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Article

Purification and Functional Characterization of a New Endoglucanase from Pleurotus djamor PLO13 Produced by Solid-State Fermentation of Agro-Industrial Waste

by
Monizy da Costa Silva
1,
Ricardo Bezerra Costa
1,
Marta Maria Oliveira dos Santos Gomes
1,
Josiel Santos do Nascimento
2,
Andreza Heloiza da Silva Gonçalves
1,
Jéssica Alves Nunes
1,
Marta Angelo dos Santos
1,
Francis Soares Gomes
1,
José Maria Rodrigues da Luz
2,
Luciano Aparecido Meireles Grillo
2 and
Hugo Juarez Vieira Pereira
1,*
1
Institute of Chemistry and Biotechnology, Federal University of Alagoas, UFAL—A. C. Simões Campus, Maceió 57072-900, Alagoas, Brazil
2
Institute of Pharmaceutical Science, Federal University of Alagoas, UFAL—A. C. Simões Campus, Maceió 57072-900, Alagoas, Brazil
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(4), 182; https://doi.org/10.3390/fermentation11040182
Submission received: 19 February 2025 / Revised: 17 March 2025 / Accepted: 18 March 2025 / Published: 1 April 2025
(This article belongs to the Special Issue Application and Research of Solid State Fermentation)
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Abstract

The increasing generation of agro-industrial waste and its improper disposal have raised significant environmental concerns, highlighting the urgent need for sustainable alternatives which would repurpose these materials. In this context, enzymes such as endoglucanase play a critical role in degrading lignin–cellulose biomass by catalyzing the breakdown of β-1,4-glycosidic bonds in cellulose, thereby converting it into fermentable sugars with diverse industrial applications. This study aimed to investigate the production, purification, and characterization of an endoglucanase produced by the fungus Pleurotus djamor PLO13, using coconut fiber, sugarcane bagasse, wheat bran, and pineapple crown as substrates. Endoglucanase activity was measured by the Miller method (1959), using 2% (w/v) carboxymethyl cellulose (CMC) as substrate. Solid-state fermentation (SSF) was found to be highly efficient for enzyme synthesis, with wheat bran emerging as the most effective substrate, yielding an enzyme production of 7.19 U after 120 h of cultivation. The endoglucanase was purified through ethanol precipitation and ion-exchange chromatography using DEAE-Sepharose, achieving a recovery rate of 110%, possibly due to removal of inhibitors present in the crude extract. The purified enzyme exhibited stability across a broad pH range and thermostability, with optimal activity at pH 5.0 and 50 °C. Furthermore, the enzyme was activated by EDTA, Mn2+, and Ca2+, while being inhibited by Mg2+. Notably, the enzyme demonstrated halotolerance, with activity increasing by 60% upon the addition of 3 M NaCl. Kinetic analysis revealed that the purified enzyme showed affinity to the CMC substrate at the analyzed parameters (pH 5.0 and 50 °C), with Km and Vmax values of 0.0997 mg/mL and 112.2 µg/min/mL, respectively. These findings suggest that the endoglucanase from P. djamor PLO13 has promising potential for biotechnological applications, underscoring the feasibility of the use of lignocellulosic waste as sustainable substrates in industrial processes.

1. Introduction

The increasing generation of agro-industrial waste and the inadequate disposal of these materials over recent decades have heightened environmental concerns, emphasizing the need for sustainable alternatives for their reuse [1,2,3]. Lignocellulosic biomass, commonly found in agro-industrial waste such as sugarcane bagasse (SB), coconut fiber (CF), pineapple crown (PC), and wheat bran (WF), is a plentiful and renewable resource composed primarily of cellulose, hemicellulose, and lignin [4,5]. Globally, more than 1.3 billion tons of such waste are generated annually through agri-food and processing activities [6]. Without proper reutilization, this biomass contributes to environmental contamination, posing significant risks to ecosystems and local communities [7].
The valorization of lignocellulosic waste through the production of high-value products—such as biofuels, organic acids, enzymes, and food additives—offers an effective strategy which can be used to mitigate environmental impacts and promote a more sustainable, fossil fuel-independent economy [8,9,10]. FES is a bioprocess widely used in the production of enzymes, including endoglucanase, in an environmentally friendly and economical way, using agro-industrial waste, such as wheat bran, as a substrate [11]. In this process, an environment with characteristics similar to the natural habitats of microorganisms is created, favoring microbial growth on solid substrates without the need for a free aqueous phase [12]. Among its advantages, the lessened need for water, the minimum generation of toxic waste, the operational simplicity, and the high production yield stand out [13].
Microorganisms are the main sources of industrial enzymes due to the high productivity, ease of cultivation, and enzymatic stability of these organisms, surpassing animal and plant sources [14]. According to Rigo [15], more than 80% of industrial enzymes come from microorganisms, with filamentous fungi and yeasts representing more than 50% of this total. Filamentous fungi are widely used in research to obtain industrial enzymes due to the ability of these fungi to grow in conditions of low water availability [15,16]. The genus Pleurotus, known for its culinary and medicinal value, is classified as a saprophyte and has a diversified enzyme complex, including cellulase [13,17,18], standing out for the secretion of efficient hydrolytic enzymes, which release essential nutrients through solid-state fermentation (SSF) [19].
Among enzymes of biotechnological importance, endoglucanases—a key component of the cellulase complex—are crucial for the hydrolysis of β-1,4 glycosidic bonds in cellulose, a process which generates fermentable sugars [20,21]. These enzymes have extensive applications across various industries, including biofuels, papers, and foods and beverages [22,23,24,25,26]. Their use in industrial processes offers an eco-friendly and efficient alternative to chemical catalysts due to their high specificity, biodegradability, and low toxicity, aligning with the Sustainable Development Goals (SDGs), which advocate for renewable energy adoption and the sustainable use of natural resources [27,28].
The purification of enzymes, such as endoglucanase, is an essential process for understanding their structural and functional properties, allowing the prediction of their industrial applications. The choice of purification techniques depends on the degree of purity required for the final use of the enzyme [29]. Among the methods used, ion-exchange chromatography stands out for its efficiency in the separation of enzymatic fractions with high specific activity, in addition to allowing the estimation of the molecular mass of the purified enzyme [11]. This method has been described in the literature by several authors [8,29,30,31], reinforcing its relevance in obtaining purified enzymes for industrial applications.
This study aimed to investigate the production, purification, and characterization of an endoglucanase enzyme produced by the fungus Pleurotus djamor PLO13, utilizing coconut fiber, sugarcane bagasse, wheat bran, and pineapple crown as alternative cultivation substrates.

