Selection and characterization of a native Pycnoporus sanguineus strain as a lignocellulolytic extract producer from submerged cultures of various agroindustrial wastes

Gutiérrez-Soto, G., Medina-González, G. E., García-Zambrano , E. A., Treviño-Ramírez, J. E., and Hernández-Luna, C. E. (2015). "Selection and characterization of a native Pycnoporus sanguineus strain as a lignocellulolytic extract producer from submerged cultures of various agroindustrial wastes," BioRes. 10(2), 3564-3576.

Abstract

A native strain of Pycnoporus sanguineus (CS2) was selected because of its lignocellulolytic potential on a color-based agar plate screening system; pectinolytic and amylolytic capabilities were also assessed. The effects of different lignocellulosic substrates under submerged cultures of selected fungi on the enzymatic production of cellulases, xylanases, amylases, and laccases were evaluated. Seven different treatments, in which different combinations of wheat straw (WS), wheat bran (WB), orange peels (OP), grapefruit peels (GP), and apple peels (AP) were used, were established. Controls of 2% (w/v) carboxymethyl-cellulose (CMC) and xylan were used in a rich medium for basidiomycetes (RMB). The highest titers were achieved using OP-based cultures, with large titers of CMCases (33.5 U), avicelases (15.7 U), ß-D-glucosidase (72.9U), and xylanases (18.3 U). The best levels of amylase and laccase activity were obtained in the RMB plus CMC (RMB-CMC) (7.2 U) and in the medium OF/AP/GP (6.4 U), respectively. The relative molecular sizes of cellulase, xylanase, and amylase were 66.2, 56.5, and 90.8 kDa, respectively. Laccase and amylase had maximum activities at 60 °C whereas cellulase and hemicellulase had maximum activities at 70 °C. The optimal pH for cellulases, xylanases, and amylases was 5.0 in every case, and more than 95% activity was observed at pH 6. These results reveal some efficient operating parameters for the application of these enzymatic extracts as adjuvants to improve animal nutrition.

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Selection and Characterization of a Native Pycnoporus sanguineus Strain as a Lignocellulolytic Extract Producer from Submerged Cultures of Various Agroindustrial Wastes

Guadalupe Gutiérrez-Soto,^a,b Guadalupe E. Medina-González,^a Eduardo A. García-Zambrano^a, José E. Treviño-Ramírez,^a and Carlos E. Hernández-Luna ^b,*

Keywords: Amylases; Cellulases; Pycnoporus sanguineus CS2; Thermostability; Xylanases

Contact information: a: Universidad Autónoma de Nuevo León, Facultad de Agronomía, Campus de Ciencias Agropecuarias, UANL, Francisco Villa S/N, ExHacienda El Canadá, Gral. Escobedo, N.L. México. CP. 66455; b: Universidad Autónoma de Nuevo León, Facultad de Ciencias Biológicas, Departamento de Química, Laboratorio de Enzimología, Av. Pedro de Alba esq. Av. Manuel Barragán, Ciudad Universitaria, San Nicolás de los Garza, N. L. México, CP 66451;

* Corresponding author: carlosehlmx@yahoo.com

INTRODUCTION

Novel techniques for reutilizing waste byproducts from the food industry are rising in popularity along with the strengthening of the green movement around the world. The main objective of these bioprocesses is to obtain compounds with high industrial value such as enzymes, carbohydrates, amino acids, lipids, and many other biomolecules from organic sources (Castillo et al. 2009; Yang et al. 2011; Cunha et al. 2012). These biotechnological processes are sustainable because the enzymes and other compounds are produced cheaply due to the abundance of lignocellulose-rich wastes (Altaf et al.2010).

Organisms like white rot fungi (WRF) yield simple sugar molecules by degrading the main components of wood’s cell wall, including lignin, hemicellulose, and cellulose, depending on their extracellular enzymes (Henrissat et al. 1998; Steffen et al. 2007). Lignocellulolytic enzymes can be classified into two groups. The first group degrades cellulose and xylan and includes -glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91), endo-1,4- -glucanase (EC 3.2.1.4), and xylanases (EC 3.2.1.8). The second group includes the entire lignin oxidative complex, composed of laccase (EC 1.10.3.2), lignin peroxidase, LiP (EC 1.11.1.14), and manganese peroxidase, MnP (EC 1.11.1.13) (Rana 2011).

