypoxylon stygium | MG649252.1 | Ascomycota | Xylariales | 0.000 |
ST15 | Sordariomycetes sp. | MF076626.1 | Ascomycota | Sordariomycetes | 0.010 |
ST16 | Colletotrichum gloeosporioides | KX197387.1 | Ascomycota | Glomerellales | 0.001 |
ST17 | Phanerochaetaceae sp. | HM595569.1 | Basidiomycota | Polyporales | 0.000 |
ST18 | Guignardia mangiferae | EU273524.1 | Ascomycota | Botryosphaeriales | 0.000 |
LV1 | Cladosporium xanthochromaticum | MF473323.1 | Ascomycota | Capnodiales | 0.000 |
LV2 | Xylaria sp. | KR093861.1 | Ascomycota | Xylariales | 0.030 |
LV3 | Phaeosphaeria podocarpi | NR_137933.1 | Ascomycota | Pleosporales | 0.000 |
LV4 | Peniophora sp. | HQ608131.1 | Basidiomycota | Russulales | 0.000 |
LV5 | Gloeotinia temulenta | DQ235697.1 | Ascomycota | Hypocreales | 0.000 |
The functionality and synthesis of laccases are influenced by the distribution of copper atoms within the binding sites and the nutrient concentrations (carbon, nitrogen, inducers, and copper) used during microbial growth and the enzyme expression process [5, 33]. In light of this, the fungi displaying the highest laccase activity (ST1, ST15, and LV2) were cultivated in a nutrient-rich medium designed explicitly for laccase production (LML). The results revealed that the extracts obtained from ST15 - Sordariomycetes sp. displayed a laccase activity of 0.040 U.mg− 1.min− 1, while those from LV2 - Xylaria sp. exhibited even higher enzymatic activity at 0.051 U.mg− 1.min− 1 (Fig. 2). Given the remarkable activity of LV2, it was further subjected to growth in a liquid medium, and specific growth conditions (pH, time, and temperature) were adjusted to enhance laccase production (Table 4).
Table 4
Enzyme activity data as a function of pH, time, and temperature
pH | Time (day) | Temperature | Specific activity (U.mg− 1.min− 1) |
8 | 5 | 35 | 0.000 |
6 | 15.7 | 29 | 0.019 |
4 | 5 | 35 | 0.000 |
6 | 9 | 39.1 | 0.000 |
6 | 9 | 18.9 | 0.002 |
8 | 13 | 35 | 0.000 |
4 | 13 | 35 | 0.000 |
6 | 9 | 29 | 0.044 |
6 | 9 | 29 | 0.041 |
8 | 13 | 23 | 0.034 |
4 | 5 | 23 | 0.000 |
4 | 13 | 23 | 0.017 |
9.36 | 9 | 29 | 0.030 |
8 | 5 | 23 | 0.000 |
6 | 2.3 | 29 | 0.000 |
2.64 | 9 | 29 | 0.000 |
Through the analysis of the response surfaces (Fig. 3), it was possible to observe that laccase production by the LV2 fungus is significantly influenced by temperature and pH. The results indicate that temperatures above 30°C and below 25°C and pH values below 5.0 and above 8.0 favor enhanced laccase production. These findings align with previous studies by Kittl et al. (2012) and Zhang et al. (2020), who also reported optimal pH conditions of approximately 5.0 and a temperature of 30°C for laccase production and activity in other fungal species [34–35].
The remarkable resemblance between our study's optimal conditions and those reported in the literature further reinforces the validity and reliability of these experimental findings. Additionally, it was possible to find that the growth of the LV2 fungus for periods exceeding 7 days was essential for achieving optimal laccase production. This prolonged cultivation period is likely attributed to the necessary activation of the fungus's metabolic system and the time required for sufficient excretion of the enzyme of interest. The extended growth period contributes to laccase yield and ensures the attainment of enzymatic activity at desirable levels. Overall, this study sheds light on the critical factors influencing laccase production in the LV2 fungus, offering valuable insights into optimizing growth conditions for enhanced laccase synthesis, which holds great promise for various biotechnological applications.
It was determined that the optimal conditions for laccase production by Xylaria sp. (LV2) were a temperature of 29°C, a pH of 6.7, and a cultivation time of 11 days, as evidenced in Fig. 4, confirming the results obtained from response surface analyses. Comparing these data with findings from the literature, a significant enzymatic activity, particularly for LV2, was observed. In studies conducted by Mtibaà et al. (2018), maximum laccase activity of 0.26 U.mg− 1 was observed after eight days of growth for the ascomycete Thielavia sp. [36]. Chaurasia et al. (2014) reported a maximum laccase activity of 0.36 U.mg− 1 after seven days for Trameteshirsuta MTCC-1171 [37]. Fillat et al. (2016) observed a maximum laccase activity of 0.25 U.L− 1 after 20 days for Neofusicoccum luteum, 0.18 U.L− 1 after 20 days for Neofusicoccum australe, and 0.15 U.L− 1 after 30 days for Hormonema sp. [28]. Our study's observed optimal conditions for laccase production by Xylaria sp. (LV2) provide valuable insights for biotechnological applications, suggesting its potential as an efficient enzyme producer under specific environmental and cultivation conditions. Further growth was performed under this condition after determining the best growth conditions of the LV2 fungus in a liquid medium to produce laccase. The enzyme extract attainment produced a chitosan biosensor for quantifying dopamine in synthetic body fluids.