2. Materials and Methods

All reagents used in the tests were of analytical-grade purity, with solutions prepared using ultrapure water obtained from a Milli-Q® system (Millipore, Burlington, MA, USA). Materials used included potato dextrose agar (Kasvi, Pinhais, Brazil, acetic acid, methanol, ethanol, butanol, t-butanol, isopropanol, propanone, acetonitrile, dinitromethane, dichloromethane, heptane, nitric acid (Dinâmica, Valparaíso, Brazil), sodium chloride, hydrochloric acid, sodium hydroxide, bromophenol blue, magnesium chloride, manganese chloride, copper sulphate, zinc chloride (Synth, Sao Paulo, Brazil), Coomassie brilliant blue, Bradford reagent, bovine serum albumin, ammonium sulphate, β-mercaptoethanol, benzamidine, silver nitrate (Sigma-Aldrich, St. Louis, MO, USA), Triton X-100, Tween-40, Tween-80, trichloroacetic acid, ethylenediaminetetraacetic acid, sodium dodecyl sulfate (SDS), calcium chloride, carboxymethyl cellulose (CMC) (Vetec, Rio de Janeiro, Brazil), and glycerol (Nuclear, Rio de Janeiro, Brazil).

2.1. Obtaining and Preparing Waste

The agro-industrial wastes used in this study—WF, CF, SB, and PC—were donated by companies located in Maceió/AL, Brazil, and the surrounding region. The samples were initially dried in an air-circulating oven (TECNAL, Niort, France) at 50 °C for 24 h. The dried components were ground using a Wiley blade mill (ACB LABOR®, Albany, NY, USA) until a particle size of 2.0 mm was achieved. The ground samples were then analyzed for crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), lignin, cellulose, and hemicellulose, following the protocols of the Association of Official Analytical Chemists [32].

2.2. Preparing the Inoculum

The Pleurotus djamor PLO13 strain was obtained from the Mycothek of the Federal University of Viçosa (UFV), Minas Gerais, Brazil. The fungal cultivation followed the protocol described by Velez [18].

2.3. Production of Endoglucanase by Solid State Fermentation

The agro-industrial waste was moistened with sterile distilled water in the following proportions: 85% for CF, 80% for SB and PC, and 60% for WF. The moistened substrates were sterilized in an autoclave at 121 °C for 20 min. After sterilization, four agar discs (1 ± 0.5 cm in diameter) containing mycelial growth of Pleurotus djamor PLO13 were inoculated into 100 mL Erlenmeyer flasks containing 5 g of agro-industrial waste. The flasks were incubated in a germination chamber (SL 222, Solab, Cambridge, MA, USA) at 27 °C for 10 days. All inoculation procedures were conducted under sterile conditions in a laminar flow hood.

2.4. Enzyme Extraction

At 24 h intervals during fermentation, 5.0 mL of sodium acetate buffer (100 mM, pH 5) was added to each 1.0 g of agro-industrial waste in the Erlenmeyer flasks. The mixtures were allowed to stand for 10 min at 25 °C, after which the contents were filtered and centrifuged at 9000× g for 15 min at 25 °C. The resulting supernatant, referred to as crude enzymatic extract (CEE), was collected, while the precipitate was discarded.

2.5. Endoglucanase Activity

Endoglucanase activity was determined by incubating a reaction mixture, with a total volume of 1 mL, consisting of 190 µL of sodium acetate buffer (0.1 M, pH 5); 100 µL of 2% carboxymethyl cellulose (CMC) solution as substrate; and 10 µL of the enzyme sample in the CEE, 25 µL in the precipitated fractions and 50 µL in the purified enzyme fraction. The reaction was carried out at 50 °C for 60 min and, at the end of this period, 200 µL portions of DNS reagent (3,5-dinitrosalicylic acid) were added, followed by incubation at 100 °C for 5 min. After cooling to room temperature (25 ± 2 °C), the reaction was terminated with the addition of 500 µL of distilled water. The absorbances of the samples were determined at 540 nm in a spectrophotometer (VARIAN CARY 50 BIO, Agilent Technologies, Santa Clara, CA, USA) [8]. The analyses were performed in triplicate, and the reducing sugars released were quantified by the Miller method [33]. An enzymatic unit was defined based on the relationship between the amount of reducing sugars (AR), as determined from the glucose standard curve; the total volume (TV); the sample volume (VC); the hydrolysis time (TH); and the factor 0.18, corresponding to 1 μmol of glucose per milligram, according to the equation presented below.
U/mL = AR × (VT/0.18 × VC × TH)

