Enzymatic cocktail obtained through fungus co-culture

Abstract

The invention relates to an enzymatic cocktail obtained from fungal extracts by co-cultivation, and its application in the hydrolysis of plant biomass to obtain carbohydrate monomers. The extracts are obtained by a low-cost process. The enzymatic extracts are used in the enzymatic hydrolysis of plant biomass to convert polysaccharides into fermentable sugars, which can be used to produce renewable fuels and chemicals, such as alcohols, organic acids, polyols, and biopolymers.

Description

1 / 11 Enzymatic cocktail obtained through co-cultivation of fungi. FIELD OF THE INVENTION [1] The present invention relates to enzymatic cocktails, derived from enzymatic extracts obtained from the co-cultivation of macrofungi with other filamentous fungi. The enzymatic extracts produced are applied in the hydrolysis of plant biomass to obtain fermentable sugars, such as glucose, xylose and other monosaccharide fractions with 5 or 6 carbons (C5 or C6), which are used for the production of renewable fuels and chemicals. DESCRIPTION OF THE STATE OF THE ART [2] Plant or lignocellulosic biomass can be used for the production of renewable fuels and chemicals. One of the ways to convert biomass into these renewable products is by fermentation of a hydrolysate rich in fermentable sugars, a process that involves the steps of pretreatment, hydrolysis or enzymatic saccharification, fermentation and separation / purification. [3] The cell wall of plant biomass is made up of structural components, which are cellulose, hemicellulose and lignin. These components are strongly associated, forming a stable and recalcitrant complex. In this way, the rigidity of the biomass structure is reduced in the pretreatment stage, making the cellulose and hemicellulose more accessible to the action of enzymes in enzymatic hydrolysis, which is the next stage of the process. [4] The enzymatic hydrolysis or saccharification step employs a set of enzymes, i.e., an enzymatic cocktail, to transform cellulose, and hemicellulose depending on the type of pretreatment used, into fermentable sugars. However, one of the bottlenecks in this step is associated with the high costs of the enzymes used in the steps to obtain fermentable sugars for the production of fuels and chemicals derived from Petition 870260077380, dated 04 / 08 / 2026, page 13 / 28 2 / 11 of plant biomass. Therefore, enzymatic hydrolysis processes require enzymes with high depolymerization capacity and low production / obtaining costs so that the products obtained are competitive with industrially widespread processes, such as ethanol obtained from sugarcane juice. [5] The enzymes used in hydrolysis or enzymatic saccharification include cellulases (endoglucanase, exoglucanase and cellobiase or beta-glucosidase), xylanases, peroxidases and laccases, among other accessory enzymes, such as lytic monooxygenases. These enzymes are of paramount importance in the industrial areas of biofuels (cellulosic ethanol), as well as in animal nutrition, since they can assist in the partial or total delignification of plant biomass (input), increasing the digestibility of the raw material, focusing mainly on the nutrition of pigs, poultry and fish (monogatric animals). Filamentous fungi secrete a wide variety of enzymes that break down the structural components of the plant cell wall, and these microorganisms are the main sources for obtaining / producing hydrolytic enzymes used in commercial enzymatic cocktails. For example, Trichoderma reesei is used for the production of enzymes on an industrial scale (BISCHOF et al., 2016). [6] Other filamentous fungi, such as macrobasidiomycetes or macrofungi, are important sources for obtaining enzymes for lignin breakdown, mainly (GUTIÉRREZ-SOTO et al., 2015). Macrofungi are filamentous fungi capable of producing fruiting bodies, known as mushrooms, and can be found within the phyla Basidiomycetes or Ascomycetes, with the majority being in the phylum Basidiomycetes. Macrofungi possess two types of extracellular enzymatic systems necessary to degrade plant biomass: (a) a hydrolytic system, responsible for the degradation of polysaccharides, consisting mainly of xylanases and cellulases, and (b) a lignolytic system, which degrades lignin, mainly comprising laccases, ligninases, and peroxidases (PERALTA et al., 2017). The main biotechnological application of macrofungi is related to the pre Petition 870260077380, dated 04 / 08 / 2026, page 14 / 28 3 / 11 treatment of plant biomass, acting as “delignifying” agents of the cell wall and thus making fermentable sugars available. The most important macrofungi in this process are the so-called “white-rot fungi” (WRF) due to their predominantly oxidative enzymatic profiles, which characterize them as the most efficient in the delignification process. This group of fungi is characterized by the complete degradation of lignin, in addition to partially degrading the cellulose and hemicellulose of plant biomass (MANALAVAN et al., 2015). [7] Producing enzymes from a single organism may not be the best strategy for obtaining the highest yield of fermentable sugars from plant biomass. For example, Trichoderma reesei produces a low amount of beta-glucosidase, although it produces a high concentration of endoglucanases and exoglucanases, making it necessary to use another organism that produces sufficient amounts of beta-glucosidase. In addition, auxiliary enzymes, such as xylanases, monooxygenases, peroxidases and laccases, can increase the efficiency of the enzymatic hydrolysis process. This means that smaller quantities of enzymes will be used, resulting in lower costs for this process in different industrial scenarios, which in turn work with the deconstruction of the structural components of the plant cell. [8] Several studies have been found in the literature that have carried out co-cultivation of fungi. For example, Trichoderma reesei RUT-C30 and Aspergillus niger NL02 were co-cultivated for the production of cellulolytic enzymes. After 5 days, the filter paper activity (FPA) and beta-glucosidase activity (BGA) of the mixed cultures were 3.30 and 1.01 IU / mL, respectively, higher than the results of the monocultures, which were 2.48 and 0.24 IU / mL for T. reesei and 0.20 and 0.70 IU / mL for monocultures (FANG et al., 2010). [9] Crude enzyme extracts were described by solid-state fermentation co-cultivation of T. reesei RUT-C30 with Aspergillus saccharolyticus AP, Aspergillus carbonarius ITEM 5010 or Aspergillus niger CBS 554.65. The co-cultivation of T. reesei and A. saccharolyticus yielded the extract Petition 870260077380, dated 04 / 08 / 2026, page 15 / 28 4 / 11 more competitive enzymatic, with the highest efficiency in the enzymatic hydrolysis of pre-treated wheat straw (KOLASA et al., 2014).

