at lower temperature than cellulases from Trichoderma reesei does?
Hernández C1, García G1, Vázquez-Marrufo G2, Alarcón E1*. 1
Universidad Veracruzana. Instituto de Biotecnología y Ecología Aplicada (INBIOTECA), Av. de las culturas veracruzanas #101 col. Emiliano Zapata CP. 91090, Xalapa Veracruz, México.
2
Universidad Michoacana de San Nicolás de Hidalgo. Facultad de Veterinaria, Centro Multidisciplinario de Estudios en Biotecnología, Morelia, Michoacán.
*Correspondence author [email protected]
Abstract
The use of bioprospections for isolate and discover novel strains capables to produce cellulases with specific characteristics is very heplful. In this work, we aimed to isolate different fungal strains in a altitudinal gradient in the Pico de Orizaba volcano (The mexican highest mountain), searching for mesophilic cellulase-producing fungi, and we compared their yields agains cellulases from Trichoderma reesei (a tropical fungi) in a temperature gradient. From the isolated strains, on CMC-Czapek Dox Agar, we selected six strains to produce cellulases in liquid media with different carbon sources. We found that lactose was the best carbon source for cellulase production (endoglucanase and β-glucosidase) for all strains. The cellulase activities of the concentrated protein were evaluated in a temperature gradient from 30 to 50 °C, using cellulases from T. reesei as control. Results indicate that fungal strains from Pico de Orizaba produce mainly mesophilic cellulases, projecting a strain belonging to the family Pleosporaceae (optimal reaction temperature of 30 and 33°C). This could be understood as an adaptation to cold- climate of the mountain.
Introduction
Cellulases are important industrial enzymes produced mainly from Ascomycota. Nowdays they are used as detergent additives, for improving the production process of beer and wine, for production of purees, for biostoning of jeans, among others (Kuhad et al., 2011). They have the potential for being used for wood-biopulping, and in large-scale production of second generation bioethanol. Cellulase is a group of hydrolytic enzymes with different molecular weight and, generally, one of the following activities: β- 1,4-endoglucanase (E.C.3.2.1.4), exoglucanase (cellobiohydrolase; E.C.3.2.1.176) or β-glucosidase (cellobiase; E.C. 3.2.1.21).
Actually, Trichoderma reesei is the principal source of industrial cellulases, mainly because produce a large amount of enzymes, its plasticity and its excellent production of exoglucanses (Esterbauer et al., 1991). Trichoderma’s cellulases (like any enzymes) have specific requirements for catalysis, pH (4.8)
and temperature (50 °C) are some important ones (Ghose, 1987; Ballesteros et al., 2004). Also, they trend to have low β-glucosidase activity, that is why the mixes with exogenous β-glucosidase are common for improve their hydrolysis potential (Fortes et al., 2010).
These characteristics are important in industry, because limits the conditions in where they can be used. In order to diversify the field of applications of cellulases, many efforts have been made to modify their structure, by mutagenesis or protein engeenering; enhancing their thermostability, thermophilicity, alkalophilicity (St-Pierre et al., 2012) or the production of β-glucosidase (Jung et al., 2012). However, despite of the advances in this field, other organisms would exist that produce cellulases with different catalystic requirements that those produced with Trichoderma reesei, and that could be used for specific industrial processes. Psycrophilic cellulases were produced from bacterial strains from Indian soils (Venkatachalam et al., 2014) and mesophilic cellulases from bacterial strains from deep Atlantic Ocean (Odisi et al., 2012). Likewise, alkaline cellulases were produced from Aspergillus sp and Penicillium sp strains from soil of the rainforest of Perú (Indira et al., 2014), and cellulases that endure halophytes conditions were produced from bacterial strains of Odisha region, in India (Vega et al., 2012).
Thermophilic cellulases have attracted the attention of many studies, because their high yields under extreme operational conditions (Acharya and Chaudhary, 2012). However, for some processes, like simultaneous fermentation and saccharification of biomass, the use of cellulases with high yields at mesophilic temperatures (30 °C) could be preferable. This because Saccharomyces cerevisiae can not survive at thermophilic conditions required for enzymatic hydrolysis of T. reesei cellulases (50 °C). The use of bioprospections for isolate and discover novel strains capables to produce cellulases with specific characteristics could be very heplful. In this work, we aimed to isolate different fungal strains in a altitudinal gradient in the Pico de Orizaba volcano (mexican highest mountain), searching for mesophilic cellulase-producing fungi, and we compared their yield agains cellulases from T. reesei (a
tropical fungi) in a temperature gradient. This in the basis that in Pico de Orizaba prevail cold temperatures, and fungi that habit their ecosystems could have some adaptations at enzymatic level.
