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Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea Hare Aplysia kurodai. Efficient glucose release from sea lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa ß-glucosidase.

Tsuji A, Tominaga K, Nishiyama N, Yuasa K - PLoS ONE (2013)

Bottom Line: Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase.Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce.These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, The University of Tokushima Graduate School, Tokushima, Japan. tsuji@bio.tokushima-u.ac.jp

ABSTRACT
Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.

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Hydrolysis of CMC, filter paper, and seaweeds by the synergistic action of cellulases and ß-glucosidases.(A) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (B) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (C) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (D) Seaweed, sea lettuce (Ulva pertusa), Eisenia bicyclis, and Lessonia nigrescens (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (E, F) TLC analysis of reaction products of sea lettuce treated with purified enzymes or Trichoderma cellulase. Control sea lettuce (E) and sea lettuce treated with steam explosion (F) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of Trichoderma cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.
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pone-0065418-g004: Hydrolysis of CMC, filter paper, and seaweeds by the synergistic action of cellulases and ß-glucosidases.(A) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (B) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (C) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (D) Seaweed, sea lettuce (Ulva pertusa), Eisenia bicyclis, and Lessonia nigrescens (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (E, F) TLC analysis of reaction products of sea lettuce treated with purified enzymes or Trichoderma cellulase. Control sea lettuce (E) and sea lettuce treated with steam explosion (F) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of Trichoderma cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.

Mentions: Sea hare cellulases can produce glucose from CMC and filter paper without cellobiohydrolase or ß-glucosidase, unlike the Trichoderma cellulolytic system [7]. Glucose release from CMC increased markedly following addition of ß-glucosidase. A synergistic effect of 45 K and 65 K cellulase on the production of glucose from filter paper was observed, although no effect was observed on CMC digestion with these enzymes (Figure 4A, 4B). When filter paper (60 mg) was digested with 10 µg of 45 K or 65 K cellulase at 37°C for 48 h in separate tubes, the total glucose released from the filter paper in each reaction was 759 µg (218+541 µg). However, when filter paper was digested with 45 K and 65 K cellulase in the same tube, there was an approximate 2-fold increase in glucose production (1380 µg). Similarly, when filter paper was digested with 21 K, 45 K, and 65 K cellulase in the same tube, there was a 2-fold increase in glucose production compared to reactions in separate tubes. The addition of 210 K or 110 K ß-glucosidase resulted in complete hydrolysis of cellobiose to glucose. To identify the best combination of ß-glucosidases and cellulases to maximize glucose productivity, the glucose-producing activities of the various enzyme mixtures on filter paper were compared (Figure 4C). Filter paper (60 mg) was hydrolyzed to glucose by a pair consisting of either 21 K or 45 K cellulase together with either 210 K or 110 K ß-glucosidase (2 µg of enzyme) at 37°C for 24 h. Compared with the same reaction using all the enzymes (0.531 mg of glucose production), approximately 70% of the glucose productivity was obtained when 3 enzymes, 210 K ß-glucosidase (1.4 units), 21 K (0.036 units), and 45 K cellulase (0.34 units), were used.


Comprehensive enzymatic analysis of the cellulolytic system in digestive fluid of the Sea Hare Aplysia kurodai. Efficient glucose release from sea lettuce by synergistic action of 45 kDa endoglucanase and 210 kDa ß-glucosidase.

Tsuji A, Tominaga K, Nishiyama N, Yuasa K - PLoS ONE (2013)

Hydrolysis of CMC, filter paper, and seaweeds by the synergistic action of cellulases and ß-glucosidases.(A) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (B) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (C) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (D) Seaweed, sea lettuce (Ulva pertusa), Eisenia bicyclis, and Lessonia nigrescens (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (E, F) TLC analysis of reaction products of sea lettuce treated with purified enzymes or Trichoderma cellulase. Control sea lettuce (E) and sea lettuce treated with steam explosion (F) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of Trichoderma cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675134&req=5