The potential of cellulases and other hydrolytic enzymes to convert cellulosic biomass has made the industrial application and design of new bioprocesses more popular in the last few years (Kuhad et al.2011). Most research has focused on either solid state fermentation (SSF) or submerged (SF) fermentation, both of which have exhibited good enzyme production (García and Torres 2003; Sinegani et al. 2005; Altaf et al. 2010; Philippoussis et al. 2011). Enzyme production under agitated or solid state fermentation depends on the characteristics of the substrates used. Best conditions involve a balance between the cellulose, xylan, and lignin contents. The characteristics of the media are translated to enzyme production depending on the organism under study.

Research on lignocellulolytic enzymes has demonstrated their potential in the food, animal feed, brewing and wine, agriculture, pulp and paper, textile, bioremediation, medical, and cosmetic industries (Elisashvili et al. 2008; Mendonça et al. 2010; Kuhad et al. 2011). Studies have shown that WRF can produce lignocellulolytic enzymes when grown in food industry by-products and other lignocellulose-rich substrates such as oak leaf litter, tea leaves, reed grass, bean stalks, wheat and pea straws, apple peels, and banana peels (Sinegani et al. 2005; Valášková and Baldrian 2006; Levin et al. 2007; Eliasashvili et al. 2008; Philippoussis et al. 2011).

The aim of the present work was to evaluate the effects of different agroindustrial residues during submerged fermentation of Pycnoporus sanguineus CS2 on the production of lignocellulolytic enzymes, including cellulases, xylanases, laccases, and amylases. Furthermore, some biochemical and operative properties, including the relative molecular weights, optimal pH, and optimal temperature were characterized.

EXPERIMENTAL

Materials

Chemicals and plant residues

All of the chemicals used were reactive grade purchased from Sigma-Aldrich (St. Louis, MO), Becton, Dickenson and Co. (Le Pont de Claix, France), or Bio-Rad (Hercules, CA). Wheat straw, oat flakes, orange peels, grapefruit peels, and apple peels were collected on the campus of Marin and La Ascensión of Facultad de Agronomía-UANL and from the metropolitan area surrounding Monterrey, N. L., Mexico. All residues were washed and chopped into small pieces about 1.0 cm in size.

Fungal strains and inocula preparation

Pycnoporus sanguineus CS2, P. sanguineus CS20, P. sanguineus CS43, and P. sanguineus CS90 strains were obtained from carpophores collected in oak forests and scrubland around Monterrey, N. L., Mexico from different ecotypes. All strains belong to the strain collection of vegetative mycelium of the Enzymology Laboratory of the Facultad de Ciencias Biológicas, UANL. Reference strains Phanerochaete chrysosporium ATCC 24785, Trametes hirsuta UAMH 8156, and Trametes versicolor UAMH 8272 were used for comparison. All strains were conserved with YMGA medium (10 g/L malt extract, 4 g/L glucose, 4 g/L yeast extract, and 15 g/L agar; Hernández-Luna et al. 2008), with periodic subcultures every three months. Inocula preparation consisted of 5 d of strain reactivation on YMGA medium. Agar cylinders (0.5-cm) cut from the periphery of the cultures were used to inoculate solid and liquid media.

Methods

Screening in solid medium assay

Identification of the main enzymatic activities involved in the modification of plant cell wall components was performed using the medium described by Sin et al. (2002). The medium was supplemented with 2% carboxymethyl (CM) cellulose (Sigma-Aldrich) to detect cellulases, with xylan (from birch wood, Sigma-Aldrich) to detect xylanases, with 0.5% pectin (from citrus peel, Sigma-Aldrich) to detect pectinases, 0.02% Poly R-478 dye for lignin modifying enzymes (LME), and 0.02% syringaldazine for laccases. Solid media were seeded with one 0.5-cm cylinder and incubated at 28 °C. Mycelium growth was recorded daily, Poly R-478 discoloration was measured for 30 d, and the syringaldazine reaction was registered until the fifth day of testing. To determine the hydrolytic activities, medium was revealed with iodine solution during the third day of growth (Sin et al. 2002). A numeric scale in which values from 0 to 3 were assigned was as follows: 3 corresponds to a reaction diameter greater than the mycelium growth, 2 corresponds to a reaction diameter similar to the growth diameter, 1 corresponds to a growth diameter larger than the reaction diameter, and 0 corresponds to no reaction or discoloration.