[10] Thus, obtaining enzyme complexes containing enzymes such as cellulases, hemicellulases and other accessory enzymes, such as monooxygenases, peroxidases and laccases, is of great importance to increase the efficiency of enzymatic hydrolysis of plant biomass. And one of the ways to obtain these enzyme complexes is by co-cultivation of two genera of fungi, including macrofungi.

[11] In the present invention, enzyme cocktails with unexpected qualitative and quantitative composition were obtained from the co-culture of macrofungi with other filamentous fungi, when compared to the monoculture of the same microorganisms, indicating an additional effect. SUMMARY OF THE INVENTION

[12] In a first aspect, the present invention relates to obtaining enzymatic cocktails for hydrolysis of plant biomass through co-cultivation by submerged fermentation of filamentous fungi and macrofungi using plant biomass as a culture medium.

[13] In a second aspect, the invention relates to enzymatic cocktails obtained according to the process described in the invention.

[14] The invention also relates to the use of these enzymatic cocktails in the hydrolysis of plant biomass to obtain soluble sugars, which are used for the production of renewable fuels and chemicals. DESCRIPTION OF THE FIGURES

[15] Figure 1. Presents the results of the enzymatic activities of the extracts obtained from the monocultures Trichoderma reesei ATCC60787 (TR), Coprinus sp. (FPB125), Laetisaria arvalis ATCC52088 (LA), Schizophyllum commune CC328 (SC), Chrysosporium lucknowense ATCC44006 (CL), Trametes versicolor Petition 870260077380, dated 04 / 08 / 2026, page 16 / 28 5 / 11 (CC124), Panus lecomtei (CC40) and Lepiota fuscipes (CC402), using decanter sludge resulting from the palm oil extraction process.

[16] Figure 2. Presents the results of the enzymatic activities of the extracts obtained from the co-cultures T. reesei with Coprinus sp. FPB125 (TR+FPB125), T. reesei with L. arvalis ATCC52088 (TR+LA), T. reesei with S. commune CC328 (TR+SC), T. reesei with P. lecomtei CC40 (TR+CC40), T. reesei with C. lucknowense ATCC44006 (TR+CL), T. reesei with T. versicolor CC124 (TR+CC124) and T. reesei with L. fuscipes CC402 (TR+CC402), of fungi using decanter sludge resulting from the palm oil extraction process.

