Artículo sometido a Enzyme and Microbial Technology (Elsevier)
El artículo reporta los resultados obtenidos del capítulo 2. Producción de celulasas y xilanasas en cultivos mixtos de hongos filamentosos, aislados de bosques veracruzanos.
Elsevier Editorial System(tm) for Enzyme and Microbial Technology Manuscript Draft
Title: Cellulase and xylanase production from different Ascomycota in mixed cultures of liquid media
Article Type: Research Paper
Keywords: endoglucanase; β-glucosidase; exoglucanase; SDSPAGE; Trichoderma; Fusarium.
Corresponding Author: Dr. Enrique Alarcón, Ph.D. Corresponding Author's Institution: Universidad Veracruzana First Author: Christian Hernández, PhD
Order of Authors: Christian Hernández, PhD; Adriane Milagres , Ph.D.; Gerardo Vázquez-Marrufo, Ph.D.; Enrique Alarcón, Ph.D.
Manuscript Region of Origin: MEXICO
Abstract: Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, β-glucosidase) and xylanase activities. A strategy that seeks to produce hydrolytic complexes with these features is the use of cocultures of filamentous fungi, which produces different enzyme isoforms, resulting in an enzymatic complex of different characteristics to those produced with monocultures. The present study aimed to explore the production of cellulases and xylanases in mixed cultures (one, two, three and four fungal strains on the same bioreactor) of wild strains of filamentous fungi from the genus Trichoderma, Aspergillus and Fusarium, in liquid media. Endoglucanase, exoglucansae, β-glucosidase and xylanase activities and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) are reported. Pearson´s correlation matrix and Principal Component Analysis (PCA) were used to analize data. According to our results, relationships of competition were stablished in mixed cultures, which produce spositive (endoglucanase and β-glucosidase productions), negative (number of extracellular protein isoforms and xylanases productions) or neutral (exoglucanase production) effects in the variables tested. We conclude that the coculture of Aspergillus niger-Fusarium oxysporum contributes to increase 19.02% endoglucanase and 6.35% β-glucosidase, compared with monocultures. The use of tricultures and polycultures (more than three different strains) trends to diminish the amount of enzymes produced, and the extracellular protein isoforms, due to the competition relationships (stressful conditions) between fungal strains.
Cellulase and xylanase production from fungal polycultures in liquid media
Hernández C.1, Milagres A.M.F.2, Vázquez- Marrufo G3, Alarcón E.1*
1Universidad Veracruzana, Instituto de Biotecnología y Ecología Aplicada (INBIOTECA) México
2Universidade de São Paulo, Escola de Engenharia de Lorena (EEL) Departamento de Biotecnología, Brasil
3Universidad Michoacana de San Nicolás de Hidalgo, Facultad de Medicina Veterinaria y Zootecnia, Centro Multidisciplinario de Estudios en Biotecnología (CMEB) México
Keywords: endoglucanase, β-glucosidase, exoglucanase, SDS-PAGE, Trichoderma, Aspergillus,
Fusarium.
Abstract
Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, β-glucosidase) and xylanase activities. A strategy that seeks to produce hydrolytic complexes with these features is the use of cocultures of filamentous fungi, which produces different enzyme isoforms, resulting in an enzymatic complex of different characteristics to those produced with monocultures. The present study aimed to explore the production of cellulases and xylanases in mixed cultures (one, two, three and four fungal strains on the same bioreactor) of wild strains of filamentous fungi from the genus Trichoderma, Aspergillus and Fusarium, in liquid media. Endoglucanase, exoglucansae, β-glucosidase and xylanase activities and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) are reported. Pearson´s correlation matrix and Principal Component Analysis (PCA) were used to analize data. According to our results, relationships of competition were stablished in mixed cultures, which produce positive (endoglucanase and β-glucosidase productions), negative (number of extracellular protein isoforms and xylanases productions) or neutral (exoglucanase production) effects in the variables tested. We conclude that the coculture of Aspergillus niger-Fusarium oxysporum contributes to increase 19.02% endoglucanase and 6.35% β-glucosidase, compared with monocultures. The use of tricultures and polycultures (more than three different strains) trends to diminish the amount of enzymes produced, and the extracellular protein isoforms, due to the competition relationships (stressful conditions) between fungal strains.
