Enhanced growth and production of laccase and peroxidases by Pleurotus ostreatus in an enriched natural medium in submerged fermentation

Abstract

Pleurotus ostreatus is a wood-decaying fungus capable of producing key enzymes for lignin degradation. Laccase (Lcc), manganese peroxidase (MnP), lignin peroxidase (LiP), and unspecific peroxygenase (UnP) activities of P. ostreatus grown on a wheat straw infusion medium enriched with malt extract and yeast extract (SMY) were studied. Growth kinetics and enzymatic parameters were also determined. Glucose medium was used as a control. The specific growth rate of P. ostreatus in SMY (0.62 h⁻¹) was more than twice as high as that shown in the control (0.24 h⁻¹). This fungus produced 5-fold higher biomass (15.7 g/L) in SMY medium than in the control (3 g/L). Lcc, MnP, LiP and UnP activities were approximately 67-,19-,11-, and 17-fold greater in SMY (3106, 4082, 1564, and 1883 U/L, respectively) than in the control (46, 213, 143, and 112 U/L respectively). P. ostreatus constitutively produced all the studied enzymes; however, the presence of wheat straw infusion components (i.e. phenolic compounds) in SMY enhanced the enzymes production. This is believed to be the first study to examine UnP production by P. ostreatus in an enriched natural medium. UnPs have been reported as promising enzymes for degradation of hazardous pollutants; however, there is still little information about their production.

Full Article

Enhanced Growth and Production of Laccase and Peroxidases by Pleurotus ostreatus in an Enriched Natural Medium in Submerged Fermentation

Ariadna Denisse Andrade-Alvarado  ,^a Angel González-Márquez  ,^b

Rosario González-Mota  ,^c and Carmen Sánchez  ,^d,*

Pleurotus ostreatus is a wood-decaying fungus capable of producing key enzymes for lignin degradation. Laccase (Lcc), manganese peroxidase (MnP), lignin peroxidase (LiP), and unspecific peroxygenase (UnP) activities of P. ostreatus grown on a wheat straw infusion medium enriched with malt extract and yeast extract (SMY) were studied. Growth kinetics and enzymatic parameters were also determined. Glucose medium was used as a control. The specific growth rate of P. ostreatus in SMY (0.62 h⁻¹) was more than twice as high as that shown in the control (0.24 h⁻¹). This fungus produced 5-fold higher biomass (15.7 g/L) in SMY medium than in the control (3 g/L). Lcc, MnP, LiP and UnP activities were approximately 67-,19-,11-, and 17-fold greater in SMY (3106, 4082, 1564, and 1883 U/L, respectively) than in the control (46, 213, 143, and 112 U/L respectively). P. ostreatus constitutively produced all the studied enzymes; however, the presence of wheat straw infusion components (i.e. phenolic compounds) in SMY enhanced the enzymes production. This is believed to be the first study to examine UnP production by P. ostreatus in an enriched natural medium. UnPs have been reported as promising enzymes for degradation of hazardous pollutants; however, there is still little information about their production.

DOI: 10.15376/biores.21.1.839-850

Keywords: Laccase; Lignin peroxidase; Manganese peroxidase; Pleurotus ostreatus; Submerged fermentation; Unspecific peroxygenase; Wheat straw medium

Contact information: a: PhD Program in Engineering Sciences, Technological Institute of Aguascalientes, Aguascalientes, 20256, Mexico; b: Postdoctoral fellow, Laboratory of Biotechnology, Research Centre for Biological Sciences, Autonomous University of Tlaxcala, Ixtacuixtla, Tlaxcala, 90120, Mexico; c: Optoelectronics Laboratory, Technological Institute of Aguascalientes, Aguascalientes, 20256, Mexico; d: Laboratory of Biotechnology, Research Centre for Biological Sciences, Autonomous University of Tlaxcala, Ixtacuixtla, Tlaxcala, 90120, Mexico; *Corresponding author: carmen.sanchezh@uatx.mx