2.6. Purification of Endoglucanase

CEE was precipitated using ice-cold absolute ethanol (99.9% v/v). In an ice bath, the extract was gently shaken and fractionated into five analytes by gradual addition of ethanol in the following proportions: 0–20%, 20–40%, 40–60%, 60–80%, and 80–100%. Each fraction was incubated for 60 min at 8 °C; this was followed by centrifugation at 15,000× g for 15 min at 4 °C. The precipitate was resuspended in 1 mL of sodium acetate buffer (0.1 M, pH 5.0), and the supernatant was subjected to further additions of ethanol until the 80–100% fraction was reached. Enzyme activity tests, as described in Section 2.5, were performed on all fractions. To eliminate the organic solvent and continue the ion-exchange chromatography, the fraction with the highest endoglucanase activity (80–100%) was subjected to rotoevaporation. Portions consisting of 10 mL of the sample were rotoevaporated for 10 min and then resuspended in 10 mL of sodium acetate buffer (100 mM, pH 5.0).
For the ion-exchange chromatography on DEAE-Sepharose, 5.0 mL of the fraction with the highest endoglucanase activity (80–100%) was loaded onto an AKTA pure M1GE chromatograph equipped with a 50 mL ion-exchange column previously equilibrated with sodium acetate buffer (100 mM, pH 5.0) (buffer A). Adsorbed proteins were eluted using a sodium acetate buffer (100 mM, pH 5.0) containing 0.5 M NaCl (buffer B), forming a concentration gradient from 0 to 100% in 10 times the column volume, with a flow rate of 0.5 mL/min. Enzyme activity in each fraction was monitored as described in Section 2.5, and the presence of proteins was determined by absorbance at 280 nm.

2.7. Protein Concentration

The protein concentration (mg/mL) was determined by the Bradford method [34], using bovine serum albumin as a standard. For the analysis, 10 μL portions of the diluted samples (1:10) were added to 790 μL of water and 200 μL of Bradford reagent. The mixture was incubated for 5 min in the dark and the absorbance measured at 595 nm in a spectrophotometer (VARIAN CARY 50 BIO, Agilent Technologies, Santa Clara, CA, USA).

2.8. Electrophoresis (SDS-PAGE)

Stacking (5%) and separation (12%) gels were prepared, and a constant voltage of 90 mV was applied. The sample buffer consisted of 0.5 M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.001% bromophenol blue. Samples were heated at 100 °C for 5 min. Protein bands were visualized using silver nitrate (AgNO3). The gel was fixed in 40% methanol and 10% acetic acid for 30 min; this was followed by incubation in 10% ethanol and 5% acetic acid for 20 min. The gel was then treated with potassium bichromate (3.4 mM) and nitric acid (3.2 mM) for 20 min. After washing, it was immersed in silver nitrate (0.012 M) for 30 min, washed again, and developed in sodium carbonate (0.28 M) and formaldehyde (1.85%) until protein bands were visible. The reaction was stopped with 5% acetic acid.

2.9. Characterization of Endoglucanase

The optimum pH of the enzyme was determined by incubating the purified enzyme in 50 mM buffers, with pH ranging from 2.0 to 13.0, and then performing enzymatic reactions as described in Section 2.5. The pH stability was assessed by incubating the enzyme in buffers with pH within the same range (2.0 to 13.0) at 50 °C for 1 h. After this period, the samples were subjected to enzymatic assays under the optimum temperature and pH conditions (50 °C and pH 5.0), and the enzymatic assay continued, according to the methodology described in Section 2.5. The results were expressed as residual activity, with the highest activity considered to be 100%.
After determining the enzyme’s optimal pH, the optimal temperature was established by incubating the purified enzyme at various temperatures (20–100 °C) for 1 h, following the methodology described in Section 2.5. To evaluate thermal stability, the isolated enzyme was pre-incubated in 190 μL of sodium acetate buffer (100 mM, pH 5.0) within a temperature range of 20–100 °C for 1 h. After incubation, the enzymatic assay (Section 2.5) was performed using the optimum temperature determined for the enzyme. Enzymatic activity tests to determine the optimum temperature and thermal stability were quantified in a spectrophotometer at 540 nm (VARIAN CARY 50 BIO, Agilent Technologies, Santa Clara, CA, USA); the results are expressed as residual activity, considering the highest activity to be 100%.
The effects of metal ions and chelating agents on endoglucanase activity were evaluated by incubating the enzyme with 10 mM solutions of various metal ions (Mg2+, Mn2+, Ca2+, Cu2+, and Zn2+) and EDTA at 50 °C for 1 h. After incubation, the substrate (CMC) was added, and the residual activity was measured as described in Section 2.5. The results are expressed as relative activity (%), with the enzyme’s activity under optimum temperature and pH conditions serving as the 100% reference mark.
The halotolerance of the purified endoglucanase from Pleurotus djamor PLO13 was determined by measuring its activity in sodium acetate buffer (100 mM, pH 5) containing NaCl at concentrations ranging from 0.01 to 7.0 M. Enzymatic reactions were conducted as described in Section 2.5. Activity in the absence of NaCl was considered to be 100%.
The kinetic parameters (Km and Vmax) of the endoglucanase were determined by incubating the enzyme with varying concentrations of CMC (0.005–300 mg/mL) in sodium acetate buffer (100 mM, pH 5.0) at 50 °C for 30 min. Km and Vmax values were calculated using Lineweaver–Burk plots.