[17] Figure 3. Shows the results of the hydrolysis of sugarcane bagasse using enzyme cocktails obtained from monocultures of Trichoderma reesei ATCC60787, Coprinus sp. FPB125 (A), Laetisaria arvalis ATCC52088 (·), Schizophyllum commune CC328 (·), Chrysosporium lucknowense ATCC44006 (), Trametes versicolor CC124 (), Panus lecomtei CC40 (·) and Lepiota fuscipes CC402 (♦), commercial enzyme Cellic® Ctec3 (x) and negative control (+).

[18] Figure 4. Shows the results of the hydrolysis of sugarcane bagasse using enzyme cocktails obtained from co-cultures of T. reesei with Coprinus sp. FPB125 (TR+FPB125, A), T. reesei with T. versicolor CC124 (TR+CC124, ), T. reesei with P lecomtei CC40 (TR+CC40, ·), T. reesei with L. fuscipes CC402 (TR+CC402, ♦), T. reesei with C. lucknowense ATCC44006 (TR+CL,), T. reesei with L. arvalis ATCC52088 (TR+LA, ·) and T. reesei with 5. commune CC328 (TR+SC, ·), in addition to Trichoderma reesei ATCC60787 monoculture (^), commercial enzyme Cellic® Ctec3 (x) and negative control (□). DETAILED DESCRIPTION OF THE INVENTION

[19] Enzymatic hydrolysis of biopolymers contained in biomass is one of the steps in converting plant biomass into renewable fuels and chemicals through fermentative processes. Due to the low efficiency and high cost of enzymes currently used, the search for improved enzymatic compositions is one of the main challenges to making this processing route viable. Petition 870260077380, dated 04 / 08 / 2026, page 17 / 28 6 / 11 biomass for different industrial purposes. The co-cultivation of fungi using submerged fermentation aims to obtain crude enzymatic extracts containing the enzyme groups necessary for enzymatic hydrolysis of plant biomass.

[20] In a first aspect, the invention relates to a process for obtaining an enzymatic cocktail for biomass hydrolysis by co-cultivation, through submerged fermentation, of filamentous fungi and macrofungi using plant biomass as a culture medium. The co-cultivation of certain filamentous fungi and macrofungi showed an additive effect with regard to the quantitative and qualitative composition of the crude enzymatic extract of the enzymes that participate in the hydrolysis of plant biomass contained in this cocktail. This composition allows for a greater release of glucose than that obtained by enzyme extracts obtained by monoculture, indicating an additional effect of the co-cultivation of these fungi. For the production of these enzymatic extracts, biomasses derived from oil palm, sugarcane, green coconut, eucalyptus, cotton, jatropha, and castor bean are used as a culture medium or carbon source. Preferably, in the present invention, biomasses derived from oil palm are used.This biomass comprises at least one of the following options: i) sludge from the decanter resulting from the palm oil extraction process; ii) palm mesocarp fiber.

[21] The process for obtaining the enzymatic cocktail of the invention comprises the co-cultivation of filamentous fungi and macrofungi by incubation in the temperature range of 25°C to 35°C for a period of 3 to 8 days. Preferably, the co-cultivation occurs with the inoculation of macrofungi prior to the inoculation of the filamentous fungus. In a preferred embodiment, the co-cultivation occurs from the inoculation of macrofungi 10 to 24 hours prior to the inoculation of the filamentous fungus. In an even more preferred embodiment, the co-cultivation occurs from the inoculation of macrofungi 14 hours before the inoculation of the filamentous fungus.

