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Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts.

Carrasco M, Villarreal P, Barahona S, Alcaíno J, Cifuentes V, Baeza M - BMC Microbiol. (2016)

Bottom Line: A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases.These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively.However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile. mario.carrasco.t@gmail.com.

ABSTRACT

Background: Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases.

Result: All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi.

Conclusions: Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.

No MeSH data available.


Related in: MedlinePlus

Enzyme activities in extracellular protein samples. Total proteins were obtained from cell-free supernatants of the analyzed yeast species cultured in YM media supplemented with glucose. The amylase (a) and cellulase (b) activity was evaluated by the well test assay at different pH and temperatures, and normalized by the total protein amount (mm/mg total protein). The gradient color scale indicated in the figure represents the lowest (L, black) to the highest (H, red) activity recorded values from culture supernatant of each yeast species
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Fig3: Enzyme activities in extracellular protein samples. Total proteins were obtained from cell-free supernatants of the analyzed yeast species cultured in YM media supplemented with glucose. The amylase (a) and cellulase (b) activity was evaluated by the well test assay at different pH and temperatures, and normalized by the total protein amount (mm/mg total protein). The gradient color scale indicated in the figure represents the lowest (L, black) to the highest (H, red) activity recorded values from culture supernatant of each yeast species

Mentions: Total secreted proteins were obtained by ammonium sulfate precipitation from supernatants from yeast cultures using YM medium supplemented with glucose, and enzyme activities in the samples were evaluated by well test assays. In all assays, denaturalized protein controls corresponding to the protein sample incubated at 100 °C for 15 min were included and in all cases, no activity was observed, as shown in well 2 from Fig. 2a. As exemplified in Fig. 2, a high amylase activity was observed in D. fristingensis (T9) samples and a lower activity was observed in samples from Leuconeurospora sp. T11cd2 cultures (Fig. 2a). Le. fragaria and Mrakia sp. culture samples showed higher cellulase activities (Fig. 2b). In addition, enzyme activities of the protein samples were tested at different pH and temperature conditions including pH 4.6, 5.4 and 6.2; and 4, 10, 15, 22, 30 and 37 °C. The protein samples from the majority of the yeasts analyzed had higher amylase activities at pH 5.4 and 6.2, and at 30 and 37 °C (Fig. 3a). The highest amylase activities were observed in protein samples from Dioszegia fristingensis (T9Df1), M. blollopis, Holtermaniella watticus and Tetracladium sp., which were higher at pH 6.2 and 37 °C. No amylase activity was observed in samples from Cryptococcus sp. and Cr. gilvescens. Cellulase activity could not be assayed at pH 4.6 because the medium in the agar-plates did not solidify at this pH. As shown in Fig. 3b, the highest cellulase activity was observed in samples from Leuconeurospora T17Cd1 at pH 6.2 and 22–30 °C. Also, high activities were observed in samples from Mrakia sp. (pH 5.4; 22–30 °C), M. blollopis (pH 5.4; 4–15 °C) and Tetracladium sp. (pH 5.4; 30 °C). No cellulase activity was observed in samples from Cr. gastricus, H. watticus, Leucosporidiella fragaria and Wickermanomyces anomalus in the assayed conditions. In general, the halos observed in the enzyme activity assays had variable degrees of turbidity. However, samples from Tetracladium sp. and M. gelida gave very clear halos in the amylase and cellulase activity assays, respectively, suggesting greater substrate degradation. Although all yeasts were able to grow in medium with SS or CMC as the sole carbon source, the corresponding extracellular enzyme activity was not observed in some of the protein samples as mentioned above. Considering that it has been previously reported that CMC has an inducing effect in the cellulase activity from the fungus Ganoderma applanatum MR-56 [27] and that amylolytic activity was induced by SS in bacterial isolates [28], it is possible that the yeasts tested in this work require the presence of the substrate to induce the expression of the corresponding genes that encode the respective enzyme activity. To evaluate this possibility, the yeasts were cultured in YM medium supplemented with SS or CMC, the