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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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Mode of action of 210 K or 110 K ß-glucosidase on CMC, laminarin, and lichenan.(A) Digestion of CMC with 210 K or 110 K ß-glucosidase in the absence or presence of 21 K or 45 K cellulase. CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 210 K or 110 K ß-glucosidase and 5 mg of 21 K or 45 K cellulose, as indicated, at 37°C for 10, 20, 30, and 60 min. Reducing sugar (1) and glucose (2) in the reaction mixture were determined. Reaction products were analyzed by TLC (3). (B) Laminarin (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min. The glucose content in the reaction mixture was then determined. Reaction products were analyzed by TLC. (C) Lichenan (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min.
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pone-0065418-g003: Mode of action of 210 K or 110 K ß-glucosidase on CMC, laminarin, and lichenan.(A) Digestion of CMC with 210 K or 110 K ß-glucosidase in the absence or presence of 21 K or 45 K cellulase. CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 210 K or 110 K ß-glucosidase and 5 mg of 21 K or 45 K cellulose, as indicated, at 37°C for 10, 20, 30, and 60 min. Reducing sugar (1) and glucose (2) in the reaction mixture were determined. Reaction products were analyzed by TLC (3). (B) Laminarin (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min. The glucose content in the reaction mixture was then determined. Reaction products were analyzed by TLC. (C) Lichenan (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min.

Mentions: The cleavage specificity of 110K and 210K ß-glucosidase toward various synthetic and natural substrates was compared. Specific activity of both ß-glucosidases toward 4MU-ß-glucoside, 4MU-ß-galactoside, cellobiose, lichenan, and laminarin is shown in Table 2. 210K ß-glucosidase exhibited higher specific activities against all substrates examined than 110K ß-glucosidase. Among the synthetic substrates examined, both ß-glucosidases were most active against 4-MU-ß-glucoside. Compared with the maximum activity toward 4MU-ß-glucoside, 110K and 210K ß-glucosidase exhibited 34–35% activity toward 4-MU-ß-galactoside. Both ß-glucosidases showed weak activity toward 4MU-ß-xyloside (110K: 0.9%, 210K: 3.5%). Neither the 110K nor the 210K enzyme exhibited activity toward 4MU-α-glucoside, 4MU-α-galactoside, or 4MU-α-mannoside. Only a small amount of glucose was released from CMC by these ß-glucosidases (Figure 2C). All cello-oligosaccharides were completely hydrolyzed to glucose by both ß-glucosidases (Figure 2D). To investigate the mode of hydrolysis of cellohexaose, the time-course of degradation of cellohexaose by 110K or 210K ß-glucosidase was analyzed (Figure 2E). Cellohexaose was hydrolyzed into cellopentaose, cellotetraose, cellotriose, cellobiose, and glucose within 20 min (210 K) and 5 min (110 K). These results suggest that 210 K ß-glucosidase prefers longer oligo-cellulose than cellotriose. It is noteworthy that both enzymes exhibited weak lactase (Figure 2F) and gentiobiose cleavage activities (data not shown). Figure 3 shows the glucose-releasing activity of 110 K and 210 K ß-glucosidase toward CMC, laminarin, and lichenan. Lichenan is a ß-D-glucan that consists of repeating glucose units linked by ß-1,4 and ß-1,3 glycosidic bonds. Laminarin is a storage polysaccharide of seaweed consisting of a ß-1,3-linked glucose main chain and ß-1,6-linked glucose branches. Although collaboration between cellulases is necessary for production of glucose from CMC by ß-glucosidase (Figure 3A), laminarin and lichenan were efficiently digested to glucose only by 210 K ß-glucosidase (Figure 3B, 3C). Glucose release from laminarin and lichenan was confirmed by a quantitative glucose assay using glucose oxidase. Cardran, a polysaccharide consisting of ß-1,6-linked glucose, was also cleaved by both ß-glucosidases. The relative activity toward cardran of 110 K and 210 K ß-glucosidase was 12% and 32%, respectively, of the activity against laminarin. These results suggest that the primary role of 110 K and 210 K ß-glucosidase is the decomposition of cello-oligosaccharides and laminarin to glucose.