Enzyme production in submerged medium

The submerged fungal cultivation was carried out in a rotary shaker at 200 rpm and 28 °C in 500-mL flasks containing 200 mL of medium. As a positive control for cellulase and xylanase production, a rich medium for basidiomycetes (RMB) supplemented with CM-cellulose (RMB-CMC) and xylan (RMB-Xyl) at a concentration of 2% (w/v) was used. The average composition as reported by Pozdnyakova et al. (2006) was 10 g of glucose, 0.5 g of yeast extract, 10 g of peptone, 0.72 g of NH₄NO₃, 1.0 g of MgSO₄, 0.5g of KCl, and 1 mL of trace element solution (1 g/L FeSO₄, 2.8 g/L ZnSO₄, and 3.3 g/L CaCl₂) per 100 mL double-distilled water. Submerged fermentation of the residues was performed using 60-mM potassium phosphate buffer at pH 6. Concentrations and combinations of residues for each medium are shown in Table 1. All media were autoclaved twice for 60 min (121 °C, 15 psi) and allowed to cool to room temperature before each flask was inoculated with three 0.5-mm diameter agar plugs of mycelium taken from the periphery of a 5-d growth fungal colony in YMGA.

Table 1. Agroindustrial Residues Based Media: Abbreviations, Combinations, and Concentrations

Enzymatic assays

A 2-mL aliquot was taken from each flask every 2 d for enzymatic assays. Determinations of the cellulases, xylanases, amylases, laccases, lignin peroxidases (LiP), and manganese-dependent peroxidases (MnP) present in cultures were performed. Cellulase, xylanase, and amylase activities were assayed by measuring the concentration of reducing sugars released according to the method described by Miller (1959). Reaction mixtures consisted of 0.5 mL of a 50-mM sodium citrate buffer at pH 5.0, 0.3 mL of CM-cellulose, Avicel, Cellobiose, Starch or D-xylan as the substrate (1% final concentration) in 2 mL of sample. The reaction mixtures were incubated for 30 min at 50 °C. At the indicated times, samples of 0.1 mL of the reaction mixture were taken and boiled with 0.1 mL of dinitrosalicylic acid (DNS) for 5 min and submerged in an ice bath for 5 min. To perform photometric determination, 1 mL of double-distilled water was added, and samples were analyzed at 540 nm using a Shimadzu UV-Vis 1240 mini spectrophotometer (Japan). Standard curves for glucose, xylose, and maltose were used to determine the cellulase, xylanase, and amylase titers, respectively. All assays were performed in duplicate and enzyme units were expressed as the amount of enzyme that catalyze the liberation of 1 mol of reducing sugar per minute. Laccase determinations were done according to the method reported by Abadulla et al. (2000), using a reaction with 2,6-dimethoxyphenol (2,6-DMP) as the substrate in a 200-mM sodium acetate buffer at pH 4.5; spectrophotometer determinations were performed at 468 nm. These assays were also used to determine the optimal pH and temperature. Laccase units were expressed as the amount of enzyme that oxidize 1 mol of DMP per minute.

SDS-PAGE and zymograms

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a 10% polyacrylamide gel according to the method detailed by Laemmli (1970) and stained with Coomassie blue. Cellulase activity was detected following the protocol of Blanco et al. (1998). After electrophoresis, the gels were soaked in 2.5% (v/v) Triton X-100 for 30 min and washed twice in 50-mM phosphate citrate buffer, pH 5.0) for 30 min at room temperature. The gels were overlaid onto a 1-mm-thick 1% agarose gel containing 0.2% substrate (CMC, xylan, or starch) and incubated at 50 °C to allow the reaction to proceed. After incubation, substrate-agarose gels were stained, depending on the substrate, with 0.1% (w/v) Congo red or iodine solution for 30 min and 1 min, respectively, and washed with 1-M NaCl until bands became visible. Congo red-stained gels were immersed in 5% (w/w) acetic acid to develop a dark blue background.

