Bioprospection and characterization of the amylolytic activity by filamentous fungi from Brazilian Atlantic Forest

Filamentous fungi are widely diverse and ubiquitous organisms. Such biodiversity is barely known, making room for a great potential still to be discovered, especially in tropical environments which are favorable to growth and species variety. Filamentous fungi are extensively applied to the production of industrial enzymes, such as the amylases. This class of enzymes acts in the hydrolysis of starch to glucose or maltooligosaccharides. In this work twenty-five filamentous fungi were isolated from samples of decomposing material collected in the Brazilian Atlantic Forest. The two best amylase producers were identified as Aspergillus brasiliensis and Rhizopus oryzae. Both are mesophilic, they grow well in organic nitrogen-rich media produce great amounts of glucoamylases. The enzymes of A. brasiliensis and R. oryzae are different, possibly because of their phylogenetical distance. The best amylase production of A. brasiliensis occurred during 120 hours with initial pH of 7.5; it had a better activity in the pH range of 3.5-5.0 and at 60-75°C. Both fungal glucoamylase had wide pH stability (3-8) and were activated by Mn2+. R. oryzae best production occurred in 96 hours and at pH 6.5. Its amylases had a greater activity in the pH range of 4.0-5.5 and temperature at 50-65oC. The most significant difference between the enzymes produced by both fungi is the resistance to thermal denaturation: A. brasiliensis glucoamylase had a T50 of 60 minutes at 70oC. The R. oryzae glucoamylase only had a residual activity when incubated at 50°C with a 12 min T50.


Introduction
Fungi are widely diverse and distributed in all terrestrial ecosystems.They are decomposers, mutualists or pathogens with crucial roles in the cycling of nutrients (Tedersoo et al. 2014).According to Blackwell (2011), it is estimated that there are about 5.1 million species of fungi around the world.Up to the present date, the dictionary of fungi counts fewer than 100,000 species (Kirk et al. 2008).
Fungus diversity is spread along a latitudinal gradient.Therefore, equatorial and tropical forests, such as the Brazilian Atlantic forest, hold a great part of this diversity (Shi et al. 2014;Tedersoo et al. 2014).This forest has one of the most diverse and threatened biotas of the world, which remains with just 11.4% to 16% of the original coverage (Ribeiro et al. 2009;Joly et al. 2014).
Filamentous fungi are known as great enzyme producers, like amylases.Starch is the main reserve carbohydrate in plants and the second most abundant carbohydrate in nature.It is present in corn, potato, rice and wheat, what accounts for a great part of the human diet.Starch is enzymatically hydrolyzed to maltose, glucose and oligosaccharide syrup (Vielle & Zeikus 2001).Amylases are widely applied in industries such as textile, paper and cellulose, detergent, baking and beverage.They account for about 30% of total enzymes commercialized (Vielle & Zeikus 2001;Van Der Maarel 2002;Souza & Magalhães, 2010).Enzymatic hydrolysis is more specific than is chemical hydrolysis and has a theoretical efficiency of 100% (Ballesteros et al. 2002).
In this research, 25 filamentous fungi present in decaying material from the Brazilian Atlantic forest were isolated.The two strains with bigger secretion of enzymes had the production optimized and the amylases of crude extract were characterized.They showed characteristics with possible industrial application.

Isolation of fungi
The fungi were isolated from decaying materials collected in the Atlantic forest in Peruíbe, SP, Brazil (Table 1).The samples were stored in sterile tubes, diluted in the ration of 1:1000 or 1:10000 and plated in oatmeal agar (2% oatmeal flour, 2% bacteriological agar, 0.5% glucose) containing traces of pentabiotic (benzathine benzylpenicillin, procain benzylpenicillin, potassium benzylpenicillin, dihydrostreptomycin sulfate, streptomycin sulfate).The Petri plates were incubated at 30°C for seven days.The strains grown were purified through successive streaking.