Materials and Methods
Sampling method and fungi isolation
Soil samples of nororiental face of Pico de Orizaba volcano (between Puebla and Veracruz states, México) were taken in four different altitudinal points: pine-oak forest (3200 msnm), abies forest (3500 msnm), arboreal limit (4000 msnm) and paramo (4300 msnm) (Figure 1.)
At each point, a transect of 50 m was charted and at each 10 m, the litter (400 cm2) was collected in plastic bags. The litter samples (20 in total) were placed into a cooler and transported to laboratory. We chose sampling litter because it is the principal habitat of cellulose-degrading fungi (Voříšková and Baldrian, 2013). Sampling was done at the end of winter, 2015.
For fungi isolation, litter fragments were placed in Czapek-Dox Agar with carboxy-methyl cellulose as unique carbon source, and 50 mg.L-1 of tetraciclin to avoid bacterial growth. Fungal morphospecies were sequencially isolated until obtain pure strains. From the strains isolated, the six that had faster growth were selected to produce cellulases in liquid cultures. The strains were named as follows: POF1 (Pine oak forest 1), AF1 (Abies forest 1), AF2 (Abies forest 2), AL1 (Arboreal limit 1), P1 (Paramo 1) and P2 (Paramo 2).
Figure 1. Distribution of the sampling sites in Pico de Orizaba volcano. Sampling sites cover an antitudinal gradient from 3200 (a), 3500 (b), 4000 (c) to 4300 (d). Image from Google Maps server®.
Cellulase production in liquid cultures
For cellulase production assessment, strains were inoculated in 150 mL of Mandels and Weber medium (1969) with the following composition per litre: yeast extract 1 g, peptone 4 g, KH2PO4 4 g, (NH4)2SO4 2.8 g, MgSO4 x 7 H2O 0.6 g, CaCl2 x 2 H2O 0.8 g. The medium was enriched with trace elements (mg.L- 1
): FeSO4 5.0 mg, ZnSO4 x 7H2O 1.4 mg, CoCl2 2.0 mg.
Because the cellulase producing fungi corresponded to new isolations, a monofactorial desing using three different carbon sources was used to induce cellulase production: lactose, carboxy methyl cellulose and sugarcane bagasse; all the carbon sources were added to culture medium at 2%. All treatments were done in triplicate; thus, a total of 54 experimental inits (Crystal flask, 250 ml) with 150 mL of culture medium were prepared, inoculated and incubated in a environmental chamber Bunker GmbH at 28° C, without agitation thought 11 days.
Samples to measure cellulase activities: endoglucanase and β-glucosidase, were taken each 48 hours. Extracellular protein amount was quantificated in order to determine the specific activities of cellulases.
Effect of temperature on cellulase activity
At the end of incubation time, the culture medium was filtered using a kitasato matrass and a vaccum pump thought Whatmann paper. After that, the crude extract was dialysated using a tubular-cellulose membrane, and poly-ethylen glycol as dehydrating agent by 18 h at 4 °C. The concentrated protein was used to determine the cellulase activities in a temperature gradient.
Endoglucanase and β-glucosidase specific activities were evaluated at mesophilic temperatures: 30, 33, 36 and 39 °C, and the termophilic temperature of 50 °C. As control, comercial cellulases of Trichoderma reesei (Sigma, USA) were evaluated in the same temperature gradient. The strains that produced cellulases that hydrolyze at mesophilic temperatures were molecular identified.
Fungal identification
Three strains were identified by amplification and analysis of the ITS rDNA region (Schoch et al., 2012). 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 and conditions described by White et al., (1990). Amplicons were sequenced at Elimbiopharm (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 more robust the 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 (MO) trees were also generated using same genetic distances. All these steps for phylogenetic analysis were conducted in Mega 6 (Tamura et al., 2013).
Analytical methods
Relative activity of 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 (Eivazi and Tabatabai, 1988). The activity was reported as U•ml-1. Relative activity of endoglucanase was determined according to Ghose (1969), quantifying the reducing sugars released from carboxymethyl cellulose by the method of dinitrosalicylic acid (DNS; Miller, 1954). 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).
Extracellular protein amount was calculated by the colorimetric method of coomassie blue at 595 nm according to Bradford (1976). The values of relative activities of cellulases and the protein amount were used to calculate the specific activities of endoglucanase and β-glucosidase (U.mg protein-1
).
Results
Effect of carbon source in cellulase production
The use of different carbon sources to induce cellulase production affects the production of endoglucanase and β-glucosidase (Figures 2 and 3). The strains AL1, P1 and POF1 produced the highest amount of β-glucosidase when lactose (AL1 = 36.67 and POF1 = 4.52 after 11 days of culture, and P1 = 22.66 ± 2.42 U.mg protein -1 after 9 days of culture) or CMC (AL1 = 28.90 and P1 = 31.42 after 11 days of culture, and POF1 = 43.65 ± 6.37 U.mg protein -1 after 9 days of culture) were used as carbon source; meanwhile, when SCB was used the activities decreased (AL1 = 18.94; POF1 = 5.32 and P1 =19.36 U.mg protein -1 after 11 days of culture).