INTRODUCTION

The filamentous wood-decaying fungi (i.e. basidiomycetes and ascomycetes) are unique organisms that break down lignocellulosic materials, which makes them essential for the carbon cycle in the earth (Sánchez et al. 2020; Corbu et al. 2023). In particular, the white rot fungus Pleurotus ostreatus is an edible mushroom belonging to the basidiomycete class. It can produce enzymes such as laccase (Lcc), manganese peroxidase (MnP), and lignin peroxidase (LiP) that are the key enzymes for lignin biodegradation (Sánchez 2009; Hernández-Domínguez et al. 2017; Martínez-Berra et al. 2018). P. ostreatus can be cultivated on several agricultural wastes (e.g. wheat straw, barley straw, rice waste, etc.) (Huang et al. 2024), since it is capable of converting complex lignin materials into utilizable carbohydrates for its growth (Sánchez 2010; Zárate-Salazar et al. 2020). Lcc is a copper-containing oxidase that catalyzes the oxidation of various compounds using molecular oxygen, generating water as a byproduct. Laccases oxidize non-phenolic, and phenolic groups such as hydroxyindoles, benzenothiols and aromatic amines, which are commonly present in lignin (Basera et al. 2024). Oxidation of phenolic acids by laccases could produce various kinds of reactive oxygen species, among them hydrogen peroxide (H₂O₂) (Huang and Yang 2022). H₂O₂ production is a crucial role for Lcc, since it is necessary to activate other enzymes (i.e., peroxidases) during lignin biodegradation. LiP (or ligninase) utilizes H₂O₂ as a cosubstrate to catalyze the oxidative cleavage of the phenolic and non-phenolic structures present in lignin (Singh et al. 2021). MnP or Mn-dependent peroxidase oxidizes Mn²⁺ to Mn³⁺ using H₂O₂ and the Mn³⁺ oxidizes a range of phenolic and non-phenolic aromatic moieties of lignin (Kumar and Arora 2022). In addition, unspecific peroxygenases (UnPs) are secreted fungal enzymes able to hydroxylate epoxidize C═C bonds, inactive C–H bonds, and sulfonate thioethers, which exhibit hybrid characteristics of P450 monooxygenases and heme peroxidases, showing a catalytic versatility (Karich et al. 2017). It has been suggested that a high nitrogen content, which can be supplied by plant-derived compounds (e.g. soybean meal or soybean peptone), may induce UnPs expression (González-Rodríguez et al. 2023). UnPs have been reported as promising enzymes for degradation of hazardous pollutants; however, there is still little information about their production. UnPs from the fungi Agrocybe aegerita and Marasmius rotula achieved conversion for 35 out of 40 U.S. EPA priority pollutants, such as nitroaromatic compounds, halogenated biphenyl ethers, polycyclic aromatic hydrocarbons and derivatives of phthalic acid and chlorinated benzenes (Karich et al. 2017). Fungi such as Chaetomium globosum, some species of Corpinopsis and Marasmius, Psathyrella aberdarensis, and Coprinellus radians have been reported as UnPs producers (Huang et al. 2024). P. ostreatus was able to secrete unspecific peroxidase (UnP) in a natural medium supplemented with polyethylene (Andrade-Alvarado et al. 2024; González-Márquez et al. 2024).

Ligninolytic enzymes have a wide range of industrial applications, such as bioremediation of pollutants, biofuel production, textile and paper pulp processing; clarification of musts and wines among others (Othman et al. 2023; Huang et al. 2024). Therefore, Lcc, MnP, LiP and UnP offer eco-friendly and sustainable alternatives for converting residues or waste lignocellulosic substrates into valuable products and can also be used as biocatalytic for detoxification of contaminated places.

In the present work, biomass production, Lcc, MnP, LiP, and UnP activities of P. ostreatus grown on a wheat straw infusion medium enriched with malt extract and yeast extract and on glucose medium (as control) were evaluated. Calculation of growth kinetics and enzymatic parameters were also carried out.

EXPERIMENTAL

Organism, Culture Media, and Fermentation Conditions

Pleurotus ostreatus was obtained from the culture collection at the Research Centre for Biological Sciences (CICB) at the Autonomous University of Tlaxcala (UATx, Mexico). Two different liquid culture media were prepared containing the following carbon sources: 1) wheat straw infusion enriched with 25 g/L of malt extract and 5 g/L of yeast extract (SMY medium), and 2) 10 g/L of glucose (control medium). Both culture media also had a mineral salts medium, which consisted of (in g/L): K₂HPO₄, 0.19; Mg(NO₃)₂, 0.2; Ca(H₂PO₄)₂, 0.5; MgSO₄, 1; FeSO₄, 0.005; ZnSO₄, 0.007; MnSO₄, 0.009; and CuSO₄, 0.011. Wheat straw infusion was prepared by boiling 100 g of chopped wheat straw in 1 L of distilled water for 1 h, then filtered through cheesecloth, and completed with additional distilled water (100 mL approx.) to 1 L (González-Márquez et al. 2024). Erlenmeyer flasks of 125 mL capacity containing 50 mL of culture medium were used. Ten mycelial fragments (8 mm in diameter) of P. ostreatus were inoculated into each flask. The flasks were incubated in an orbital shaker (Prendo, Mexico) at 27 °C and 120 rpm for 26 d. Samples were taken in triplicate every 24 h.