Selection of strains
The isolated strains were grown in conical tubes containing oatmeal medium.An aqueous suspension of conidia was obtained by scraping the surface of each one of these cultures with 10 mL distilled water.A volume of 1 mL of the conidia suspension was inoculated in 25 mL Adams modified medium (AM) as described by Peixoto et al. (2003) during 72 hours at 30°C.Then, the cultures were vacuum-filtered and dialyzed overnight in distilled water at 4°C for removal of residual reducing sugars.After this process, the amylolytic activity was measured.

Enzymatic assay
Amylolytic activity was determined with 3,5-Dinitrosalicylic acid (DNS) (Miller 1959).The assay was composed of 50 µL crude extract and 50 µL 1% soluble starch in 50 mM sodium citrate buffer, pH 5.5.The mixture was incubated at 60°C for 5 min, interrupted by the addition of 100 µL DNS reagent, and boiled for 5 minutes.After cooling, 1 mL distilled water was added and the assay was read at 540 nm in SpectraMax Plus 384 Microplate Reader.The blank was consisted the enzyme inactivated by DNS prior to the addition of the substrate.One unit of enzyme activity was defined as that catalyzing the conversion of 1 µmol glucose, per minute, in the assay conditions.

Optimization of culture
The cultures were carried out in 125 mL Erlenmeyer flasks containing 25 mL of one of the following liquid media: AM (Adams 1990, Peixoto et al. 2003); Khanna (Khanna et al. 1995), Segato Rizzatti (SR, Rizzatti et al. 2001) or Vogel (Vogel 1964).The pH of the medium was adjusted to 6.5 and it was supplemented with 1% soluble starch, as carbon source.A volume of 10 5 spores per mL of culture medium was inoculated and the incubation occurred in bacteriological incubator (static condition) or shaker (100 rpm) for 3 days, at 30ºC.
In order to determine the best temperature for fungal growth, cultures were held at 25, 30, 35 and 40ºC during 72 hours in the culture medium previously selected (AM and SR).In order to determine the effect of the initial pH in the enzymatic production, a pH range of 6.5 to 8.5 was tested for Aspergillus brasiliensis and 5.5 to 7.5 for Rhizopus oryzae.Time course was performed up to 144 hours to select the time of higher enzymatic secretion.

Characterization of the crude extract
In order to determine the pH stability, the crude extract was incubated in McIlvaine buffer between 2.5 and 8.0 during 24 hours at 25°C (McIlvaine 1921).The resistance to thermal denaturation was determined at 50, 60 and 70 °C during 10 to 300 minutes.
In order to estimate the most favorable temperature zone and pH for enzymatic activity, an experimental design 2 2 was proceeded with three central point repetitions at p<0.5%.The results were analyzed with the Statistica 12 software.The points were composed of the variation of pH in sodium citrate buffer 50 mM, range 3.5 to 7.5, and the temperature in a range of 40 to 70°C.
Several carbon sources (starch, wheat bran, glucose, maltose, barley bagasse, ground corn and oatmeal flour), at 1% concentration, were added to culture media of both fungi aiming to determine the type of amylase synthetized in optimal conditions of the microorganism development.After incubation, the samples were collected, dialyzed and incubated with 1% starch in sodium citrate buffer, for 20 minutes.The enzymatic assays were stopped by boiling.The end-products of the enzymatic assays were applied in a Thin-Layer Chromatography (TLC) and a solution of 1 mg/mL glucose, maltose, maltotriose, maltotetraose, and maltopentose was carried as control.The chromatography was developed in a solution of n-butanol, ethanol and distilled water (5:3:2).After the plates were developed and dried, they were sprayed with a solution containing 18 mL methanol, 2 mL sulfuric acid, and 0.04 g orcinol, and it was revealed at 100°C until the spots came clear.
The following compounds were added in the enzymatic assays at concentrations of 2 and 5 mM to determine the effect on the activity:

Phylogenetic analysis
The Phylogenetic analysis with Rhizopus oryzae and Aspergillus brasiliensis was based on the regions of Internal Transcribed Spacer (ITS) 1 and 4. A multiple alignment of sequences was obtained with the MUSCLE 3.6 software (Edgar 2004).Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 6 (Tamura et al. 2013).The phylogenetic trees were based on the Maximum Likehood using the Neighbor-Joining method with 500 robustness non-parametric bootstrap replicates.The similarity rate between the sequences was shown in percentage considering the number of conserved bases and the total number of aligned bases.

Selection and identification
Twenty-five strains of filamentous fungi were isolated from samples of organic material in decomposition (Table 1).The strains were cultivated for 3 days in AM liquid medium.The amylolytic activities of the crude extracts were measured and are expressed on Table 1.Two strains stood out: the fungus 6B (3.5 U/mL) and the fungus 8A (4.2 U/mL).
The regions ITS 1-4 were amplified and sequenced for the identification of fungi (Table 2).The fungus 8A was identified as Rhizopus oryzae Went & Prinsen Geerligs.The fungus 6B showed a great similarity to the fungus Aspergillus brasiliensis Varga, Frisvad & Samson and Aspergillus niger van Tiegh.Hence, the amplification of the fragment of β-tubulin and calmodulin genes was performed, concluding the identification as Aspergillus brasiliensis.

Optimization of culture
The fungi selected were cultured in Khanna, AM, SR and Vogel media for 3 days, at 30°C, either in stirring at 100 rpm or in static condition, with initial pH adjusted to 6.5.The inoculum was composed of 10 5 spores per mL of culture medium. A. brasiliensis had a greater secretion in the SR medium under static fermentation, which saves more energy in comparison with the agitation condition (Figure 1).The SR medium contains yeast extract (0.45%) as the main source of nitrogen and it was the only medium tested with peptone (0.02%).The salt compositions of the culture medium are monobasic potassium phosphate in low concentration (0.015%), 0.012% magnesium sulfate heptahydrate and 0.05% monobasic ammonium phosphate.The latter can be also considered as a source of inorganic nitrogen.
R. oryzae had the best secretion of amylases in AM medium under agitation.AM medium has the biggest amount of yeast extract between the media tested (0.8%).In the Vogel medium, its activity was very low, suggesting a low assimilation of inorganic nitrogen.Besides carbon and nitrogen sources, the AM medium is composed by only two salts: monobasic potassium phosphate (0.3%) and magnesium sulfate heptahydrate (0.05%), both in a much higher concentration than the SR medium.
The fungi were grown in several temperatures in the previously standardized medium conditions for 3 days.The extracellular amylolytic activity was greater at 30°C for both fungi, suggesting that both are to be considered as mesophilic (Figure 2A).
In order to determine the ideal initial pH, the culture media were adjusted in the range 6.5 -8.5 to the fungus A. brasiliensis, and in the range of 5.5 to 7.5 for R. oryzae.The initial pH of 7.5 favored the production of amylases by A. brasiliensis (Figure 2B).The final pH in all conditions tested was much lower than the initial one (around 3.5).In contrast, the best initial pH to R. oryzae was 6.5 (Figure 2C).The final pH in every condition was higher than was it in the initial one (about 8.0).Time-course of amylase production was carried until 144 hours (Figure 2D).The period of 120 hours had the greater amylolytic secretion to A. brasiliensis.It was also noted that the pH of the culture medium began decreasing on the second day and stabilized after 72 hours.On the other hand, the culture of 96 hours was more favorable to R. oryzae.The pH of the culture medium began increasing on the second day and stabilized after 72 hours.The surface (Figure 3A) plot shows that the ideal assay temperature was in the range of 60 -75°C, and the pH of 3.5 -5.Replacing the values correspondent to 70°C (0.943) and pH 4 (-0.977), the activity of 6 U/mL was obtained.An assay in triplicate was carried out in the same parameters and an activity of 6.7 was obtained with a standard variation of ± 0.7, as expected to the model, with an r 2 of 0.96 and a calculated F 18.2 times higher than the tabulated F.