On the other hand, lactose was the prefer carbon source for the production of endoglucanase (Figure 3). And in the same way that β-glucosidase, the strains P1 (after 9 days of culture) and POF1 (after 4 days of culture) produced the highest amount of endoglucanase (30.41 ± 9.22; 38.60 ± 10.28 U.mg protein -1, respectively). At the end of the incubation time, the enzymes of all strains, produced with lactose, were concentrated and the effect of temperature on its activities was evaluated.
Figure 2. β-glucosidase activity in cultures of fungi isolated from Pico de Orizaba volcano, using three different carbon sources: a) lactose, b) carboxymethyl cellulose c) sugarcane bagasse. Middle points indicate the mean and bars the standard error, n = 3.
Figure 3. Endoglucanase activity in cultures of fungi isolated from Pico de Orizaba volcano, using three different carbon sources: a) lactose, b) carboxymethyl cellulose c) sugarcane bagasse. Middle points indicate the mean and bars the standard error, n = 3.
Figure 4. Cellulases activities of concentrated proteins of the different fungi isolated from Pico de Orizaba volcano in a temperatura gradient. Fit = weighted least square.
Effect of temperature on cellulase activities
Cellulases produced in liquid media with fungi from cold ecosystems of Pico de Orizaba volcano showed different optimal reaction temperature (ORT) (Figure 4). Specific endoglucanase activities of the strains isolated from pine-oak forest and Abies forest (POF1, AF1 and AF2) showed highest activities between 33 to 39 °C, decreasing at 50 °C. The strain AL1, isolated at 4000 m over sea level in the arboreal limit, showed its highest activity at 33 °C (452.10 U.mg protein-1), and decreased when temperature increased. Contrary, endoglucanase from T. reesei showed its highest endoglucanase activity at 50 °C (233.98 U.mg protein-1), and decreased when temperature decreased, until 35.35 U.mg protein-1 at 30 °C.
Specific β-glucosidase activities of AL1, and AF2 showed its highest activities at 30 and 33 °C, respectively. Thus, the enzymes produced by those strains could be considered as mesophilic β- glucosidases. Contrary, β-glucosidases of POF1, AF1 and P1 had high activity at 39 °C, and P2 at 50°C (Figure 4).
Our results inducate that cellulases produced with different fungal strains from forestal areas of Pico de Orizaba, hydrolyze at temperatures from 30 to 39 °C, better than at 50 °C. Strains from Paramo produced cellulases that hydrolyze better at 39 to 50 °C, in the same way that cellulases from T. reesei.
Phylogenetic analysis of the fungal isolates
We selected AL1 as a strain with potential to produce mesophilic cellulases, but the strains POF1 and P1, which had high cellulase production were also identified. Blastn search of amplicons obtained from fungal isolates P1 and POF1 show maximal identity with sequences of Trichoderma harzianum complex, whereas isolates AL1 shows maximal identity with Pleosporaceae species (genus Alternaria and
Ulocladium). Phylogenetic reconstruction of the ITS region from strains P1 and POF1 groups with its corresponding sequences T. harzianum complex with a bootstrap value of 100%, clearly separated from all other species within the genus (Figure 5). Strain AL1 grouped with genus Alternaria and Ulocladium, both genus are polyphyletic and paraphyletic related taxa of the Pleosporaceae family, and phylogenetic between those two genus are commonly not-resolved (Xue and Zhang, 2007; Runa et al., 2009) (Figure 5).
Figure 5. Phylogenetic reconstruction (Maximum parsimony) of the three cellulase most-producing fungi, isolated from Pico de Orizaba cold ecosystems.
Discussion
Bioprospection, is a key way to find new organisms with potential biotechnological uses. In this work, we aimed to find evidence that litter fungi of cold mountain ecosystems have cellulase adapted to hydrolyze at lower temperatures (mesophilic cellulases) than cellulases of the tropical fungi T. reesei
(thermophilic cellulases: 50 °C). Several studies reported that themophilic bacteria produce thermotolerant cellulases, which can be used for many industrial processes (Arora et al., 2015; Norashirene et al., 2014); however, mesophilic cellulases have recibed less attention in the bioprospection field, in spite of they could be used for simultaneous saccharification and fermentation processes, insted of cellulases of T. reesei or themotolerant yeast (Kádár et al., 2004).