extracellular proteins were obtained and the activities measured (Additional file 1). Figure 4 shows the enzyme activities of protein samples from yeast cultivated in medium supplemented with glucose, SS or CMC, at the pH and temperature at which the highest activity was observed in each case. An amylase activity inducing effect by SS was observed only in samples obtained from four yeast species, most notably in Cr. gilvescens and M. robertii (Fig. 4a). In the other species, the amylase activity was higher in samples from yeast cultivated in medium supplemented with glucose; even more for H. watticus, Rh. glacialis T8Rg and Rh. glacialis T11Rg, no amylase activity was detected when the yeasts were grown in media supplemented with SS. For Cryptococcus sp., no amylase activity was observed in the protein samples obtained from cultures in both conditions. In the analyses of cellulase activities, an inductor effect by CMC was observed only in samples from Cr. gastricus, H. watticus and Le. fragaria, whereas in the samples from the other yeasts species, the highest cellulase activities were observed when yeasts were cultivated in medium supplemented with glucose (Fig. 4b). No cellulase activity was observed in samples from W. anomalus grown in either condition. As indicated in each column of Fig. 4, for almost all yeast species, the total protein amounts were higher in samples from cultures supplemented with SS or CMC than with glucose; however, this increase in total protein content was not correlated with an increase of either amylase or cellulase activity in the samples. Although Cryptococcus sp. and W. anomalus were able to grow using SS and CMC as the sole carbon source, and amylase and cellulase activities were previously detected in yeast colony assays, neither activity was detected in extracellular protein samples from yeast cultures in either condition. It is possible that the corresponding enzymes are anchored to the cell wall, as this has been described for amylase-like enzymes in two species of Aspergillus [29, 30]. There is limited data regarding amylase activity in the yeast genera described in this work; amylase activity was described in Cryptococcus sp. [31] and Cr. flavus [32], the latter of which was successfully expressed in S. cerevisiae [33], Mrakia blollopis [34], Rhodotorula svalbardensis sp. nov [35] and Tetracladium setigerum [36]. No reported data were found in literature about amylase activity in the genera Dioszegia, Leuconeurospora and Holtermaniella. Cellulase activity has been described in Cryptococcus laurentii and Cryptococcus nemorosus [37], Tetracladium [36], and in Mrakia species isolated from Arctic puddles [38]. No data were found in the literature regarding cellulase activity for the genera Dioszegia, Leuconeurospora, Leucosporidiella and Wickermamomyces. The yeast studied in this work were originally isolated from soil samples of King George Island [26] in which there is a considerable input of organic materials, including vegetation. Furthermore, the temperatures in this region are warmer than the rest of Antarctica, having seasonal changes and that can reach up to 20 °C in summer [26, 39–41]. According to this, the yeasts studied that showed extracellular cellulase and amylase activities, can use the complex carbon sources, suggesting an active role in vegetable residues recycling and mineralization in this cold environment. Furthermore, the amylase and cellulase activities were detected in a wide range of temperature (4 to 30 °C) and the majority showed higher activities at moderate temperatures (22 or 30 °C). However, these temperatures are lower than those described for these enzymes in mesophilic organisms, which generally show optimal activities at temperatures over 40 °C [42, 43]. This last point reflects the adaptations of the yeasts studied here to the conditions of the sub-Antarctic region where they thrive.Fig. 2


Screening and characterization of amylase and cellulase activities in psychrotolerant yeasts.

Carrasco M, Villarreal P, Barahona S, Alcaíno J, Cifuentes V, Baeza M - BMC Microbiol. (2016)

Enzyme activities in extracellular protein samples. Total proteins were obtained from cell-free supernatants of the analyzed yeast species cultured in YM media supplemented with glucose. The amylase (a) and cellulase (b) activity was evaluated by the well test assay at different pH and temperatures, and normalized by the total protein amount (mm/mg total protein). The gradient color scale indicated in the figure represents the lowest (L, black) to the highest (H, red) activity recorded values from culture supernatant of each yeast species
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4759947&req=5