A wheat straw infusion was prepared by boiling 100 g of chopped wheat straw in 1 L of distilled water for 1 h, filtering through cheesecloth, and bringing the final volume to 1 L with distilled water

Biomass Production and Supernatant Collection

The mycelial biomass from the liquid cultures was separated by vacuum filtration using pre-dried filter paper (Whatman No. 4) at 60 °C for 48 h with a vacuum pump (Millipore, Merck, Germany) (Ríos-González et al. 2019). Biomass production (X) was quantified by subtracting the original weight of the filter paper from the filter paper with the dried biomass. The supernatant was placed into Eppendorf tubes and stored in a freezer for later use.

Growth Kinetics of P. ostreatus

The specific growth rate (μ) was determined by fitting the logistic equation into the experimental data of biomass production over time (Ahuactzin-Pérez et al. 2016). X_max corresponds to the maximum biomass produced during the fermentation expressed in g/L.

Enzyme Activity of Laccases and Peroxidases, and Enzymatic Parameters

Lcc, MnP, LiP and UnP activities were evaluated as previously reported (Ocaña-Romo et al. 2024). Briefly, Lcc activity was measured using 2,6-dimethoxyphenol (DMP) as the substrate at 468 nm. The reaction mixture contained DMP and 100 µL of supernatant. MnP activity was determined using guaiacol as the substrate at 334 nm. The reaction mixture had guaiacol, tartrate buffer, MnSO₄, hydrogen peroxide, and 10 µL of supernatant. LiP activity was determined using veratryl alcohol as the substrate at 310 nm in a reaction mixture composed of veratryl alcohol, hydrogen peroxide, tartrate buffer, and 20 µL of supernatant. UnP activity was measured using veratryl alcohol as the substrate at 310 nm. The reaction mixture contained veratryl alcohol, hydrogen peroxide, citrate buffer, and 10 µL of supernatant. Enzymatic activity measurements were carried out using a UV-Vis spectrophotometer Jenway 7305 (Stone, Staffs, UK). In all cases, one enzyme unit (U) was defined as the amount of enzyme required to catalyze the conversion of 1 μmol of substrate per minute.

The enzymatic parameters, namely, the yield of enzyme per unit of biomass (Y_E/X) was determined by dividing the maximum enzyme activity (E_max) by the maximum biomass (X_max) produced by the fungus during the fermentation. Enzyme productivity (P_RO) was calculated by dividing E_max by the time to which it was achieved. Specific rate of enzyme production (q_p) was obtained by multiplying the μ value by the Y_E/X value (Ahuactzin-Pérez et al. 2016).

Statistical Analysis

All data were analyzed to determine normality and homogeneity of variances using the Shapiro-Wilk and Levene tests, respectively. Statistical differences between groups were determined using one-way ANOVA, followed by Tukey’s post-hoc test for multiple comparisons. The analyses were performed using the Statistical Analysis System (SAS® Free software Trials) program.

RESULTS AND DISCUSSION

Biomass Production and Kinetics of Growth

Biomass production of P. ostreatus was evaluated in glucose medium (control) and in SMY medium (Fig. 1). P. ostreatus showed the maximum biomass production in glucose medium (3 g/L) (Fig. 1a) and in SMY medium (15.7 g/L) (Fig. 1b) at 14 d and 10 d of growth, respectively. In both media, the fungus entered the exponential phase after 2 d and reached the stationary phase in glucose medium and in SMY medium at 15 d and 11 d, respectively. The biomass production of P. ostreatus was 5-fold higher in SMY medium than in the control medium. Studies on the composition of wheat straw infusion after soaking it in hot water showed that it contained glucose, xylose, lignin, protein, and essential minerals such as N, P, K and Ca (Mejía and Albert 2012; Tozluoğlu et al. 2015). The lignin detected in these studies might be traces of low molecular weight lignin. Therefore, the wheat straw infusion provided the fungus with a nutrient-rich natural medium that favored its growth. Similarly, Mejía and Albertó (2012) reported that the use in a culture medium of the residual water derived from wheat straw significantly enhanced mycelial growth in P. ostreatus.