pone-0065418-g004: Hydrolysis of CMC, filter paper, and seaweeds by the synergistic action of cellulases and ß-glucosidases.(A) CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with various combinations of purified enzymes (2 µg) as indicated at 37°C for 1 h. Reaction products were analyzed by TLC. (B) Filter paper (60 mg) was digested with various combinations of purified enzymes (10 µg) as indicated at 37°C for 48 h, and reaction products were analyzed by TLC. (C) Filter paper (60 mg) was digested with 21 K and 45 K cellulase (2 µg) in the presence of 110 K or 210 K ß-glucosidase (2 µg) at 37°C for 16 h. Reaction products were analyzed by TLC. (D) Seaweed, sea lettuce (Ulva pertusa), Eisenia bicyclis, and Lessonia nigrescens (20 mg in 50 mM acetate, pH 5.5) were incubated with purified enzymes (10 µg) at 37°C for 24 h. Glucose and reducing sugar content were then determined. (E, F) TLC analysis of reaction products of sea lettuce treated with purified enzymes or Trichoderma cellulase. Control sea lettuce (E) and sea lettuce treated with steam explosion (F) (20 mg in 50 mM acetate, pH 5.5) were incubated with purified cellulase (20 µg) in the presence of ß-glucosidase (20 µg) at 37°C for 15 h. As controls, 50 µg of Trichoderma cellulases, meicelase, and onozuka R-10 were used. The data shown are from one of three independent experiments with similar results.
Mentions: Sea hare cellulases can produce glucose from CMC and filter paper without cellobiohydrolase or ß-glucosidase, unlike the Trichoderma cellulolytic system [7]. Glucose release from CMC increased markedly following addition of ß-glucosidase. A synergistic effect of 45 K and 65 K cellulase on the production of glucose from filter paper was observed, although no effect was observed on CMC digestion with these enzymes (Figure 4A, 4B). When filter paper (60 mg) was digested with 10 µg of 45 K or 65 K cellulase at 37°C for 48 h in separate tubes, the total glucose released from the filter paper in each reaction was 759 µg (218+541 µg). However, when filter paper was digested with 45 K and 65 K cellulase in the same tube, there was an approximate 2-fold increase in glucose production (1380 µg). Similarly, when filter paper was digested with 21 K, 45 K, and 65 K cellulase in the same tube, there was a 2-fold increase in glucose production compared to reactions in separate tubes. The addition of 210 K or 110 K ß-glucosidase resulted in complete hydrolysis of cellobiose to glucose. To identify the best combination of ß-glucosidases and cellulases to maximize glucose productivity, the glucose-producing activities of the various enzyme mixtures on filter paper were compared (Figure 4C). Filter paper (60 mg) was hydrolyzed to glucose by a pair consisting of either 21 K or 45 K cellulase together with either 210 K or 110 K ß-glucosidase (2 µg of enzyme) at 37°C for 24 h. Compared with the same reaction using all the enzymes (0.531 mg of glucose production), approximately 70% of the glucose productivity was obtained when 3 enzymes, 210 K ß-glucosidase (1.4 units), 21 K (0.036 units), and 45 K cellulase (0.34 units), were used.

Bottom Line: Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase.Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce.These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science and Technology, The University of Tokushima Graduate School, Tokushima, Japan. tsuji@bio.tokushima-u.ac.jp

ABSTRACT
Although many endo-ß-1,4-glucanases have been isolated in invertebrates, their cellulolytic systems are not fully understood. In particular, gastropod feeding on seaweed is considered an excellent model system for production of bioethanol and renewable bioenergy from third-generation feedstocks (microalgae and seaweeds). In this study, enzymes involved in the conversion of cellulose and other polysaccharides to glucose in digestive fluids of the sea hare (Aplysia kurodai) were screened and characterized to determine how the sea hare obtains glucose from sea lettuce (Ulva pertusa). Four endo-ß-1,4-glucanases (21K, 45K, 65K, and 95K cellulase) and 2 ß-glucosidases (110K and 210K) were purified to a homogeneous state, and the synergistic action of these enzymes during cellulose digestion was analyzed. All cellulases exhibited cellulase and lichenase activities and showed distinct cleavage specificities against cellooligosaccharides and filter paper. Filter paper was digested to cellobiose, cellotriose, and cellotetraose by 21K cellulase, whereas 45K and 65K enzymes hydrolyzed the filter paper to cellobiose and glucose. 210K ß-glucosidase showed unique substrate specificity against synthetic and natural substrates, and 4-methylumbelliferyl (4MU)-ß-glucoside, 4MU-ß-galactoside, cello-oligosaccharides, laminarin, and lichenan were suitable substrates. Furthermore, 210K ß-glucosidase possesses lactase activity. Although ß-glucosidase and cellulase are necessary for efficient hydrolysis of carboxymethylcellulose to glucose, laminarin is hydrolyzed to glucose only by 210K ß-glucosidase. Kinetic analysis of the inhibition of 210K ß-glucosidase by D-glucono-1,5-lactone suggested the presence of 2 active sites similar to those of mammalian lactase-phlorizin hydrolase. Saccharification of sea lettuce was considerably stimulated by the synergistic action of 45K cellulase and 210K ß-glucosidase. Our results indicate that 45K cellulase and 210K ß-glucosidase are the core components of the sea hare digestive system for efficient production of glucose from sea lettuce. These findings contribute important new insights into the development of biofuel processing biotechnologies from seaweed.

Show MeSH
Related in: MedlinePlus