Fig3: Enzyme activities in extracellular protein samples. Total proteins were obtained from cell-free supernatants of the analyzed yeast species cultured in YM media supplemented with glucose. The amylase (a) and cellulase (b) activity was evaluated by the well test assay at different pH and temperatures, and normalized by the total protein amount (mm/mg total protein). The gradient color scale indicated in the figure represents the lowest (L, black) to the highest (H, red) activity recorded values from culture supernatant of each yeast species
Mentions: Total secreted proteins were obtained by ammonium sulfate precipitation from supernatants from yeast cultures using YM medium supplemented with glucose, and enzyme activities in the samples were evaluated by well test assays. In all assays, denaturalized protein controls corresponding to the protein sample incubated at 100 °C for 15 min were included and in all cases, no activity was observed, as shown in well 2 from Fig. 2a. As exemplified in Fig. 2, a high amylase activity was observed in D. fristingensis (T9) samples and a lower activity was observed in samples from Leuconeurospora sp. T11cd2 cultures (Fig. 2a). Le. fragaria and Mrakia sp. culture samples showed higher cellulase activities (Fig. 2b). In addition, enzyme activities of the protein samples were tested at different pH and temperature conditions including pH 4.6, 5.4 and 6.2; and 4, 10, 15, 22, 30 and 37 °C. The protein samples from the majority of the yeasts analyzed had higher amylase activities at pH 5.4 and 6.2, and at 30 and 37 °C (Fig. 3a). The highest amylase activities were observed in protein samples from Dioszegia fristingensis (T9Df1), M. blollopis, Holtermaniella watticus and Tetracladium sp., which were higher at pH 6.2 and 37 °C. No amylase activity was observed in samples from Cryptococcus sp. and Cr. gilvescens. Cellulase activity could not be assayed at pH 4.6 because the medium in the agar-plates did not solidify at this pH. As shown in Fig. 3b, the highest cellulase activity was observed in samples from Leuconeurospora T17Cd1 at pH 6.2 and 22–30 °C. Also, high activities were observed in samples from Mrakia sp. (pH 5.4; 22–30 °C), M. blollopis (pH 5.4; 4–15 °C) and Tetracladium sp. (pH 5.4; 30 °C). No cellulase activity was observed in samples from Cr. gastricus, H. watticus, Leucosporidiella fragaria and Wickermanomyces anomalus in the assayed conditions. In general, the halos observed in the enzyme activity assays had variable degrees of turbidity. However, samples from Tetracladium sp. and M. gelida gave very clear halos in the amylase and cellulase activity assays, respectively, suggesting greater substrate degradation. Although all yeasts were able to grow in medium with SS or CMC as the sole carbon source, the corresponding extracellular enzyme activity was not observed in some of the protein samples as mentioned above. Considering that it has been previously reported that CMC has an inducing effect in the cellulase activity from the fungus Ganoderma applanatum MR-56 [27] and that amylolytic activity was induced by SS in bacterial isolates [28], it is possible that the yeasts tested in this work require the presence of the substrate to induce the expression of the corresponding genes that encode the respective enzyme activity. To evaluate this possibility, the yeasts were cultured in YM medium supplemented with SS or CMC, the extracellular proteins were obtained and the activities measured (Additional file 1). Figure 4 shows the enzyme activities of protein samples from yeast cultivated in medium supplemented with glucose, SS or CMC, at the pH and temperature at which the highest activity was observed in each case. An amylase activity inducing effect by SS was observed only in samples obtained from four yeast species, most notably in Cr. gilvescens and M. robertii (Fig. 4a). In the other species, the amylase activity was higher in samples from yeast cultivated in medium supplemented with glucose; even more for H. watticus, Rh. glacialis T8Rg and Rh. glacialis T11Rg, no amylase activity was detected when the yeasts were grown in media supplemented with SS. For Cryptococcus sp., no amylase activity was observed in the protein samples obtained from cultures in both conditions. In the analyses of cellulase activities, an inductor effect by CMC was observed only in samples from Cr. gastricus, H. watticus and Le. fragaria, whereas in the samples from the other yeasts species, the highest cellulase activities were observed when yeasts were cultivated in medium supplemented with glucose (Fig. 4b). No cellulase activity was observed in samples from W. anomalus grown in either condition. As indicated in each column of Fig. 4, for almost all yeast species, the total protein amounts were higher in samples from cultures supplemented with SS or CMC than with glucose; however, this increase in total protein content was not correlated with an increase of either amylase or cellulase activity in the samples. Although Cryptococcus sp. and W. anomalus were able to grow using SS and CMC as the sole carbon source, and amylase and cellulase activities were previously detected in yeast colony assays, neither activity was detected in extracellular protein samples from yeast cultures in either condition. It is possible that the corresponding enzymes are anchored to the cell wall, as this has been described for amylase-like enzymes in two species of Aspergillus [29, 30]. There is limited data regarding amylase activity in the yeast genera described in this work; amylase activity was described in Cryptococcus sp. [31] and Cr. flavus [32], the latter of which was successfully expressed in S. cerevisiae [33], Mrakia blollopis [34], Rhodotorula svalbardensis sp. nov [35] and Tetracladium setigerum [36]. No reported data were found in literature about amylase activity in the genera Dioszegia, Leuconeurospora and Holtermaniella. Cellulase activity has been described in Cryptococcus laurentii and Cryptococcus nemorosus [37], Tetracladium [36], and in Mrakia species isolated from Arctic puddles [38]. No data were found in the literature regarding cellulase activity for the genera Dioszegia, Leuconeurospora, Leucosporidiella and Wickermamomyces. The yeast studied in this work were originally isolated from soil samples of King George Island [26] in which there is a considerable input of organic materials, including vegetation. Furthermore, the temperatures in this region are warmer than the rest of Antarctica, having seasonal changes and that can reach up to 20 °C in summer [26, 39–41]. According to this, the yeasts studied that showed extracellular cellulase and amylase activities, can use the complex carbon sources, suggesting an active role in vegetable residues recycling and mineralization in this cold environment. Furthermore, the amylase and cellulase activities were detected in a wide range of temperature (4 to 30 °C) and the majority showed higher activities at moderate temperatures (22 or 30 °C). However, these temperatures are lower than those described for these enzymes in mesophilic organisms, which generally show optimal activities at temperatures over 40 °C [42, 43]. This last point reflects the adaptations of the yeasts studied here to the conditions of the sub-Antarctic region where they thrive.Fig. 2