Optimum pH and temperature
The enzymatic activity of the crude extract of R. oryzae can be determined by the following equation: Activity (U/mL) = 2.52 -0.68.pH -0.96.(T°C) 2 -0.68.(pH) 2 According to the surface plot (Figure 3B), the ideal assay temperature was in the range of 50-65°C and the pH of 4.0-5.5.Replacing the values of 60°C (0) and pH 4.0 (-0.977), 2.54 U/mL was obtained.An assay in the same conditions obtained 2.94 ± 0.58 U/mL, as expected to the model, with an r 2 of 0.9 and a calculated F 5.9 times higher than the tabulated F.

Stability and effect of compounds
The crude extracts were incubated in McIlvaine buffers for 24 hours (Figure 4A) at 25°C.The amylase of A. brasiliensis kept the activity above 90% in the range of pH 3.0-6.5 and above 88% in the range of pH 7.0-8.0.The amylase of R. oryzae kept 100% of the activity in the range of pH 3.5-8.0.At pH 3.0, it kept 91% of its activity and 75% at pH 2.5.
The thermal denaturation of amylases of both fungi was analyzed at several times and temperatures (Figure 4B).Amylases of A. brasiliensis were completely stable at 50°C, for 120 minutes.The enzyme was completely stable at 60°C, for 100 minutes.However, at 70°C the half-life was 32 minutes.R. oryzae amylases, among all assayed conditions, just had residual activity after exposure at 50°C, with a half-life of 12 minutes.
The influence of several compounds upon the amylolytic activity was tested (Table 4).The Al 3+ ions had little influence upon A. brasiliensis but, in contrast, at 5 mM, it completely inhibited the R. oryzae amylase.Ag 2+ decreased the amylase of both fungi extracts, but had a greater effect over R. oryzae, with no residual activity at the highest concentration.The amylase activity of R. oryzae was 55% inhibited with 5 mM Cu 2+ but increased 9% in A. brasiliensis.The 2 mM Ba 2+ decreased the activity in A. brasiliensis and a slight increase (7%) was observed with 5 mM of this compound; the opposite happened with R. oryzae, which was activated at 2 mM and inhibited at 5 mM.On the other hand, Co 2+ ions had little effect over A. brasiliensis, but decreased 42% at 5 mM in R. oryzae.Both extracts were not affected by Pb 2+ .The stronger enzymatic activation was obtained with 5 mM Mn 2+ for A. brasiliensis and at 2 mM for R. oryzae.The glucoamylases of both extracts were not considerably influenced by β-mercaptoethanol and EDTA.

Hydrolysis products of amylase on starch revealed by TLC
Amylases produced by both fungi in culture media supplemented with several carbon sources (1% starch; wheat bran; maltose; glucose; barley bagasse; ground corn and oatmeal flour) were assayed with starch and the   end-products were applied in TLC.The crude extract of A. brasiliensis in all sources enabled a great release of glucose and a small amount of oligosaccharides during the hydrolysis (Figure 5A), signaling for a higher secretion of glucoamylase.There was the production of glucoamylases even in the culture with glucose as an only carbon source, suggesting that it is a constitutive enzyme.The fungus R. oryzae also secreted more glucoamylase in all media, but there was the release of some more oligosaccharides, in small quantities, suggesting also the presence of α-amylases (Figure 5B).The glucoamylase of R. oryzae is also constitutive.