Introduction
Cellulases are specialized enzymes in the degradation of cellulose produced in high quantities by filamentous fungi (mainly Ascomycota), which have been particularly studied in species of the genus
Trichoderma [1, 2]. Cellulase activity [FPU; 3], results of three enzymatic activities: exoglucanase (1, 4- β-D-glucan-cellobiohydrolase, EC 3.2.1.91), endoglucanase (endo-1, 4-β-D-glucanase, EC 3.2.1.4) and β-glucosidase (β-D-glucoside glucanohydrolase, cellobiase, EC 3.2.1.21); the synergy of those three activities is able to degrade cellulose into glucose [4]. Trichoderma spp. are well known for producing high amounts of endo- and exo-glucanases, but are generally poor producers of β-glucosidase [5]; thus, for biotechnology purposes, the addition of this enzyme is needed for an effective hydrolysis of cellulose [6]. Other fungi genera capable to produce cellulase in high amounts are Aspergillus [7], Penicillium [8, 9], and Fusarium [10, 11].
Aspergillus is a genus known for the production of β-glucosidase [12], and these enzymes added to those produced by Trichoderma can contribute to the construction of efficient cellulase complexes [5]. However, a mix of those enzymes added a posteriori of the production process is not the only strategy to generate cellulase complexes more efficient than those produced in monocultures. The use of cocultures of species belonging to to different genera of filamentous fungi seeks to induce the production of cellulase enzymes with a balance between exo, endo and β-glucosidase activities, more favorable than those produced by monocultures [13]. Several studies have been conducted in this field [e.g. 14, 15, 16], mainly in solid state fermentation. This area of research has been increased in last years in response to the growing demand for cellulases, because of their great importance in various industries that use lignocellulose as feedstock, like the production of second generation bioethanol [17].
In natural ecosystems, like forest or dessert soils [18], as well as in agricultural residues, like the piles of sugar cane (Saccharum officinarum L.) baggase produced by sugar industry [19], the lignocellulose is degraded through the combined action of many microorganisms. The synergistically degradation of lignocellulose observed by the action of different fungal or bacterial species, is due to the secretion of different degrading enzymes, like cellulases, xylanases and accessory enzymes [20, 21]. This offers the possibility of the use of fungi consorcium to produce enzymatic complexes with great potential to enhance lignocellulose degrading performance [5], or for some specific aims, like the production of thermotolerant enzymes [19]. The use of cocultures of different fungal strains, or fungi- bacteria combinations [22], are good attemps to desing enzymatic complexes with better features than those produced in monocultures; however, more research is needed to understand how the production of lignocellulose degrading enzymes is affected by the interactions of more than two microorganisms.
The increase of Filter Paper Unit (FPU) activity in Trichoderma-Aspergillus cocultures has been attributed to an increase in β-glucosidase; but also has been hypothesized that an increase in the number
of isoforms of cellulases in these cultures are occurring [23], contributing to a better procesing of the substrate by the enzyme complex (synergistic effects). However, lower cellulase activity of the
Trichoderma reesei – Aspergillus terreus cocultures than those of monocultures has also been reported [24]. The isoforms of cellulase produced in soil could change according to different management systems [18], and this modifies the Kmof the cellulase system. However, it is unknown that the use of
cocultures, or cultures of more than two strains of fungi, could cause positive or negative effects in the pattern of enzyme isoforms that were produced.