Bottom Line: A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases.These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively.However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Ciencias Ecológicas, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile. mario.carrasco.t@gmail.com.

ABSTRACT

Background: Amylases and cellulases have great potential for application in industries such as food, detergent, laundry, textile, baking and biofuels. A common requirement in these fields is to reduce the temperatures of the processes, leading to a continuous search for microorganisms that secrete cold-active amylases and cellulases. Psychrotolerant yeasts are good candidates because they inhabit cold-environments. In this work, we analyzed the ability of yeasts isolated from the Antarctic region to grow on starch or carboxymethylcellulose, and their potential extracellular amylases and cellulases.

Result: All tested yeasts were able to grow with soluble starch or carboxymethylcellulose as the sole carbon source; however, not all of them produced ethanol by fermentation of these carbon sources. For the majority of the yeast species, the extracellular amylase or cellulase activity was higher when cultured in medium supplemented with glucose rather than with soluble starch or carboxymethylcellulose. Additionally, higher amylase activities were observed when tested at pH 5.4 and 6.2, and at 30-37 °C, except for Rhodotorula glacialis that showed elevated activity at 10-22 °C. In general, cellulase activity was high until pH 6.2 and between 22-37 °C, while the sample from Mrakia blollopis showed high activity at 4-22 °C. Peptide mass fingerprinting analysis of a potential amylase from Tetracladium sp. of about 70 kDa, showed several peptides with positive matches with glucoamylases from other fungi.

Conclusions: Almost all yeast species showed extracellular amylase or cellulase activity, and an inducing effect by the respective substrate was observed in a minor number of yeasts. These enzymatic activities were higher at 30 °C in most yeast, with highest amylase and cellulase activity in Tetracladium sp. and M. gelida, respectively. However, Rh. glacialis and M. blollopis displayed high amylase or cellulase activity, respectively, under 22 °C. In this sense, these yeasts are interesting candidates for industrial processes that require lower temperatures.

No MeSH data available.


Related in: MedlinePlus