RESULTS AND DISCUSSION

Pycnoporus sanguineus is a worldwide distributed basidiomycete known for its high lignolytic potential. This potential has been associated with production of high-redox potential laccases as well as low-molecular weight mediators (Eggert et al. 1996; 1997). Sigoillot et al. (2002) reported that the lignocellulolytic and hemicellulolytic activities of Pycnoporus cinnabarinus are thermostable, and the potential of this fungus for use in different industrial and biotechnological processes has been explored, which takes advantage of agroindustrial residues for enzyme or bioethanol production (Quiroz-Castañeda et al. 2011). The lignolytic, cellulolytic, and hemicellulolytic potentials on solid media, of four native strains isolated from different ecotypes of Northeast Mexico were evaluated in this study. Taking into account the versatility of these fungi as producers of hydrolytic enzymes, the pectinolytic and amylolytic capabilities were also assessed.

Screening in Solid Plate Assay

From a collection of 120 native strains from different Northeast Mexico ecotypes, the lignocellulolytic, pectinolytic, and amylolytic potentials of four Pycnoporus sanguineus isolates were explored. Among them, a color-based agar plate system was established and the enzymatic capabilities of these autochthonous strains were compared to those of three well-studied international reference strains, as exemplified in Fig. 1. In general (Table 2), the four Pycnoporus sanguineus strains were good cellulase producers and exhibited better production rates than the reference strains Phanerochaete chrysosporium ATCC 24785, Trametes hirsuta UAMH 8156, and Trametes versicolor UAMH 8272. Strains P. sanguineus CS2 and P. sanguineus CS43 exhibited higher hydrolytic activity over pectin and starch than the rest of the strains evaluated. All strains had lower capability for xylanase production, but P. sanguineus CS2, P. sanguineus CS43, and Trametes hirsuta UAMH 8156 strains were the best producers.

P. sanguineus CS2 and P. sanguineus CS43 had the highest lignolytic activities. Both strains had entirely discolored the growth area on Poly R-478 plates starting on the fifth day of growth. The rest of the strains evaluated had moderate activity. The same behavior was observed in the medium supplemented with syringaldazine, in which the positive reaction diameter was larger than the growth diameter. In a similar study with 37 thermophilic fungal strains, isolated from different lignin-rich ecotypes (Younes et al. 2011), it was observed that isolates with the highest lignolytic potential had low hydrolytic potential and that the strains with the best hydrolytic activities did not produce lignolytic enzymes. These findings highlight the limitations of studied isolates in the simultaneous degradation of plant cell wall components. On the other hand, filamentous fungi have been reported to be the best cellulase producers. Under similar conditions of hydrolase activity detection to those in this work, the best filamentous strains exhibit hydrolysis areas as large as the growth area (Khokhar et al.2012). The native basidiomycetes evaluated in the present study had hydrolysis areas twice as large as the growth area. In the case of pectinases, some studies of basidiomycetes (Pericin et al. 1992; Pericin et al.1997; Levin and Forchiassim 1998; Xavier-Santos et al. 2004) report P. sanguineus as the best polygalacturonase producer.

Fig.1. Color-Based System for Screening on Agar Plates. Figure shows a series of representative results for Pycnoporus sanguineus CS2, in comparison with Trametes hirsuta UAMH8156. Enzymatic potentials were estimated as described in methods.

Table 2. Lignocellulolytic Profiles of Pycnoporus sanguineus Native Strains and Reference Strains