3. Results and Discussion

3.1. Physicochemical Analysis, Selection of Lignocellulosic Waste, and Fermentation Profile

The physicochemical analysis of the lignocellulosic substrates revealed significant variations in composition (Table 1). Among the residues analyzed, WF exhibited the highest percentage of total protein, as well as elevated levels of NDF and hemicellulose (Table 1). Consistent with these findings, Gomes and collaborators reported low lignin content (7.27%) and high protein content (19.46%) in WF samples [5]. Conversely, CF, SB, and PC residues demonstrated lower protein levels (3.98, 2.67, and 7.77, respectively) alongside higher concentrations of NDF, ADF, and lignin, indicating a more complex composition (Table 1). The high lignin content in these residues poses a challenge for the conversion of cellulose and hemicellulose into fermentable sugars by filamentous fungi [35]. Lower lignin content facilitates the degradation of polymers, thereby enhancing the secretion of lignocellulolytic enzymes [8]. Agro-industrial waste holds substantial potential as a growth medium for microorganisms in SSF processes due to its abundance of nutrients and carbon, as well as its ability to provide optimal conditions for fungal growth [5,36].
The fungus Pleurotus djamor PLO13 exhibited distinct growth and enzyme production profiles on the four agro-industrial residues (Figure 1). Among the substrates analyzed, wheat bran (WF) proved to be the most efficient in fungal growth and endoglucanase production, reaching 2.7 U/mL after 120 h of cultivation (Figure 1), with a total activity of 7.19 (Table 2). This performance can be attributed to its lower structural complexity and its balanced cellulose and hemicellulose content combined with a low lignin content (Table 1), factors that favor enzymatic conversion and endoglucanase secretion. In comparison, residues such as sugarcane bagasse (SB) and coconut fiber (CF) showed lower levels of fungal growth and enzymatic activity, due to their greater structural complexity and higher lignin content (Table 1), factors which hinder the conversion of carbohydrates into fermentable sugars (Figure 1). These factors justify the choice of WF as the main substrate for enzyme production, which is in line with sustainable approaches to reuse agro-industrial waste without the need for chemical pre-treatments.
Enzyme production is closely linked to nutrient availability in the medium, often exhibiting an initial increase followed by a decline during the SSF process [13,37]. The endoglucanase activity obtained from Pleurotus djamor PLO13 in this study is consistent with the activities reported for other organisms, such as P. roqueforti (0.879 U/mL) and A. flavus (0.540 U/mL), which also produced endoglucanase using lignocellulosic substrates [5,38]. It is worth noting that experimental conditions may vary between studies, and this may influence the observed results.
Lignocellulosic waste with high lignin content typically requires physical or chemical pretreatment to facilitate degradation. However, these treatments significantly increase operational costs and accelerate equipment wear in bioethanol-related industries [8]. Furthermore, such pre-treatments can produce phenolic compounds, which are toxic to certain microorganisms and inhibit their growth [8,39]. Unlike these conventional approaches, the present study demonstrated a sustainable alternative by excluding pre-treatments and medium supplementation, and relying solely on lignocellulosic waste, water, and microorganisms. This method resulted in efficient and environmentally viable enzyme production, highlighting the potential of this method for industrial applications.

3.2. Ethanol Precipitation of Endoglucanase

The CEE was precipitated using ethanol, an effective method for concentrating proteins. Ethanol promotes biomolecule aggregation by reducing protein solubility and interacting with water, thereby lowering the water’s availability for protein interactions in solution [40].
The 80–100% ethanol precipitation fraction showed the highest endoglucanase activity, even after the rotoevaporation process, with a recovery of 94% (Table 2). Recoveries close to 100% indicate high enzyme retention efficiency, suggesting that the ethanolic fraction efficiently concentrates the enzyme of interest, minimizing losses during the purification process. No detectable endoglucanase activity was observed in the earlier fractions, further confirming the efficiency of the method in isolating endoglucanase from Pleurotus djamor PLO13. Moreover, the enzyme demonstrated high stability in high ethanol concentrations, an advantageous property for industrial applications such as second-generation (2G) ethanol production. Similarly, Paul and collaborators used ethanol precipitation to partially purify endoglucanases from filamentous fungi, including T. harzianum, A. niger, and A. flavus [41]. Farinas and collaborators also achieved optimal endoglucanase and xylanase activities from A. niger in an 80% ethanol fraction, with recoveries of 23% and 40%, respectively [42].