Phylogenetic analysis
The phylogenetic comparison of species close to R. oryzae and A. brasiliensis was based on the regions of ITS 1-2 (Internal Spacer).The sequences were obtained on GenBank and aligned with MUSCLE 3.6.From the alignment, an unrooted dendrogram that shows the proximity of species was built (Figure 6).The analysis showed similarity between the sequences of A. nidulans (Eidam) G. Winter and A. fumigatus Fersen of 64.5%; 69.8% between A. brasiliensis and A. awamori; 71.1% between A. oryzae (Ahlb.)Cohn and A. flavus Link.The high rate of similarity between the species of Aspergillus can be observed with Castrillo et al. (2012), where A. brasiliensis and A. awamori belong to the same clade, and with Geiser et al. (2000), who demonstrate that A. oryzae and A. flavus form a paraphyletic group.
R. oryzae and R. delemar Boidin ex Wehmer & Hanzawa have a high rate of similarity (72.9%) and constitute a monophyletic group.R. niveus M. Yamaz. is closer to R. oryzae and R. delemar, with a high bootstrap value (100).Between the fungi studied, Rhizopus microsporus var.

Discussion
According to Domsch et al. (2007), the fungus distribution is related with the weather, soil, vegetation and the kind of organic matter.Fungi of the genera Aspergillus and Rhizopus are frequently isolated from the Atlantic forest biome (Costa et al. 2012, Schoenlein-Crusius et al. 2006, Tauk Tornisielo et al. 2005, Schoenlein-Crusius & Milanez 1998).The isolation methodology was not intended to recover a maximum number of species, but strains that were easily cultivated in laboratory conditions and prospective amylase producers.
The amylases have several applications in industry, and each of these bioprocesses requires enzymes with diverse characteristics of pH,  2013) model.The tree with the highest log likelihood (-2874.0908) is shown.The numbers show the bootstrap value analysis of 500 repetitions and the percentage higher than 70% of trees in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 10 nucleotide sequences.There was a total of 776 positions in the final dataset.Evolutionary analyses were conducted in MEGA6.The species names are followed by the GenBank accession number.(B) by Neighbor-Joining method.The optimal tree with the sum of branch length = 1.00924206 is shown.The percentage of replicate trees in which the associated taxa is clustered together in the bootstrap test (500 replicates) with the percentage higher than 70% are shown next to the branches.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using the Tajima & Nei (1984) method and are in the units of the number of base substitutions per site.The rate variation among sites was modeled with a gamma distribution (shape parameter = 2).The analysis involved 10 nucleotide sequences.All ambiguous positions were removed for each sequence pair.There was a total of 776 positions in the final dataset.Evolutionary analyses were conducted in MEGA6.The species names are followed by the GenBank accession number.temperature and stability.The importance of bioprospection lies in the discovery of novel enzymes, with unique features, that can contribute to a more efficient process (Dhali et al. 2016, Singh et al. 2014).Coutinho & Reilly (1997) divided the glucoamylases following their evolutionary history.Fungi of Aspergillus genera are part of the most derivative group of glucoamylases, with a high efficient design.They have a well-developed starch-binding domain and a longer linker region.Fungi from the genera Rhizopus are part of a group with a more primitive glucoamylase structure, which reflects in its efficiency and stability.
The time-course of amylase production showed distinct characteristics of A. brasiliensis and R. oryzae.The pH of crude extract differed due to the production of secondary metabolites, probably the production of glycolic and citric acid by A. brasiliensis and ammonia by R. oryzae, as previously observed in R. oligosporus Saito when in nitrogen-rich culture media (Varga et al. 2007, Sparringa & Owens 1999).
A. fumigatus showed similar stability of temperature (Silva & Peralta 1998).The pH stability results were similar to the A. niger glucoamylases and superior to the stability of A. awamori (Nakaz.)(range pH 6.0-9.0)(Gudi et al. 2013, Pestana & Castilho 1985).The R. oryzae strain isolated in this work produced glucoamylases with pH and temperature of activity with characteristics similar to the same species strain of Roch-chui & Hang (1990).R. delemar has glucoamylases that act better in pH within the range of R. oryzae (4.5) and lower optimum temperature (40°C) (Soccol et al. 1994).R. niveus has the activity pH in a higher range (4.5-6) and the same optimum temperature than R. oryzae (Saha & Ueda 1983).The tolerance from thermal denaturation of the glucoamylase in the absence of the substrate was extreme low.The pH stability results were again like the strain in the work of Roch-chui & Hang (1990).Rhizopus microsporus var.rhizopodiformis, a thermo-tolerant strain, showed lower stability of pH (approximately 80% of residual activity in the range of 2.5-7.5, after only 2 hours of incubation) (Peixoto et al. 2003).
Therefore, from environmental samples from the Brazilian Atlantic Forest, it was possible to isolate filamentous fungi strains that produced high amounts of amylases with distinct biochemical characteristics.These differences reflect upon the evolutionary history of R. oryzae, a basal filamentous fungus that alkalinizes the culture media during growth and secretes a less stable glucoamylase, with half-life of 12 minutes at 50°C; A. brasiliensis, a derivative species that produces a highly stable, glucoamylase with half-life of 1 hour at 70°C, and an acidic one, with great activity at pH 3.5, so that it has interesting characteristics for future large-scale applications.
An experimental design was performed to determine the optimum pH and temperature for the enzymatic hydrolysis according the points in Table3.The experimental design was composed of 11 assays with 3 repetitions at the central point.According to the results, the enzymatic activity of the crude extract of A. brasiliensis was determined by the following equation: Activity (U/mL) = 5.55 -1.99.pH -1.(T°C.pH)-1.42.(T°C) 2 -1.14.(pH) 2