On the other hand, xylanases are another group of enzymes that are involved in the degradation of lignocellulose [25]. Xylanase activity represents a group of enzymes that act together to degrade xylan, the main structural component of the hemicellulose. Just as cellulases, filamentous fungi also produce xylanases, but there is a scarcity of knowledge about their production in mixed cultures. Although, in natural ecosystems the same substrate (lignocellulose) can be simultaneously degraded by a whole community of microorganisms [26, 27], few attemps have been made to describe the production of cellulase and xylanases in liquid-media polycultures (more than three strains).
With this in mind, the first objective of the present study was to isolate and identify wild strains of filamentous fungi able to produce cellulases (exoglucanases, endoglucanases, β-glucosidases) and xylanases. The second aim was to characterize the production of these enzymes’ activities in monocultures and mixed cultures (2, 3 and 4 strains at the same reactor) of the isolated strains. Finally, we seek to characterize the profile of extracellular proteins produced for each type of fungal combination. According to the background, we expected an increase in cellulase and xylanase activities in mixed cultures, as well as an increase in the number of isoforms in polycultures, compared with those found in monocultures.
Materials and methods
Strains isolation and identification
The strains here studied were isolated directly from litter of Pinus patula at the Cofre de Perote National Park, (19°30' N: 97° 9' O; Veracruz, México) and litter of a Citrus sp. plantation at Tuzamapan town (19°24' N: 96°52' O; Veracruz, México). For strain isolation the selective medium Czapek-Dox (Agar- carboxy methyl cellulose) was added with tetracicline (50 mg.l-1) to avoid bacterial contamination [28]. A qualitative analysis for the presence of cellulases by direct Congo red staining was done [29]. Four of the isolated strains were used to produce cellulases and xylanases in mixed cultures, these strains were identified by amplification and analysis of the ITS rDNA region [30]. DNA of the selected strains was isolated using the Fastprep (MP Biomedicals, LLC, USA) a kit for fungal DNA isolation, as described by the supplier. PCR reactions of the ITS region were conducted with the primers (ITS1/ITS4) and
conditions described by White et al., [31]. Amplicons were sequenced at Elimbiopharm (Hayward CA, USA) and the sequences obtained were subjected to Blastn search in Genbank. Those sequences of Genbank with maximum identity with amplicons obtained were retrieved and used for phylogenetic analysis. Additional ITS sequences of the same genus obtained in Blastn search were selected and retrieved from Genbank in order to make a more robust phylogenetic analysis of each studied strain. The sequences were aligned by ClustalW and edited by hand. Genetic distances were calculated by the Kimura- 2 parameters and then the phylogenetic reconstruction was conducted by using the Maximum Likelihood criteria with 1000 bootstrap replicates. Maximum Parsimony (MP) trees were also generated using same genetic distances. All these steps for phylogenetic analysis were conducted in Mega 6 [32]. Obtained sequences were deposited in Genbank with the accession numbers KX253665 (strain PS1- G06), KX253666 (strain PS3), KX253667 (strain Asp 546), KX253668 (strain C02-E06). See strains code below.
Table 1. Different strain combinations used for cellulase and xylanase production in liquid media
Treatment code Culture type Strains
M1 Monocultures F. oxisporum M2 T. harzianum (1) M3 T. harzianum (2) M4 A. niger C1 Cocultures F. oxisporum + T. harzianum (1) C2 F. oxisporum + T. harzianum (2) C3 F. oxisporum + A. niger C4 T. harzianum (1) + T. harzianum (2) C5 T. harzianum (1) + A. niger T1 Tricultures
F. oxisporum + T. harzianum (1) + T. harzianum (2)
T2 F. oxisporum + T. harzianum (1) + A. niger
T3 F. oxisporum + T. harzianum (2) + A. niger
T4 T. harzianum (1) + T. harzianum (2) + A. niger
P1 Polycultures F. oxisporum + A. niger + T. harzianum (1) + T. harzianum
(2)
Experimental design
To evaluate the effect of growing different strains of filamentous fungi together on the production of cellulases and xylanases, a monofactorial design was performed, where the factor was the type of combination, i.e. monoculture, coculture, triculture or polyculture (Table 1). The response variables were the activities of exoglucanase, endoglucanase, β-glucosidase and xylanase, and the profile of
extracellular proteins produced.