3 = High activity, 2 = Mild activity, 1 = Weak activity, and 0 = No activity

LME, lignin modifying enzymes

Enzyme Production in Submerged Medium

Once the best lignocellulolytic enzyme producer, Pycnoporus sanguineus CS2 strain, for enzymatic production was selected, the submerged fermentation process was evaluated. A rich medium and agroindustrial residues based on a potassium phosphate buffer were used. Cellulase (CMCases, Avicelases, and β-D-glucosidase) as well as xylanase, amylase, and laccase production curves were determined. Under all tested conditions, β-D-glucosidase had the highest titers (43 to 73 U/flask) and amylase and laccase had lower titers. Amylase production ranged from 1 to 7 U/flask and laccase production ranged from 1 to 6 U/flask. The CMCases were more abundant than the Avicelases. This was not the case in WB and WB/AP/GP media in which CMCase titers were in the range of 20 to 30 U/flask. Avicelases exhibited a similar production pattern as CMCases with respect to the media used, but their titers were lower, ranging from 12 to 15 U/flask. All results are shown in Table 3. The highest enzymatic activities were observed when using WS/OP medium, except in the case of laccases. However, lignolytic activity could be inhibited by the polyphenols present in orange peels. This theory is supported by the results obtained under the same condition but when replacing orange peels with grapefruit peels, in which higher laccase titers (6.4 U/flask) were observed. Amylases were present at higher levels in RMB-CMC and RMB-Xyl. The highest titers for all enzymatic activities were detected in WB media.

Table 3. Lignocellulolytic Enzyme Production of Pycnoporus sanguineus CS2

* Liquid media on flask = 200 mL total volume

With regards to cellulase production via submerged fermentation, the greatest production was in WS/OP medium. In general, a variation of titers as a function of the culture media occurred. This was similar to the results obtained by Pukahuta et al. (2004) in their study of 19 strains of Lentinus polychrous cultivated in media supplemented with agroindustrial residues. They observed that, on different isolates of the same species, activity titers varied between 0.017 and 1.041 U/mL, and cellulases were not detected at all in five strains. These values are low compared to those reported by Elisashvili et al. (2009), a study in which Pseudotremella gibbosa IBB22 strain was selected as the best producer with 122 U/mL. In the case of the Trametes strains assayed, varied titers from 2 to 74 U/mL were observed. For Pycnoporus genus, it has been reported that cellulase production reached 20 U/mL from a P. coccineus IBB310 strain via the submerged fermentation of tangerine peels (Elisashvili et al. 2011). For P. sanguineus PF-2, 0.06 U/mL production via the submerged fermentation of milled corn cobs was reported (Falkoski et al. 2012). These results are very close to those of the present work.

Xylanases were produced at contents ranging from 4 to 18 U/flask, titers similar to those of exocellulases and lower than those of endocellulases. Production variability was once again a function of the media used. Even though these titers are lower than those reported for P. coccineus (17 to 32 U/mL), a similar behavior was observed using different residues in solid-state fermentation. In this case, banana peels were better natural inductors than leaf litter, wheat straw, or apple peels (Elisashvili et al. 2008). For P. sanguineus PF-2 under submerged fermentation in media supplemented with milled corncobs, the level detected was 10 U/mL (Falkoski et al. 2012), much higher than that in the present study for P. sanguineus CS2. The difference in these values may be due to media composition, culture conditions, or the origin of the strains.

With regard to ß-glucosidase production, both P. sanguineus PF-2 and P. sanguineus CS2 strains showed similar production levels at 0.20 and 0.36 U/mL, respectively.

In the present work, the amylase production curves of P. sanguineus CS2 were obtained under different culture conditions, where the greatest titles were detected in RMB-CMC and RMB-Xyl media (0.035 and 0.032 U/mL, respectively). Regarding amylase production via submerged fermentation, P. sanguineus IBAMA-Brazil has been examined using starch or wheat bran as the carbon source. This is the first use of this fungus as a new amylase source reported by Siqueira et al.(1997). The greatest production of this strain was on the fourth day of culture, whereas for P. sanguineus CS2, the greatest production occurred on the fifth day.

Regarding laccase production from P. sanguineus CS2, maximum production of 7.5 U/mL in a Kirk medium modified with 350-μM cupric sulphate and 3% ethanol as inducers was reported (Salcedo-Martínez et al. 2013). Under the culture conditions used in this study, laccase titers were notably lower. This could be due to the presence of pigments and phenolic compounds in the lignin rich-substrates used, which could inhibit laccase activity while stimulating cellulase, hemicellulose, and pectinase activities (Elisashvili et al. 2009).