[22] In the aforementioned invention, the production of enzymatic cocktails using submerged fermentation is carried out by co-cultivating a filamentous fungus with Petition 870260077380, dated 04 / 08 / 2026, page 18 / 28 7 / 11 at least one macrofungus selected from species of the genera Coprinus, Laetisaria, Schizophyllum, Chrysosporium, Panus, Trametes and Lepiota. Preferably, the enzymatic cocktails of the present invention are obtained by co-cultivation of a filamentous fungus with at least one macrofungus selected from: Coprinus sp. FPB125 (deposited in the Embrapa AleloMicro database under number BRM050072), Laetisaria arvalis ATCC52088, Schizophyllum commune CC328 (deposited in the Embrapa AleloMicro database under number BRM050073), Panus lecomtei CC40 (deposited in the Embrapa AleloMicro database under number BRM044603), Trametes versicolor CC124 (deposited in the Embrapa AleloMicro database under number BRM047439) and Lepiota fuscipes CC402 (deposited in the Embrapa AleloMicro database under number BRM050074).

[23] The co-culture using submerged fermentation of the invention includes a filamentous fungus of the genus Trichoderma, preferably of the species Trichoderma reesei. In a more preferred embodiment, the filamentous fungus used in the invention includes the strain Trichoderma reesei ATCC 60787.

[24] In a further preferred embodiment of the invention, the process for obtaining the enzymatic cocktail comprises the co-cultivation of the macrofungus Lepiota fuscipes CC402 and the fungus Trichoderma reesei ATCC 60787.

[25] In another preferred embodiment of the invention, the process for obtaining the enzymatic cocktail comprises the co-cultivation of the macrofungus Panus Lecomtei CC40 and the fungus Trichoderma reesei ATCC 60787.

[26] The invention also relates to the enzymatic cocktail for biomass hydrolysis obtained according to the fungal co-cultivation process described above. This cocktail comprises at least one of the following enzymes: cellulases, hemicellulases, peroxidases and laccase. These enzymatic extracts are used directly in liquid form. The enzymatic extracts can be concentrated from a liquid stream containing the crude enzymatic extract, with the excess water being separated by membranes, drying or lyophilization. Petition 870260077380, dated 04 / 08 / 2026, page 19 / 28 8 / 11

[27] The invention also relates to the use of the enzymatic cocktail described in the present invention in the hydrolysis of plant biomass. Preferably, the plant biomass is selected from: sugarcane, elephant grass, sweet sorghum bagasse, empty bunches and mesocarp fibers of the oil palm fruit and corn residues. In a more preferred embodiment, the enzymatic cocktail of the invention is used in the hydrolysis of sugarcane bagasse. The use of the enzymatic cocktail in the hydrolysis of plant biomass showed an additive effect on glucose release, superior to that obtained by fungal extracts obtained by monoculture. EXAMPLES

[28] Example 1: Monoculture of fungi

[29] In 250 mL Erlenmeyer flasks, 1.250 g of palm biomass (decanter sludge - BD or mesocarp fiber - FM), previously ground and dried at 65°C for two days, 50 mL of Mandels and Weber (1969) MW medium with the following composition (gL-1): Urea 0.3 g, yeast extract 0.25 g, bacteriological peptone 0.75 g, (NH4)2SO4 2 g, MgSO4.7H2O 0.3 g, CaCl2.2H2O 0.4 g, ZnSO4.7H2O 1.4 mg, FeSO4.7H2O 5.0 mg, CoCl2.6H2O 2.0 mg, MnSO4.H2O 1.04 mg and PEG6000 0.1% (w / v). As inoculum, 5 mycelial discs (5 mm in diameter) of agar were used, fully colonized with the fungi Panus lecomtei CC40, Trametes versicolor CC124, Lepiota fuscipes CC402, Schizophyllum commune CC328, Coprinus sp. FPB125, Chrysosporium lucknowense ATCC44006, Laetisaria arvalis ATCC52088 and Trichoderma reesei ATCC60787 separately.

[30] Transferred to incubation in a shaker at 150 rpm and 28°C for five days. After five days of culture, the contents of the Erlenmeyer flasks were filtered and centrifuged at 10000 RPM at 5°C for 10 minutes. The supernatant was collected, and sodium azide solution was added to a final concentration of 0.02% (w / v) to avoid contamination. FPase (total cellulase), β-glucosidase, xylanase, laccase, and total peroxidase activities were determined. Petition 870260077380, dated 04 / 08 / 2026, page 20 / 28 9 / 11

[31] The results of the enzymatic activities of the enzymatic extracts obtained in the monocultures of Trichoderma reesei ATCC60787 (TR), Coprinus sp. (FPB125), Laetisaria arvalis ATCC52088 (LA), Schizophyllum commune CC328 (SC), Chrysosporium lucknowense ATCC44006 (CL), Trametes versicolor (CC124), Panus lecomtei (CC40) and Lepiota fuscipes (CC402) using decanter sludge are presented in Figure 1.