As an experimental unit, Erlenmeyer 250 ml flasks were used which contained 150 ml of basal medium [33], with the following composition (per liter): 1 g of KH2PO4, 0.26 g of NaH2PO4, 0.317 g of (NH4)2SO4, 0.5 g of MgSO4 7H2O, 0.5 mg of CuSO4, 74 mg of CaCl2, 6 mg of ZnSO4, 5 mg of FeSO4, 5 mg of MnSO4, 1 mg of CoCl2, supplemented with sugarcane bagasse as carbon source (2 % w/v). The flasks were inoculated with 1 square (0.25 cm2) of agar-malt medium, colonized with mycelium of 6 days of culture. Each treatment had three experimental replicates. The cultures were kept in darkness at 30° C without agitation and sampling was performed every 4 days for 20 days.
Analytical methods
The exoglucanase, endoglucanase and xylanase activities were evaluated according to Ghose [34], quantifying the reducing sugars released from specific substrates [i.e avicel (cellulose microcristalline), carboxymethyl cellulose and birchwood xylan, respectively] by the method of dinitrosalicylic acid [DNS; 35]. The enzymatic reaction was performed at 50 ° C in a water bath, stopped with DNS reagent, read in a spectrophotometer at 540 nm and converted to μM•ml-1 by comparing with a standard curve of glucose; enzyme activity was reported as U•ml-1 (μM•min-1•ml-1).
β-glucosidase activity was evaluated by quantifying the amount of p-nitro phenol liberated from a solution of p-nitro phenyl-β-D-glucopyranoside, 0.1 mM spectrophotometrically at 412 nm [36]. The activity was reported as U•ml-1.
The profile of extracellular proteins was evaluated by electrophoresis in polyacrylamide gel (5% stacking gel, 12% separating gel) under denaturing conditions (SDS-PAGE) [37, 38]. For this, 50 ml of culture medium were concentrated 100-fold by lyophilization and resuspended in 500 µl of dH2O. Electrophoresis was performed with the concentrated protein, at the end, the gel was stained with a solution of Coomassie blue-methanol-acetic acid, the molecular weight of the detected isoforms was determined by its coefficient of migration (Rf) and compared with a low molecular weight marker (14- 97 kDa).
Statistical analysis
In order to identify if the presence/absence of the strains cultured at different combinations had relation (positive or negative) with the enzymatic production, a correlation analysis (Pearson α = 0.05) and Principal Component Analysis (PCA) was done. The PCA was carried out from a correlation matrix (Pearson) between different culture combinations (treatments) and response variables; they were also
plotted in a triplot together with the different strains as complementary variables. A second PCA was done using a matrix of presence/absence of protein isoforms categorized by its molecular weight, and the matrix of enzymatic activity; in order to identify if the activity of the different strains and combinations had any relation with specific isoforms. Analyses were made in the software XLSTAT v2014.
Results
Phylogenetic analysis of the fungal isolates
Blastn search of amplicons obtained from fungal isolates PS1-G06 and PS3 show maximal identity with sequences of Trichoderma lixii (Hypocrea lixii)/Trichoderma harzianum complex, whereas isolates Asp546, and C02-E06 shows maximal identity with Aspergillus niger, and Fusarium oxysporum ITS sequences, respectively. Phylogenetic reconstruction of the ITS region from strains PS1-G06 and PS3 groups with its corresponding sequences of the H. lixii/T. harzianum complex with a bootstrap value of 95%, clearly separated from all other species within the genus (Figure 1a). The strain C02-E06 grouped with a bootstrap value of 99% in the clade of Fusarium oxysporum and its teleomorph Gibberella moniliformis (Figure 1b). The strain Asp546 was grouped with a bootstrap value of 100% in the clade of
Aspergillus niger, Aspergillus tubingensis and Aspergillus awamori (Figure 1c).