3.3. Enzyme Purification: Ion-Exchange Chromatography on DEAE-Sepharose

After precipitation with 80–100% ethanol, the enzyme solution retained 6.75 U of total activity and 0.95 mg of total protein, corresponding to a recovery of 94% (Table 2). This fraction was then subjected to ion-exchange chromatography on DEAE-Sepharose. The chromatogram (Figure 2A) revealed that the enzyme was eluted exclusively in fraction 7, further increasing the total activity to 7.91 U, yielding a recovery of 110% and a specific activity of 13.18 U/mg of protein (Table 2). Recoveries greater than 100% suggest the removal of inhibitory compounds or partial activation of the enzyme during the purification steps. This result highlights the efficiency of single-step chromatographic enzyme isolation, a technique which enhances recovery rates while reducing process costs. Comparable studies, such as those of Dos Santos and collaborators [8] and Chaudhary and Grover [30], utilized DEAE-Sepharose and DEAE-cellulose, respectively, to purify endoglucanases from P. sanguineus and T. viride.
Electrophoretic analyses via SDS-PAGE under denaturing and reducing conditions (Figure 2B, channel 4), revealed a single protein band with an estimated molecular mass of approximately 26 kDa. According to the literature, fungal endoglucanases typically have molecular masses ranging from 25 to 70 kDa [8,43,44]. Detailed SDS-PAGE analysis indicated that the crude extract (CEE, Figure 2B, channel 2) exhibited a complex protein profile with multiple bands. In contrast, the 80–100% ethanol fraction (Figure 2B, channel 3) displayed a significantly reduced number of protein bands, confirming the effectiveness of ethanol precipitation in removing protein contaminants and concentrating endoglucanase. This streamlined purification process enabled enzyme isolation in a single chromatographic step. Comparable molecular masses have been reported in the literature for endoglucanases purified from other fungi. For instance, the endoglucanase purified from T heterothallicus PA2S4T had a molecular mass of 36.3 kDa [23]. Similarly, P. sanguineus and B. ricini URM 5627 had molecular masses of 76 kDa [8] and 39 kDa [44], respectively.