In order to evaluate the production of cellulase from the fungal strains isolated on CMC-Czapek Dox agar, we performed liquid media with three different carbon sources, i.e. lactose, CMC and SCB, and spite of all carbon sources induced cellulase production, the best results were obtained with lactose. Lactose is considered a good inducer or cellulases, even if the mechanism of induction is not complety understood (Karaffa et al., 2006; Porciuncula et al., 2013); our results point that lactose and Mendel- Weber medium, could be useful for the production of cellulases with novel fungal strains.
Of the isolated strains, all produced endoglucanases with mesophilic ORT (33-39 °C) with exception of P1 wich had an optimal reaction temperature (ORT) of 50 °C (thermophilic). The same was observed for β-glucosidase, all strains produced mesophilic β-glucosidases with exception of P2. We consider this as a evolutival characteristic of the fungi that lives in cold ecosystems, in the same way that tropical or thermophilic organisms have their cellulases adapted to catalyze at high temperatures.
Strains P1 and P2, were isolated at the highest sampling point (4300 m above sea level; Paramo), the ITS phylogeny revealed that belong to Trichoderma harzianum (P1) and Phoma herbarum (P2, tree don’t shown). T. harzianum are known to produce cellulase with ORT between 37 to 40 °C (Thrane et al., 1997); meanwhile, P. herbarum, a pathogenous fungi of plants and salmonids, have attracted the attention for its capacity to produce xylanases/cellulases, among other chemicals (Alvarez-Navarrete et al., 2015). However, few is known about the characteristics of their cellulases. In this work we found that P. herbarum could be a source of thermophilic (50 °C) β-glucosidases, however its production was very low.
Themophilic cellulases are characterized by hydrophobic protein core and polarity at the surface; compact structure with lesser internal voids; higher content of proline and leseer content of asparagines, glutamine, methionine and cysteine; increased level of H-bonding, isoelectric points and salt bridges (Arora et al., 2015; Taylor and Vaisman, 2010; Li et al., 2011). On the other hand, exist cold-active cellulases, produced by psychrophilic or psychrotolerant organisms (e.g. from polar regions or marine sediments). Those cellulases have been propossed for the production of algae-derivated bioethanol
(Wang et al., 2015); and are considered important for specific industrial uses (Gerday et al., 2000). Cold-active cellulases from Pseudoalteromonas sp. (isolated from Antartic region) have their ORT at 30 °C (Wang et al., 2015), as well as the cellulases produced with our strain AL1 who belong to Pleosporaceae family (isolated in the arboreal limit, at 4000 m above sea level). The optimization to low temperature is reached via destabilization of the structures bearing the active site, this improves the dynamic of the active site in the cold (Ferrer, 2013). Phylogeny based on ITS sequence did not clarify the identity at specie level of the strain AL1; however, pointed that the strain belong to genus Alternaria or Ulocladium (family Pleosporaceae). Some authors have reported that there is not a clear difference between those related taxa, and in spite of the efforts, the phylogeny still unresolved (Xue and Zhang, 2007; Runa et al., 2009).
Both, Alternaria and Ulocladium species are commonly leaf phytopathogens and saprotrophic, and their production of cellulases have been proved. It has been reported that the temperature of incubation for
Alternaria species (A. alternata and A. citri), affects their cellulase production, and that at 30 °C the production of cellulases was optimal (El-Said et al., 2014). Here, we observed that AL1 has its ORT at these temperature; however, other studies from Pakistan region (where predominates warm climate), indicates that cellulase from Alternaria sp have an ORT of 45 °C for β-glucosidase and 50 °C for
endoglucanase (Sohail et al., 2011).
On the other hand, Ulocladium is a potent mycotoxin-free cellulase-producer (Pedersen et al., 2009), but reports pointed that its cellulases (U. botrytis, endoglucanases) have its maximal activity at 60 °C (Abo- Elmagd and Housseiny, 2012), contrary to our AL1 cellulases. These previous data, could suggest that AL1 strain of Pico de Orizaba volcano, belonging to Pleosporaceae family, have developed a cellulase adapted to function at lower temperatures than other strains of Alternaria or Ulocladium, and that its a potential source of mesophilic cellulases. However, structural studies of their cellulases are required to proved this hypothesis.
Further studies using the cellulases obtained from AL1 for simultaneous fermentation and saccharification, or other biotechnological processes at mesophilic temperatures, shed light on the suitability of their use compared with cellulases from other more-used fungi, like T. reesei or A. niger.
Conclusion
Fungi isolated from Pico de Orizaba volcano produce mainly mesophilic cellulases (endoglucanases and β-glucosidases), with exception to those belonging to Trichoderma or Phoma genus. This could be understood as an adaptation to cold-climate of the mountain.
Among all strains, AL1, who belong to Pleosporaceae family, had high cellulase production when lactose was used as carbon source. Also, its cellulases have an ORT between 30 and 33 °C, which makes them
good candidates for biotechnological uses, developed at mesophilic temperatures.
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