Enzymes production in monocultures
The four fungal strains used in this study showed different enzymatic production profiles (Figure 2).
Aspergillus niger produced the highest amount of cellulase and xylanase activities for all monocultures tested, being 6.36 ± 1.33 U•ml-1 for endoglucanase, 5.98 ± 0.20 U•ml-1 for β-glucosidase, and 2.33 ± 0.06 U•ml-1 for xylanase after 8 days of culture; and 1.6 ± 0.06 U•l-1 for exoglucanase after 20 days of culture. Trichoderma strain 1 (PS1-G06) showed nearly a half of endoglucanase activity reached by A. niger (5.58 ± 1.22 U•ml-1 at day12), similar exoglucanase activity (1.40 ± 0.17 U•l-1 at day 16) and was deficient in β-glucosidase (0.41 ± 0.35 U•ml-1 at day 4) and xylanase activity (0.78 ± 0.03 U•ml-1 at day 20). Trichoderma strain 2 (PS3) was similar to Trichoderma strain 1, producing 3.10 ± 1.68 U•ml-1 of endoglucanase (at day 8), 0.70 ± 0.02 U•l-1 of exoglucanase (at day 16), 1.89 ± 0.14 U•ml-1 of β- glucosidase (at day 4), and 0.69 ± 0.01 U•ml-1 of xylanase (at day 20). On the other hand, Fusarium oxysporum had the lowest values of enzymatic activities of monocultures, its highest enzymatic activities were: endoglucanase (0.42 ± 0.19 U•ml-1 at day 12), exoglucanase (0.80 ± 0.09 U•l-1 at day 16), β- glucosidase (0.75 ± 0.31 U•ml-1 at day 12) and xylanase (0.04 ± 0.005 U•ml-1 at day 4).
Figure 1. Phylogenetic trees of the isolated strains. 1a) Strains PS1-G06 and PS3 groups with its corresponding sequences of the H. lixii/T. harzianum complex; 1b) Strain C02-E06 grouped with a bootstrap value of 99% in the clade of Fusarium oxysporum and its teleomorph Gibberella moniliformis; 1c) Strain Asp546 was grouped with a bootstrap value of 100% in the clade of Aspergillus niger,
Figure 2. Enzymatic activities produced by the four fungal strains in monocultures
Enzyme production in polycultures
The use of more than one fungal strain affects the activity of the enzymatic complex produced in the cultures (Table 2). Endoglucanase (7.57 ± 0.98 U•ml-1 at day 8) and β-glucosidase (6.36 ± 0•46 U.ml-1 at day 16) showed higher activity values in the coculture C3 (A. niger + F. oxysporum) than in the A. niger’s monoculture. In the same way, coculture C5 [A. niger + Trichoderma strain (1)] showed higher endoglucanase activity (6.12 ± 1.21 U•ml-1) in a shorter time (day 4) than monocultures. Mixed cultures with A. niger mantained high β-glucosidase production (Table 2); however, xylanase production was severely affected when A. niger was cultured with other fungal strains. In general, exoglucanase was not affected when fungal combinations were used.
The PCA, constructed from Pearson correlation matrix, using enzymatic activities as variables, fungal strains as complementary variables and data of whole sampling time as observations, indicates a relation between the fungal combination used and the enzyme activity observed. Axis F1 (counted for 49.49% of explained variability) was significatively (P < 0.05) related with endoglucanase (r = 0.84), xylanase (r = 0.86), and β-glucosidase (r = 0.68); and close related with A. niger (r = 0.51). All other fungal strains were negative correlated with this axis. Meanwhile, F2 (counted for 27.74% of the explained variability) was related with exoglucanase (r = 0.90), and weakly correlated with Trichoderma strainstrains (r = 0.02
and r = 0.06; Figure 3a). These two axes (biplot) condense the 77.23% of the explained variability (Figure 3b).