3.4. Characterization of Endoglucanase

Since enzymatic activity is strongly influenced by pH, we first determined the optimal pH and stability range before proceeding with thermal characterization. The endoglucanase from Pleurotus djamor PLO13 exhibited optimal activity at pH 5, maintaining approximately 95% of its activity between pH 4.0 and 7.0 and 55% at pH 8.0 (Figure 3A). Enzyme activity declined gradually in more alkaline conditions, with the enzyme retaining 45% and 30% of its activity at pH 9.0 and 10.0, respectively, and becoming completely inactivated at pH 12.0 (Figure 3A).
These findings corroborate studies by Silva and collaborators [44] and Liang and Xue [45], which reported optimal pH values of 5.0 for endoglucanases from B. ricini URM and A. niger. Similarly, Patel and Shah [46] observed a pH optimum of 4.8 for the endoglucanase from F. meliae CFA 2. Although the optimal pH for endoglucanases varies depending on the microorganism, most studies indicate that these enzymes function best in acidic environments, often at around pH 5.0 [8,47].
The pH stability of the enzyme was evaluated across a range of 2.0 to 13.0 over 1 h (Figure 3B). The enzyme retained its activity across a wide pH range, maintaining approximately 85% of its activity at pH 4.0 and 7.0. Activity decreased to 75% at pH 8.0 and 55% at pH 9.0, and 30% at pH 10.0 and 11.0, with complete inactivation resulting above 12.0 (Figure 3B). Patel and Shah [46] reported similar stability for the endoglucanase from F. meliae CFA 2 at pH 4.0 and 5.0, while dos Santos and collaborators [8] found that the endoglucanase from P. sanguineus retained more than 50% of its activity between pH 5.0 and 8.0. Endoglucanases that exhibit stability at pH 8.0 are classified as neutral to slightly alkaline and are particularly useful in detergent and textile industries. The enzyme characterized in this study retained 55% of its activity at pH 8.0 (Figure 3B), making it a potential candidate for such applications.
Once the optimal pH and stability range were established, the next step was to determine the optimal temperature and thermal stability of the enzyme. The effect of temperature on the endoglucanase from Pleurotus djamor PLO13 was evaluated across a range of 20–100 °C (Figure 4A), with optimal activity observed at 50 °C. The enzyme retained approximately 85% of its maximum activity between 40 and 60 °C, 75% at 70 °C, and 40% at 80 °C. From 80 °C onwards, a marked reduction in activity was observed, with the enzyme becoming practically inactive only at temperatures close to 90–100 °C (Figure 4A). These properties highlight its potential for industrial applications. Generally, endoglucanases exhibit optimal temperatures between 50 and 70 °C, except for those derived from extremophilic microorganisms, which may function at temperatures as high as 80 °C [8]. For instance, Narra and collaborators [43] and Hmad and collaborators [48] reported an optimum temperature of 50 °C for endoglucanases from A. terreus and S. microspora, respectively. Conversely, enzymes from A. ochraceus MTCC 1810 and A. spergillus flavus showed optimal activity at 40 °C [49,50], while the endoglucanase from F. meliae CFA 2 maintained 100% of its activity at 70 °C [46]. These findings illustrate the diversity of temperature optima for endoglucanases across different microbial sources. These thermal properties, combined with its pH stability, suggest that the endoglucanase from Pleurotus djamor PLO13 has potential for industrial applications.
Thermal stability is a critical attribute for enzymes used in industrial processes, enabling sustained activity under varying temperature conditions. The pure endoglucanase from Pleurotus djamor PLO13 demonstrated high thermostability, retaining approximately 90% of its activity between 40 and 60 °C and 80% at 70 °C, while losing all activity at temperatures above 80 °C (Figure 4B). The loss of activity at higher temperatures is likely due to alterations in the enzyme’s three-dimensional structure, which disrupt covalent bonds and catalytic function. These results align with findings for other endoglucanases, such as F. meliae CFA 2, which is stable between 50 and 70 °C [46]; A. terreus, which retains 100% of its activity at 30–50 °C and 10% at 70 °C [43]; P. sanguineus, stable from 20 to 55 °C [8]; and B. ricini URM 5627, stable between 39 and 60 °C [44]. Due to their thermostability, endoglucanases are valuable in industries such as textiles, cellulose processing, detergents, beverages, and biofuels. These enzymes enhance efficiency and reduce costs in certain applications, like biopolymerization, deinking, juice purification, and bioethanol production, during simultaneous saccharification and fermentation [22,51,52].
The impacts of EDTA and various metal ions on endoglucanase activity are shown in Figure 5. EDTA increased the activity of the pure enzyme by approximately 20%. As a chelating agent, EDTA binds to metal ions, stabilizing the enzyme and creating an environment favorable for its catalytic function. Notably, EDTA is commonly used as an additive in soap and detergent formulations [53]. The effects of EDTA vary among enzymes; for instance, Pham and collaborators [54] observed a 35% increase in the endoglucanase activity of A. niger VTCC-F021, while Naika and Tiku [55] reported a similar effect on the endoglucanase of A. aculeatus. In contrast, Marques and collaborators [56] found that EDTA inhibited enzyme activity in extracts of Penicillium roqueforti ATCC 10110.
Among the metal ions tested, Mn2+ was the most effective, increasing enzyme activity by approximately 20%, followed by Ca2+, which promoted an increase of around 15% (Figure 5). In contrast, Mg2+ inhibited enzyme activity by approximately 40%, while Zn2+ and Cu2+ had negligible effects (Figure 5). Similar trends were observed by Silva and collaborators [44], who reported that the endoglucanase from B. ricini URM 5627 was activated by Mn2+ but inhibited by Mg2+. These results highlight the complexity of the interactions between enzymes and metal ions, which depend on the enzyme’s structure and the environmental conditions.
The activity of the purified endoglucanase from Pleurotus djamor PLO13 was evaluated at NaCl concentrations ranging from 0.01 to 7 M. The enzyme exhibited stability across all salt conditions tested, with maximum activity observed at 3 M NaCl—an increase of approximately 60% compared to the control (Figure 6). A slight loss of activity (~25%) was observed at 6 M (Figure 6). These findings classify the enzyme as halophilic, as it demonstrated enhanced enzyme activity under high-salt conditions. Similar halophilic properties have been reported for other endoglucanases, such as those from P. sanguineus, which tolerate up to 5 M NaCl [8], and B. ricini URM 5627, which is stable at up to 4 M NaCl [44].
Enzymes that show increased activity in high-salt concentrations are classified as halophilic. Most of these enzymes are associated with halophilic microorganisms [57]. The ability of endoglucanases to maintain stability and activity in saline environments is a valuable characteristic for industrial applications, especially in processes that require resistance to extreme saline conditions [8]. The maximum salt concentration that each enzyme can withstand varies according to the specific properties of the enzyme, and this resistance is a competitive advantage that increases the biotechnological potential of endoglucanases, compared to other enzymes in the same class [51,58].
The purified endoglucanase from Pleurotus djamor PLO13 showed a Km value of 0.0997 ± 0.0063 mg/mL and a high Vmax of 112.2 ± 1.09 µg/min/mL, indicating that it obtained affinity and catalytic efficiency for the CMC substrate under the experimental conditions (pH 5 and 50 °C). These units were chosen due to the heterogeneous nature of CMC and its non-uniform molar mass, making mass-based measurements (mg/mL and µg/min/mL) more suitable for kinetic evaluation. Kinetic studies of other endoglucanases have revealed significant variations in affinity and enzyme activity and have also demonstrated that the use of different units may be appropriate to express these results. For example, the endoglucanase of C. thermophilum from described by Hua et al. [59] showed Km and Vmax values of 79.19 mg/mL and 59.64 ± 8.25 μg/min/m, respectively. The endoglucanase from P. sanguineus showed a Km of 3.18 mg/mL and Vmax of 0.097 mol/min/mL [8], while that from B. ricini URM 5627 showed a Km of 0.1299 mg/mL and Vmax of 0.097 mol/min/mL [44]. The endoglucanase from Streptomyces sp. demonstrated a Km of 6.37 mg/mL and Vmax of 0.056 μmol/min [60]. These results indicate that the enzyme response to the substrate can vary considerably between different sources, suggesting that the endoglucanase from Pleurotus djamor PLO13 has potential for biotechnological applications. Furthermore, the feasibility of using WF as sustainable substrate in industrial processes is highlighted.