P. ostreatus grown in a medium containing glucose, peptone, yeast extract, and mineral salts had a biomass production of 16.7 g/L, after 26 d in submerged fermentation(Argyropoulos et al. 2022). In the present research, P. ostreatus had a similar biomass production in a shorter period (10 d). In addition, P. ostreatus showed a biomass yield of 39.2 g/L in a medium containing xylose and corn steep liquor (Papaspyridi et al. 2010). Bakratsas et al. (2024) reported that P. ostreatus had a biomass production of 21 g/L when grown in a medium supplemented with wine lees and glucose. These findings show that agro-industrial by-products are rich in nutrients that can enhance the growth of P. ostreatus.

Fig. 1. Biomass production of P. ostreatus grown in glucose medium (a) and SMY medium (b) in submerged fermentation

Table 1 shows that the μ value for SMY medium (0.62 h⁻¹) was more than twice as high as that showed in the control (0.24 h⁻¹), indicating that the nutrient composition of SMY favored the growth of P. ostreatus. Bakratsas et al. (2023) reported μ values ranging from 0.8 to 1.8 d⁻¹ (0.033-0.075 h⁻¹ approximately) for P. ostreatus grown in media enriched with glucose and xylose. Furthermore, Bettin et al. (2010) observed a μ of 0.038 h⁻¹ for Pleurotus sajor-caju cultivated in a stirred-tank bioreactor using sucrose and casein. The present results show that SMY represents a rich nutritional medium that enhanced mycelial growth, due to the presence of wheat straw hydrolysates in the wheat straw infusion.

Table 1. Growth Parameters of P. ostreatus Grown in Glucose Medium and SMY Medium Under Submerged Fermentation Conditions

Enzyme Production of P. ostreatus

The Lcc production by P. ostreatus grown in SMY medium and in control medium is shown in Fig. 2. In control cultures, Lcc was not produced during the first 5 d of growth, while a very low production level was observed from 6 to 12 d. Lcc production showed an increase after 13 d, reaching the highest level at 14 d (46.4 U/L) of fermentation (Fig. 2a). In SMY cultures, Lcc was produced after 3 d, showing an increase after 5 d, having the highest activity at 10 d (3110 U/L) (Fig. 2b).

Fig. 2. Laccases activity of P. ostreatus grown in glucose medium (a) and in SMY medium (b) in submerged fermentation

An et al. (2021) reported that P. ostreatus grown on Populus beijingensis residues showed a Lcc activity of 304 U/L in solid-state fermentation, which is a lower activity than the reported in the present research in SMY. In addition, Ergun and Urek (2017) observed that P. ostreatus had a Lcc production of 6710 U/L using potato residues in solid-state fermentation. Furthermore, Bakratsas et al. (2024) found that P. ostreatus had a Lcc activity of 74000 U/L in a medium containing 20% wine lees, glucose, and yeast extract in liquid fermentation. In addition, Deshmukh et al. (2025) reported that P. ostreatus had a Lcc activity of 2040 U/L in a medium added with sucrose, yeast extract, and malt extract, and it also showed a Lcc activity of 23900 U/L in a medium supplemented with cane molasses, corn steep liquor, yeast extract, and inducers. It is shown that agro-industrial residues and lignocellulosic materials induce laccase production, which is attributed to the components present in these materials (Mejía and Albertó 2012; Tozluoğlu et al. 2015).

MnP production by P. ostreatus in glucose medium and in SMY medium is shown in Fig. 3. In glucose medium, P. ostreatus showed a low MnP activity, which varied during the fermentation, having the highest MnP production (213 U/L) at 8 d (Fig. 3a). In SMY cultures, MnP activity increased continuously, showing the highest activity (4080 U/L) at 10 d, and then decreased at the end of the fermentation (Fig. 3b). Ergun and Urek (2017) reported that P. ostreatus had a MnP activity of 2050 U/L in a medium containing fresh potato peel in solid-state fermentation. Whereas Pleurotus eryngii showed a MnP activity of 4770 U/L in a medium added with peach peel residues (Akpinar and Urek 2022). It is suggested that lignin-based materials, which had phenolic compounds are crucial for the induction of MnP in this fungus.

Fig. 3. Manganese peroxidase activity of P. ostreatus grown in glucose medium (a) and in SMY medium (b) in submerged fermentation

Figure 4 shows the LiP production by P. ostreatus grown in glucose medium and in SMY medium. In glucose medium, P. ostreatus produced a low activity of LiP, exhibiting its maximum activity (143.4 U/L) at 12 d, which then decreased at the end of the fermentation (Fig. 4a). In SMY cultures, this fungus showed low LiP activity during the first 9 d of growth, reaching the highest activity (1560 U/L) at 10 d of growth, which decreased at the end of the fermentation (Fig. 4b). The present results showed a better performance of LiP production by P. ostreatus in SMY medium as compared to other studies. For example, Ergun and Urek (2017) reported that P. ostreatus had a LiP activity of 231 U/L when grown in a medium added with potato peel residues in solid-state fermentation. Whereas Inonotus obliquus showed a maximum LiP activity of 123 U/mL in a medium containing wheat straw under submerged fermentation conditions (Xu et al. 2017).