Figure 4 .
Figure 4. Stability of glucoamylases to pH, after 24 hours, at 25°C (A) and thermostability in optimal conditions of enzymatic assay (B) of A. brasiliensis and R. oryzae.

Figure 3 .
Figure 3. Zone of optimum pH and temperature to (A) A. brasiliensis; (B) R. oryzae.

Figure 6 :
Figure 6: Molecular Phylogenetic analysis (A) by Maximum Likelihood method.The evolutionary history was inferred by using the Maximum Likelihood method based on the Tamura et al. (2013) model.The tree with the highest log likelihood (-2874.0908) is shown.The numbers show the bootstrap value analysis of 500 repetitions and the percentage higher than 70% of trees in which the associated taxa clustered together is shown next to the branches.The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.The analysis involved 10 nucleotide sequences.There was a total of 776 positions in the final dataset.Evolutionary analyses were conducted in MEGA6.The species names are followed by the GenBank accession number.(B) by Neighbor-Joining method.The optimal tree with the sum of branch length = 1.00924206 is shown.The percentage of replicate trees in which the associated taxa is clustered together in the bootstrap test (500 replicates) with the percentage higher than 70% are shown next to the branches.The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using theTajima & Nei (1984) method and are in the units of the number of base substitutions per site.The rate variation among sites was modeled with a gamma distribution (shape parameter = 2).The analysis involved 10 nucleotide sequences.All ambiguous positions were removed for each sequence pair.There was a total of 776 positions in the final dataset.Evolutionary analyses were conducted in MEGA6.The species names are followed by the GenBank accession number.

Table 1 .
Georeferencing means of isolated fungi and amylase activity

Table 3 .
Points used at the experimental design for the crude extract of A. brasiliensis

Table 4 .
Liou et al. (2007)s at enzymatic activity of A. brasiliensis and R. oryzae.Stalpers was the most singular of the species, forming an isolated taxon from other fungi of the genera; it also has the lowest rate of similarity (49.6% if compared to R. delemar).However, R. rhizopodiformis has the necessary apomorphy to be classified close to other Rhizopus.Liou et al. (2007)had similar results when they showed linages of R. rhizopodiformis among the main glucoamylase-producing species, what demonstrates the need for more information on this species.