Molecular Weights, Optimal pH, and Temperature Estimation

The relative molecular sizes, optimal pH, and temperature for each enzymatic activity are shown in Table 4. Only one band of protein with laccase activity was detected on zymograms and SDS-PAGE gels copolymerized with CMC and xylan. For this band, a relative molecular size of 61 kDa was estimated. Amylase activity was associated with an activity band with a relative size of 91 kDa. Two protein bands with sizes of 66.2 and 56.5 kDa were detected for CMCase and xylanase activities, respectively. In the case of CMCase, the estimated weight for P. sanguineus CS2 was heavier than reported for Pycnoporus sanguineus CEIBMD01 (25 and 50 kD). However, native xylanase was smaller than reported for this strain with values between 80 and 90 kDa (Quiroz-Castañeda et al.2009). With regard to size estimation for amylases, no reports of the molecular weight of P. sanguineus were found. Nevertheless, the enzyme from the strain used in the present work (90.8 kDa) was larger than those of reports for bacteria (20 to 30 kDa), fungi, and yeast (40 to 68 kDa) (Gupta et al. 2003).

Laccase activity was detected by component analysis with a relative molecular weight of 61 kDa, close to that reported for other laccases produced by other Pycnoporus sanguineus strains (Garcia et al.2006; Acosta-Urdapilleta et al. 2010). Even though this is smaller than reported for this strain in another study (Salcedo-Martínez 2013), this may be due to adaptations of the techniques for the other enzymes of this work. All molecular weights reported here are relative.

The optimal pH for laccase production over 2,6-DMP was 3.5. The CMCases, xylanases, and amylases had an optimal pH of 5.0 with more than 95% of their activity conserved at pH 6.0. These results were similar to those reported for other P. sanguineus enzymes (Quiroz-Castañeda et al. 2009; Lomascolo et al. 2011).

The optimal temperature for laccase and amylase was 60 °C, whereas CMCase and xylanase had an optimal activity at 70 °C. In general, hydrolytic enzymes preserve more than 95% of their activity at 90 ºC (100% for cellulases, 99% for xylanases, and 98% amylases, as shown in Fig. 2). The optimal activity for CMCases and xylanases was higher than previously reported for other strains (Falkoski et al. 2012). The apparent increase in activity at 90 °C may be due to the increased physical accessibility of the remaining active enzymes in between branches of the polysaccharide structure that were somehow separated from one another at the elevated temperature. This phenomenon can be explained as a combination of two important factors, substrate solubility and enzyme thermostability.

Table 4. Molecular Weights, pH, and Temperature of P. sanguineus CS2 Extracellular Enzymes

* Detected with 2, 6-DMP (2-mM)

Fig. 2. Effects of temperature on enzyme activity. All assays were performed in triplicate and determined using the DNS method. Results showed a relative standard deviation lower than 5%.

All results allowed the strain P. sanguineus CS2 to be selected for the development of new adjuvants to improve animal nutrition for both ruminants and monogastric animals (Ribeiro et al. 2012). This can be further improved if it is considered that all degrading activities of the plant cell wall components are produced by one organism and, thanks to its plasticity, any enzyme production can be intensified by taking advantage of the agroindustrial residues generated in the region. This possibility will be explored in further studies.

CONCLUSIONS

Pycnoporus sanguineus CS2 is a thermostable cellulase, xylanase, and amylase producer that retains greater than 90% of its activity at 90 °C.
Because of its high lignocellulolytic, amylolytic, and pectinolytic potential, Pycnoporus sanguineus CS2 could be used for digestive adjuvant development in animal nutrition.
As a result of its metabolic plasticity and depending on the culture medium, it is possible to obtain high concentrations of all enzymes studied or to increase the production of specific enzymes. This would lend itself well to a broad range of applications in various industrial processes.

ACKNOWLEDGMENTS

The authors are grateful for the financial support provided by the Programa del Mejoramiento del Profesorado (PROMEP 103.5/12/7884) and the Programa de Apoyo a la Investigación Cientifica y Tecnológica de la UANL (CA953-11). We also thank PhD. Anadulce Hernández Herrera for her critical revision and comments on the manuscript.

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Article submitted: December 22, 2014; Peer review completed: February 24, 2015; Revisions received and accepted: April 16, 2015; Published: April 27, 2015.

DOI: 10.15376/biores.10.2.3564-3576