PCA showed (Pearson correlation; Figure 3c) that the presence of A. niger in the cultures was positive correlated with xylanase (r = 0.35), endoglucanase (r = 0.19) and strongly correlated with β- glucosidase (r = 0.82) activities. The other fungal strains had negative correlations with enzymatic activities, except for F. oxysporum who was positive correlated with β-glucosidase (r = 0.10); however,
this value was not significant (P > 0.05).
A distance biplot indicates that the treatment M2 (monoculture of Trichoderma strain 1) was related to exoglucanase production, mainly after 16 days of culture (Figure 3a, Black arrow). On the other hand, M4 (monoculture of A. niger) was related to xylanase and endoglucanase production, since 8 to 16 days of culture (Figure 3a, Green arrow). Finally, coculture C3 (A. niger + F. oxysporum) and triculture T3 (F. oxisporum + Trichoderma strain (2) + A. niger) were related to β-glucosidase
production (Figure 3a, Blue arrows).
Table 2. Maximum values of enzymatic activity recorded for the different treatments. The three highest values are distinguished by *
Endoglucanase Exoglucanase β-glucosidase Xylanase
Treatment U.ml-1 Day U.l-1 Day U.ml-1 Day U.ml-1 Day
Monocultures M1 0.426 12 0.8 16 0.754 12 0.043 4 M2 5.581 12 1.40** 16 0.414 4 0.787* 20 M3 3.108 8 0.7 16 1.898 4 0.698 20 M4 6.365** 16 1.60*** 20 5.987** 8 2.334*** 8 Cocultures C1 2.619 4 0.4 8 0.246 16 0.355 20 C2 1.464 4 0.6 8 1.190 4 0.209 16 C3 7.579*** 8 0.6 4 6.366*** 16 0.798** 4 C4 2.919 16 0.8* 20 1.150 4 0.654 20 C5 6.120* 4 0.7 4 4.456 8 0.580 20 Tricultures T1 2.031 8 1.10** 20 0.484 12 0.285 16 T2 3.018 12 0.7 12 4.659 16 0.381 12 T3 2.614 8 0.6 20 4.192 16 0.466 20 T4 3.300 8 0.7 16 5.304* 16 0.459 20 Polyculture P1 1.684 8 0.4 12 4.454 16 0.428 20
Figure 3. a) PCA showing the realtion between enzimatic activities and fungal combinations. Strains presence are showed with blue lines, as complementary variables. b) Accumulated variability and eigenvalues by components. c) Table of correlations between enzymatic acivities and strain presence.
Protein profile produced by polycultures
The A. niger monoculture produced the highest amount of extracellular proteins isoforms of all treatments tested (Table 3). Both Trichoderma strains produced an equal protein profile, and by its side,
F. oxysporum produced the lowest amount of protein isoforms of monocultures (Figure 4b). A reduction in the number of isoforms produced was observed when more than one strain was cultured in the same bioreactor (i.e. cocultures, tricultures, polycultures). Values of molecular weight of the produced proteins are shown in Table 3.