The extract enriched in laccase was applied to produce the biosensor matrix. Therefore, the polymeric matrix of chitosan cross-linked with SMTP was obtained, and later, the enzyme was immobilized. Analyzing the FTIR spectrum (Fig. 5A), the reduction of the band referring to the O-H bond after the synthesis of the matrix and after the immobilization of the enzyme at 3,400 cm− 1 is verified. This reduction is due to the presence of interactions between chitosan hydroxyls and both STMP and laccase. It is also possible to verify a significant increase in the band referring to the C-N bond at approximately 1,100 cm− 1 due to the presence of the laccase enzyme immobilized in the matrix, as well as at about 990 cm− 1 referring to out-of-plane angular deformations of the N-H bonds of the amino acids present in the laccase enzyme. It is also possible to identify a band in the region between 600 and 700 cm− 1 referring to the C-S bond as well as a band referring to the presence of the thiocarbonyl group at approximately 1,280 cm− 1, which confirms the incorporation of STMP to chitosan due to interactions between the molecules. These data and the enzymatic activity for laccase after obtaining the matrix used to produce the biosensor confirm the effectiveness of enzymatic immobilization.
Analyzes via DRX (Fig. 5B) corroborate the data obtained via FTIR, verifying that crystallinity increased after matrix synthesis and reduced after laccase enzyme immobilization. This fact can be explained by introducing a salt (trisodium trimetaphosphate-STMP) into the matrix, which is responsible for forming cross-links between the chitosan chains. However, it is possible to note that the crystallinity index of the matrix decreased with the incorporation of the enzyme laccase due to the breaking of the chemical bonds between the chitosan chains and the modification of the functional groups of the material. These modifications may result in a breakage or modification in the crystalline structure of chitosan, leading to a decrease in the crystallinity index (%). Lacase can also interact with the chitosan matrix through non-covalent bonds or Van der Waals forces. These interactions can disturb the organization of chitosan chains, making the matrix less crystalline and more amorphous. A crystallinity percentage of 25.41% was verified for chitosan, 45.46% for chitosan after modification with STMP (CH-STMP), and 37.06% after laccase immobilization (CH-STMP-laccase).
The matrix obtained was used in the biosensor production, and during the optimization stage of the analysis conditions, its sensitivity was evaluated as a function of pH and temperature changes. Analyzing the results of cyclic voltammograms (Fig. 6A), it is verified that both variables interfere with increasing the response signal referring to the cathodic reaction (I, µA); however, the temperature interferes more significantly in the anodic response (I, µA). It is also possible to confirm that the analysis at pHs from 5.0 to 6.0 showed the most incredible sensitivity. The best results were achieved at temperatures between 40 and 50ºC (Fig. 6B). It is also important to emphasize that the temperature favors the increase in the intensity of the response signal referring to the anodic reaction (oxidation). However, a reduction in the signal intensity related to the cathodic reaction can also be verified, making it difficult to control the enzymatic catalysis, leading to errors in the quantifications.
During the analysis of the voltammograms, it is possible to verify a positive redox potential with a value of approximately 0.2 V due to the movement of two electrons after the oxidation of dopamine. A reduction peak of approximately − 0.25 V was also verified during cathodic scanning but with lesser intensity, which suggests that the reaction has low reversity. Data analysis shows that, with increasing pH, there is a shift in the signal referring to the cathode peak (reduction reaction) to more negative potentials due to the increased difficulty of proton transfer at basic pH during dopamine analysis, which indicates that the dopamine oxidation reaction is pH-dependent. The separation between the anodic and cathode peak potentials was approximately 0.45 V, and the ratio of the anodic to cathode peak currents was about 0.53 A, which corroborates the low reversity of the observed reaction. It is also possible to follow that with increasing temperature, there is a reduction in the peak of the cathodic response, indicating that, with increasing temperature, the occurrence of the reverse reaction becomes more difficult.
In this context, the best condition for dopamine quantification with the produced biosensor was also established (pH = 5.06 and temperature = 47.8ºC) (Fig. 7). This condition allowed the quantification of dopamine in concentrations up to 0.30 µmol.L− 1 (Fig. 8). Lower concentrations did not show satisfactory results. Li et al. (2021) verified the best biosensor responses at pH 7.3 and a temperature of 44.3ºC [38]. Molinnus et al. (2018) also showed greater sensitivity of laccase biosensors when using a pH equal to 7.4 [39]. These works corroborate the obtained results and confirm the biosensor’s efficiency for quantifying dopamine at low concentrations.