[32] Example 2: Co-cultivation of fungi

[33] In 250 mL Erlenmeyer flasks, 1,250g of previously ground and dried palm biomass (decanter sludge - BD), 50 mL of MW medium and 0.1% (w / v) PEG6000 were added. As inoculum, 5 mycelial discs (5 mm in diameter) of agar fully colonized with the fungi Panus lecomtei CC40, Trametes versicolor CC124, Lepiota fuscipes CC402, Schizophyllum commune CC328, Coprinus sp. FPB 125, Chrysosporium lucknowense ATCC, Laetisaria arvalis ATCC52088 were used separately. After 14 hours, the media were inoculated with two discs of Trichoderma reesei ATCC60787 mycelium, also separately, based on the co-culture method used in MA & RUAN (2015).

[34] Transferred to incubation in a shaker at 150 rpm and 28°C for five days. After five days of culture, the contents of the Erlenmeyer flasks were filtered and centrifuged at 10000 RPM at 5°C for 10 minutes. The supernatant was collected, and sodium azide solution was added to a final concentration of 0.02% (w / v) to avoid contamination. FPase (total cellulase), β-glucosidase, xylanase, laccase, and total peroxidase activities were determined. The results of the co-cultures of T reesei with Coprinus sp. FPB125 (TR+FPB125), T. reesei with L. arvalis ATCC52088 (TR+LA), T. reesei with S. commune CC328 (TR+SC), T. reesei with P. lecomtei CC40 (TR+CC40), T. reesei with C. lucknowense ATCC44006 (TR+CL), T. reesei with T. versicolor CC124 (TR+CC124) and T. reesei with L. fuscipes CC402 (TR+CC402) are shown in Figure 2.

[35] According to the results, the co-culture of T. reesei with P. lecomtei CC40 (TR+CC40) showed higher laccase and total peroxidase activity (1111,44 and Petition 870260077380, dated 04 / 08 / 2026, page 21 / 28 10 / 11 599.74 U.mL-1, respectively) than in monocultures, higher FPase activity (1.17 U.mL-1) than in the monoculture extract of P. lecomtei CC40 and lower than that of T. reesei ATCC60787. β-glucosidase and xylanase activities were higher in the monoculture extracts of P. lecomtei CC40, but lower than in the monoculture of T. reesei ATCC60787.

[36] Example 3: Enzymatic hydrolysis of sugarcane bagasse using enzyme extracts (enzyme cocktail)

[37] Hydrolysis was performed in 24-well “DeepWell” plates. In each well, 0.8 g of plant biomass (5% - dry weight), the enzyme (monoculture extracts, co-cultures or commercial enzyme Cellic® Ctec3) at a protein concentration of 25 mg.mL-1 and 0.1 M citric acid / sodium citrate buffer solution pH 5.0 in sufficient volume to adjust the protein concentration were placed. The plates were immediately sealed with plastic film and transferred to incubation for 48 hours, 50°C and 280 rpm.

[38] During the reaction, 1 mL aliquots were taken at 0, 4, 8, 24, 32, and 48 h, placed on ice to stop the reaction, and centrifuged at 14000 rpm for 5 minutes. The resulting supernatant liquid was used to determine soluble sugars (glucose). The release of sugars from the extracts obtained in the monocultures was compared with two experimental controls: a positive control with the commercial enzyme Cellic® Ctec3 from Novozymes and a negative control with Milli-Q® water. The results of the enzymatic hydrolysis are expressed in terms of the concentration of glucose released.

[39] The results for enzymatic hydrolysis with extracts of monocultures of Trichoderma reesei ATCC60787 (♦), Coprinus sp. FPB125 (A), T. versicolor CC124 (), P lecomtei CC40 (·), L. fuscipes CC402 (♦), C. lucknowense ATCC44006 (), L. arvalis ATCC52088 (·), S. commune CC328 (·), in addition to the commercial enzyme Cellic® Ctec3 (x) and Negative Control (+), are shown in Fig. 3.