4. Conclusions

This study showed that the production of endoglucanase by the fungus P. djamor PLO13 reached its maximum at 120 h of cultivation, using wheat bran as a substrate without any required pre-treatment or additives. Wheat bran proved to be the most efficient substrate for microbial growth and enzyme production. The purified endoglucanase exhibited halotolerant, thermostable, and halophilic properties, with an approximate molecular mass of 26 kDa and a recovery of 110% following purification through organic precipitation and anion-exchange chromatography. The enzyme showed optimal activity at pH 5.0 and 50 °C, maintaining stability across broad pH and temperature ranges. Endoglucanase activity was enhanced by EDTA, Mn2+, and Ca2+, but inhibited by Mg2+. Kinetic analysis revealed a Km value of 0.0997 mg/mL and Vmax of 112.2 µg/min/mL, indicating substrate affinity and catalytic efficiency in the hydrolysis of specific substrates. These findings underscore the potential of the endoglucanase from P. djamor PLO13 for a range of biotechnological applications.

Author Contributions

M.d.C.S.: Investigation, Writing—original draft. R.B.C.: Investigation, Writing—review and editing. M.M.O.d.S.G.: Investigation, Writing—review and editing. J.S.d.N.: Investigation. A.H.d.S.G.: Writing—review and editing. J.A.N.: Investigation. M.A.d.S.: Writing—review and editing. F.S.G.: Investigation. J.M.R.d.L.: Investigation. L.A.M.G.: Writing—review and editing. H.J.V.P.: Supervision, Writing—review and editing, Project administration, Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All relevant data are within the paper and in the references.

Acknowledgments

The authors are grateful to the Brazilian Ministry of Education’s Coordination for the Improvement of Higher Education Personnel (CAPES), the Brazilian National Council for Scientific and Technological Development (CNPq), and the Alagoas State Research Foundation (FAPEAL) for funding this research.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Endoglucanase production profile (U/mL) during solid-state fermentation using Pleurotus djamor PLO13 with WF, PC, CF, and SB waste at 27 °C. Humidity was adjusted for each waste; the substrate concentration was 5.4 mg/mL.
Figure 1. Endoglucanase production profile (U/mL) during solid-state fermentation using Pleurotus djamor PLO13 with WF, PC, CF, and SB waste at 27 °C. Humidity was adjusted for each waste; the substrate concentration was 5.4 mg/mL.
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Figure 2. Purification of endoglucanase from Pleurotus djamor PLO13. (A) Ion-exchange chromatographic profile (DEAE-Sepharose, 50 mL). The eluted protein profile was monitored at 280 nm (blue line), and endoglucanase activity is represented in red. (B) Protein profile on polyacrylamide gel stained with silver nitrate. Channel 1: molecular mass marker; Channel 2: crude extract; Channel 3: 80–100% ethanolic fraction; Channel 4: purified endoglucanase.
Figure 2. Purification of endoglucanase from Pleurotus djamor PLO13. (A) Ion-exchange chromatographic profile (DEAE-Sepharose, 50 mL). The eluted protein profile was monitored at 280 nm (blue line), and endoglucanase activity is represented in red. (B) Protein profile on polyacrylamide gel stained with silver nitrate. Channel 1: molecular mass marker; Channel 2: crude extract; Channel 3: 80–100% ethanolic fraction; Channel 4: purified endoglucanase.
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Figure 3. Optimum pH and pH stability of purified endoglucanase from Pleurotus djamor PLO13. (A) Optimum pH: Activity of purified endoglucanase at different pH values. The maximum activity, observed at pH 5.0, was set as 100%. (B) pH stability: Stability profile of the purified endoglucanase after incubation at different pH values (2.0 to 13.0). Reaction conditions: temperature—50 °C, reaction time: 1 h, and CMC (2.0% w/v). Experiments were performed in triplicate and following the methodology for determination of enzymatic activity (Section 2.5).
Figure 3. Optimum pH and pH stability of purified endoglucanase from Pleurotus djamor PLO13. (A) Optimum pH: Activity of purified endoglucanase at different pH values. The maximum activity, observed at pH 5.0, was set as 100%. (B) pH stability: Stability profile of the purified endoglucanase after incubation at different pH values (2.0 to 13.0). Reaction conditions: temperature—50 °C, reaction time: 1 h, and CMC (2.0% w/v). Experiments were performed in triplicate and following the methodology for determination of enzymatic activity (Section 2.5).
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Figure 4. Optimum temperature and thermostability of purified endoglucanase from Pleurotus djamor PLO13. (A) Optimum temperature: Activity of the purified endoglucanase at different temperatures. The maximum activity, observed at 50 °C, was set at 100%. (B) Thermostability profile: Activity of the purified endoglucanase after incubation at different temperatures. The highest activity was set at 100%. Reaction conditions: pH—5.0; and CMC (2.0% w/v). Experiments were performed in triplicate and following the methodology for determination of enzymatic activity (Section 2.5).
Figure 4. Optimum temperature and thermostability of purified endoglucanase from Pleurotus djamor PLO13. (A) Optimum temperature: Activity of the purified endoglucanase at different temperatures. The maximum activity, observed at 50 °C, was set at 100%. (B) Thermostability profile: Activity of the purified endoglucanase after incubation at different temperatures. The highest activity was set at 100%. Reaction conditions: pH—5.0; and CMC (2.0% w/v). Experiments were performed in triplicate and following the methodology for determination of enzymatic activity (Section 2.5).
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Figure 5. Activity of purified endoglucanase from Pleurotus djamor PLO13 in the presence of different metal salts. The enzyme activity without additives was used as a control and set as 100%. Reaction conditions: metal salt concentration—10 mM; temperature—50 °C; pH—5.0; reaction time: 1 h. Experiments were performed in triplicate, following the methodology for determining enzyme activity (Section 2.5).
Figure 5. Activity of purified endoglucanase from Pleurotus djamor PLO13 in the presence of different metal salts. The enzyme activity without additives was used as a control and set as 100%. Reaction conditions: metal salt concentration—10 mM; temperature—50 °C; pH—5.0; reaction time: 1 h. Experiments were performed in triplicate, following the methodology for determining enzyme activity (Section 2.5).
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Figure 6. Effect of NaCl concentration on the activity of purified endoglucanase from Pleurotus djamor PLO13. CMCase activity was assessed in the presence of different NaCl concentrations (0 to 7.0 M). Enzyme activity in the absence of NaCl was used as a control and set as 100%. Reaction conditions: temperature—50 °C; pH—5.0; reaction time: 1 h. Experiments were performed in triplicate, following the methodology for determining enzyme activity (Section 2.5).
Figure 6. Effect of NaCl concentration on the activity of purified endoglucanase from Pleurotus djamor PLO13. CMCase activity was assessed in the presence of different NaCl concentrations (0 to 7.0 M). Enzyme activity in the absence of NaCl was used as a control and set as 100%. Reaction conditions: temperature—50 °C; pH—5.0; reaction time: 1 h. Experiments were performed in triplicate, following the methodology for determining enzyme activity (Section 2.5).
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Table 1. Chemical composition (% g/100 g) of wheat bran (WF), coconut fiber (CF), sugarcane bagasse (SB), and pineapple crow (PC) waste in natura.
Table 1. Chemical composition (% g/100 g) of wheat bran (WF), coconut fiber (CF), sugarcane bagasse (SB), and pineapple crow (PC) waste in natura.
ComponentsWheat Bran (WF)Coconut Fiber (CF)Sugarcane Bagasse (SB)Pineapple Crown (PC)
Crude protein19.46 ± 0.533.98 ± 0.622.67 ± 0.097.77 ± 0.21
NDF51.93 ± 1.9479.09 ± 0.4995.8 ± 0.1289.38 ± 0.17
FDA13.32 ± 0.8971.37 ± 0.3763.17 ± 0.2044.38 ± 0.32
Lignin3.37 ± 0.4630.24 ± 0.1910.24 ± 1.084.96 ± 0.24
Cellulose9.94 ± 0.9841.12 ± 0.2452.92 ± 0.1539.42 ± 0.09
Hemicellulose38.86 ± 1.657.72 ± 0.6132.63 ± 0.0844.9 ± 0.19
NDF—neutral detergent fiber, and ADF—acid detergent fiber.
Table 2. Summary of purification of endoglucanase from Pleurotus djamor PLO13 produced in WF.
Table 2. Summary of purification of endoglucanase from Pleurotus djamor PLO13 produced in WF.
Purification StepsTotal Activity
(U)		Total Protein
(mg)		Specific Activity
(U/mg of Protein)		Recovery
(%)
Crude extract7.196.91.04100
Fraction 80–100%6.750.957.1094
DEAE-Sepharose7.910.6013.18110
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Silva, M.d.C.; Costa, R.B.; Gomes, M.M.O.d.S.; Nascimento, J.S.d.; Gonçalves, A.H.d.S.; Nunes, J.A.; Santos, M.A.d.; Gomes, F.S.; Luz, J.M.R.d.; Grillo, L.A.M.; et al. Purification and Functional Characterization of a New Endoglucanase from Pleurotus djamor PLO13 Produced by Solid-State Fermentation of Agro-Industrial Waste. Fermentation 2025, 11, 182. https://doi.org/10.3390/fermentation11040182