Fig. 4. Lignin peroxidase activity of P. ostreatus grown in glucose medium (a) and SMY medium (b) in submerged fermentation

Figure 5 shows the UnP production by P. ostreatus grown in glucose medium and in SMY medium. This fungus showed low and changing UnP activity during the fermentation, reaching the highest activity (112 U/L) at 15 d, which then decreased to 87 U/L at the end of the fermentation in medium added with glucose. In SMY medium, P. ostreatus produced low UnP activity during the first 7 d of growth, which increased to 1880 U/L at 10 d and then decreased to 500 U/L approximately at 16 d, reaching 1800 U/L at the end of the fermentation. The present results indicated that the composition of SMY medium significantly enhanced the UnP activity in P. ostreatus, which was higher than that activity (331 U/L) reported for A. aegerita grown in vinasse-supplemented medium after 23 d (González-Rodríguez et al. 2023), and that activity (530 U/L) showed by Chaetomium globosum in a rich glucose-based medium after 21 d (Kiebist et al. 2017).

Fig. 5. Unspecific peroxygenase activity of P. ostreatus grown in glucose medium (a) and in SMY medium (b) in submerged fermentation

Enzymatic Parameters of Lcc, MnP, LiP and UnP

The enzymatic parameters E_max, Y_E/X, P_RO, and q_p were evaluated for Lcc, MnP, LiP and UnP (Table 1). P. ostreatus showed the greatest enzymatic parameters in the SMY medium for all enzymes. In SMY medium, this fungus presented the highest E_max, Y_E/X, P_RO, and q_p values for MnP (4082 U/L, 260 U/gX, 17 U/L/h, and 162.5 (U/gX/h), respectively), followed by Lcc, UnP, and LiP. In glucose medium, P. ostreatus also exhibited the highest E_max, Y_E/X, P_RO, and q_p values for MnP (213.1 U/L, 71 U/gX, 1.1 U/L/h, and 17.7 U/gX/h, respectively), followed by UnP, LiP, and Lcc. In fact, E_max for Lcc, MnP, LiP and UnP were 67-,19-,11-, and 17-fold greater in SMY than in the control. These results show that P. ostreatus constitutively produced all the studied enzymes, and the presence of wheat straw infusion components such as lignin (probably traces of low molecular weight lignin) and phenolic compounds (Mejía and Albertó 2012; Tozluoğlu et al. 2015) in SMY induced enzyme production. González-Márquez and Sánchez (2024) evaluated Lcc enzymatic parameters for both P. ostreatus and A. aegerita grown in a malt extract medium and found higher E_max (106.5 U/L), Y_E/X (11.6 U/gX), P_RO (0.34 U/L/h) and q_p (0.25 U/gX/h) for P. ostreatus than for A. aegerita. However, P. ostreatus and A. aegerita did not show MnP, LiP, and UnP activities in malt extract medium (González-Márquez and Sánchez, 2024). It is shown that SMY is a more favorable medium for producing Lcc, MnP, LiP and UnP than malt extract medium.

Table 2. Enzymatic Yield Parameters of P. ostreatus Grown in Glucose Medium and in SMY Medium in Submerged Fermentation Conditions

CONCLUSIONS

1. Agro-industrial by-products are rich in nutrients that can enhance the growth of P. ostreatus.
2. P. ostreatus constitutively produced Lcc, MnP, LiP and UnP; however, the presence of wheat straw infusion components in SMY enhanced enzyme production.
3. The highest Lcc, MnP, LiP and UnP activities were observed at that time to which the maximum biomass was achieved (10 d) in SMY medium.
4. SMY is a more favorable medium for producing Lcc, MnP, LiP, and UnP than glucose medium.

ACKNOWLEDGMENTS

The authors express their sincere gratitude to the Secretariat of Science, Humanities, Technology and Innovation (SECIHTI) of Mexico for granting a postdoctoral fellowship to AGM (No. 555469) and a PhD scholarship to ADAA (No. 1170027).

Conflict of Interest

The authors declare that they have no conflicts of interest.

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Article submitted: October 6, 2025; Peer review completed: November 22, 2025; Revised version received: November 28, 2025; Accepted: November 30, 2025; Published: December 10, 2025.

DOI: 10.15376/biores.21.1.839-850