Tabla 3. Molecular weights of the isoforms of extracellular protein produced by the different fungal combinations tested (KDa)
Monocultures Cocultures Tricultures Polyculture
Num. Isoform M1 M2 M3 M4 C1 C2 C3 C4 C5 T1 T2 T3 T4 P1 1 63.15 123.28 123.28 184.37 72.72 123.28 142.97 38.29 112.19 38.29 38.29 129.35 53.27 53.27 2 55.52 101.18 101.18 142.97 57.89 101.18 89.87 20.39 38.29 20.76 22.79 53.27 43.26 43.26 3 37.42 72.72 72.72 115.08 42.68 72.72 66.10 16.49 22.35 18.40 20.76 46.20 23.70 23.70 4 21.20 60.42 60.42 84.99 20.75 51.25 53.32 13.39 13.39 13.74 18.40 35.19 21.96 21.96 5 - 49.32 49.32 66.10 - 41,26 28.87 - - - 13.74 32.51 14.11 14.11 6 - 41.26 41.26 53.32 - 36.27 22.14 - - - - 22.14 - - 7 - 36.27 36.27 28.13 - 23.16 19.50 - - - - 13.92 - - 8 - 20.75 20.75 24.83 - 20.75 13.85 - - - - 9 - 12.80 12.80 22.14 - 12.80 - - - - 10 - - - 19.11 - - - - 11 - - - 13.85 - - - -
A second PCA (from Pearson correlations) was constructed with the data of the 8 culture days (maximum enzymatic activity) incorporating the data of the molecular weight of the isoforms detected on SDS-PAGE (Figure 4b). Axis F1 (counted for 37.95 % of the explained variability) was related to xylanase (r = 0.91), β-glucosidase (r = 0.74) and endoglucanase (r = 0.74) activities; to A. niger strain (r = 0.59); and to isoforms of 10-20 kDa (r = 0.54), 20-30 kDa (r = 0.79), 50-60 kDa (r = 0.34), 80-90 kDa (r = 0.93), 140-150 kDa (r = 0.93) and 180-190 kDa (r = 0.78). Axis F2 (17.97 % of the explained variability) was related to exoglucanase (r = 0.35) but neither strain showed positive correlation with this axis; however, positives correlations were observed between F2 and isoforms 40-50 kDa (r = 0.70), 60- 70 kDa (r = 0.57), 70-80 kDa (r = 0.74), 110-120 kDa (r = 0.79) and 120-130 kDa (r = 0.76). Both axes condense 55.92 % of the explained variability (Figure 4c).
Biplot showed that monocultures M2 and M3 (Trichoderma strains), and coculture C2 [F. oxisporum +
Trichoderma strain(2)] were close related to the production of protein isoforms of 40-50 and 70-80 kDa, and with exoglucanase activity (Figure 4a). Isoforms of 70-80 kDa were correlated with exoglucanase activity (r = 0.27), thus, Trichoderma strains could be a producer of exoglucanase of 72.72 kDa, according to molecular-weight isoforms data (Table 3). On the other hand, β-glucosidase activity was closely related to C3 and A. niger, and correlated with isoforms of 80-90 kDa (r = 0.58; Figure 4a,d). Thus, it could be possible to assume that the isoform of 84.99 kDa is a β-glucosidase subunit produced by A. niger.
Xylanase activity was correlated with isoforms of low molecular weight (10-20 and 20-30 kDa), and significant correlations were found too between xylanases and these isoforms (r = 0.42 and r = 0.72; respectively). Also, in the biplot of the PCA, this enzyme was related to A. niger (M4) and to isoforms of 80-90 and 140-150 kDa. We observed that the strip of 24.84 kDa is repeated in the treatments M4, C3 and T3 (Figure 4b), and that this is related with the cultures with the highest xylanase activity, and with
A. niger presence.
A second PCA (from Pearson correlations) was constructed with the data of the 8 culture days (maximum enzymatic activity) incorporating the data of the molecular weight of the isoforms detected on SDS-PAGE (Figure 4b). Axis F1 (counted for 37.95 % of the explained variability) was related to xylanase (r = 0.91), β-glucosidase (r = 0.74) and endoglucanase (r = 0.74) activities; to A. niger strain (r = 0.59); and to isoforms of 10-20 kDa (r = 0.54), 20-30 kDa (r = 0.79), 50-60 kDa (r = 0.34), 80-90 kDa (r = 0.93), 140-150 kDa (r = 0.93) and 180-190 kDa (r = 0.78). Axis F2 (17.97 % of the explained variability) was related to exoglucanase (r = 0.35) but neither strain showed positive correlation with this