[40] Results of enzymatic hydrolysis using enzymatic extracts from co-cultures of T. reesei with Coprinus sp. FPB125 (TR+FPB125, A), T. reesei Petition 870260077380, dated 04 / 08 / 2026, page 22 / 28 11 / 11 with T. versicolor CC124 (TR+CC124, ), T. reesei with P. lecomtei CC40 (TR+CC40, ·), T. reesei with L. fuscipes CC402 (TR+CC402, ♦), T. reesei with C. lucknowense ATCC44006 (TR+CL, ), T. reesei with L. arvalis ATCC52088 (TR+LA, ·) and T. reesei with S. commune CC328 (TR+SC, ·), in addition to Trichoderma reesei ATCC60787 monoculture (^K), commercial enzyme Cellic® Ctec3 (x) and Negative Control (□) are shown in Fig. 4.

[41] The results of enzymatic hydrolysis show that four extracts with significant glucose concentration values, in ascending order, are TR-CC402 (T. reesei with L. fuscipes CC402) with 12.3 ±1.2 gL-1, TR-CC40 (T. reesei with P. lecomtei CC40) with 12.1 ± 0.6 gL-1, TR-CC124 (T. reesei with T. versicolor CC124) with 9.0 ± 0.4 gL-1, and TR-F125 with 4.2 ±0.1 gL-1. The extract from the T. reesei monoculture obtained a glucose concentration of 8.5 gL-1. The glucose values ​​of TR-CC402 and TR-CC40, which did not show significant differences, had a substantial increase when compared to the response of hydrolyses performed with extracts from L. fuscipes CC402 and P. lecomtei CC40 monocultures (14 and 10 times greater, respectively), including the glucose released in the hydrolysis using crude enzymatic extract of T. reesei ATCC60787 (44.7% greater). Petition 870260077380, dated 04 / 08 / 2026, page 23 / 28

Claims

1 / 2 CLAIMS 1. Process for obtaining an enzymatic cocktail for hydrolysis of plant biomass, characterized by the submerged fermentation co-cultivation of a filamentous fungus of the species Trichoderma reesei and a macrofungus selected from Panus lecomtei CC40 and Lepiota fuscipes CC402 using oil palm-derived biomass as a culture medium, and by occurring through the incubation of macrofungi for a period of 14 hours before the inoculation of the filamentous fungus.

2. Process for obtaining an enzymatic cocktail, according to claim 1, characterized in that the biomasses derived from palm oil comprise at least one of the following options: i) sludge from the decanter resulting from the palm oil extraction process, ii) palm oil mesocarp fiber.

3. Process for obtaining an enzymatic cocktail, according to any one of claims 1 to 2, characterized in that the co-cultivation occurs through incubation in the temperature range of 25 to 35°C for a period of 3 to 8 days.

4. Process for obtaining an enzymatic cocktail, according to any one of claims 1 to 3, characterized in that the filamentous fungus corresponds to the strain Trichoderma reesei ATCC 60787.

5. Process for obtaining an enzymatic cocktail, according to any one of claims 1 to 4, characterized in that the co-culture consists of the macrofungus Lepiota fuscipes CC402 and the fungus Trichoderma reesei ATCC 60787. Petition 870260077380, dated 04 / 08 / 2026, pp. 27 / 28 2 / 2 6. Process for obtaining an enzymatic cocktail, according to any one of claims 1 to 4, characterized in that the co-culture consists of the macrofungus Panus lecomtei CC40 and the fungus Trichoderma reesei ATCC 60787.

7. Enzymatic cocktail for biomass hydrolysis characterized by being obtained according to the process defined in any one of claims 1 to 6 and comprising the enzymes cellulases, hemicellulases, peroxidases and laccases, and sodium azide solution at a final concentration of 0.02% (w / v).

8. Use of an enzymatic cocktail defined in claim 7, characterized by its application in the hydrolysis of sugarcane bagasse. Petition 870260077380, dated 04 / 08 / 2026, page 28 / 28

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