AMA Style

Silva MdC, Costa RB, Gomes MMOdS, Nascimento JSd, Gonçalves AHdS, Nunes JA, Santos MAd, Gomes FS, Luz JMRd, Grillo LAM, et al. Purification and Functional Characterization of a New Endoglucanase from Pleurotus djamor PLO13 Produced by Solid-State Fermentation of Agro-Industrial Waste. Fermentation. 2025; 11(4):182. https://doi.org/10.3390/fermentation11040182

Chicago/Turabian Style

Silva, Monizy da Costa, Ricardo Bezerra Costa, Marta Maria Oliveira dos Santos Gomes, Josiel Santos do Nascimento, Andreza Heloiza da Silva Gonçalves, Jéssica Alves Nunes, Marta Angelo dos Santos, Francis Soares Gomes, José Maria Rodrigues da Luz, Luciano Aparecido Meireles Grillo, and et al. 2025. "Purification and Functional Characterization of a New Endoglucanase from Pleurotus djamor PLO13 Produced by Solid-State Fermentation of Agro-Industrial Waste" Fermentation 11, no. 4: 182. https://doi.org/10.3390/fermentation11040182

APA Style

Silva, M. d. C., Costa, R. B., Gomes, M. M. O. d. S., Nascimento, J. S. d., Gonçalves, A. H. d. S., Nunes, J. A., Santos, M. A. d., Gomes, F. S., Luz, J. M. R. d., Grillo, L. A. M., & Pereira, H. J. V. (2025). Purification and Functional Characterization of a New Endoglucanase from Pleurotus djamor PLO13 Produced by Solid-State Fermentation of Agro-Industrial Waste. Fermentation, 11(4), 182. https://doi.org/10.3390/fermentation11040182

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