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)

Mode of action of 210 K or 110 K ß-glucosidase on CMC, laminarin, and lichenan.(A) Digestion of CMC with 210 K or 110 K ß-glucosidase in the absence or presence of 21 K or 45 K cellulase. CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 210 K or 110 K ß-glucosidase and 5 mg of 21 K or 45 K cellulose, as indicated, at 37°C for 10, 20, 30, and 60 min. Reducing sugar (1) and glucose (2) in the reaction mixture were determined. Reaction products were analyzed by TLC (3). (B) Laminarin (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min. The glucose content in the reaction mixture was then determined. Reaction products were analyzed by TLC. (C) Lichenan (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675134&req=5

pone-0065418-g003: Mode of action of 210 K or 110 K ß-glucosidase on CMC, laminarin, and lichenan.(A) Digestion of CMC with 210 K or 110 K ß-glucosidase in the absence or presence of 21 K or 45 K cellulase. CMC (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 210 K or 110 K ß-glucosidase and 5 mg of 21 K or 45 K cellulose, as indicated, at 37°C for 10, 20, 30, and 60 min. Reducing sugar (1) and glucose (2) in the reaction mixture were determined. Reaction products were analyzed by TLC (3). (B) Laminarin (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min. The glucose content in the reaction mixture was then determined. Reaction products were analyzed by TLC. (C) Lichenan (1 mL, 1% in 50 mM acetate, pH 5.5) was incubated with 2 µg of 110 K or 210 K ß-glucosidase at 37°C for 10, 20, and 60 min.
Mentions: The cleavage specificity of 110K and 210K ß-glucosidase toward various synthetic and natural substrates was compared. Specific activity of both ß-glucosidases toward 4MU-ß-glucoside, 4MU-ß-galactoside, cellobiose, lichenan, and laminarin is shown in Table 2. 210K ß-glucosidase exhibited higher specific activities against all substrates examined than 110K ß-glucosidase. Among the synthetic substrates examined, both ß-glucosidases were most active against 4-MU-ß-glucoside. Compared with the maximum activity toward 4MU-ß-glucoside, 110K and 210K ß-glucosidase exhibited 34–35% activity toward 4-MU-ß-galactoside. Both ß-glucosidases showed weak activity toward 4MU-ß-xyloside (110K: 0.9%, 210K: 3.5%). Neither the 110K nor the 210K enzyme exhibited activity toward 4MU-α-glucoside, 4MU-α-galactoside, or 4MU-α-mannoside. Only a small amount of glucose was released from CMC by these ß-glucosidases (Figure 2C). All cello-oligosaccharides were completely hydrolyzed to glucose by both ß-glucosidases (Figure 2D). To investigate the mode of hydrolysis of cellohexaose, the time-course of degradation of cellohexaose by 110K or 210K ß-glucosidase was analyzed (Figure 2E). Cellohexaose was hydrolyzed into cellopentaose, cellotetraose, cellotriose, cellobiose, and glucose within 20 min (210 K) and 5 min (110 K). These results suggest that 210 K ß-glucosidase prefers longer oligo-cellulose than cellotriose. It is noteworthy that both enzymes exhibited weak lactase (Figure 2F) and gentiobiose cleavage activities (data not shown). Figure 3 shows the glucose-releasing activity of 110 K and 210 K ß-glucosidase toward CMC, laminarin, and lichenan. Lichenan is a ß-D-glucan that consists of repeating glucose units linked by ß-1,4 and ß-1,3 glycosidic bonds. Laminarin is a storage polysaccharide of seaweed consisting of a ß-1,3-linked glucose main chain and ß-1,6-linked glucose branches. Although collaboration between cellulases is necessary for production of glucose from CMC by ß-glucosidase (Figure 3A), laminarin and lichenan were efficiently digested to glucose only by 210 K ß-glucosidase (Figure 3B, 3C). Glucose release from laminarin and lichenan was confirmed by a quantitative glucose assay using glucose oxidase. Cardran, a polysaccharide consisting of ß-1,6-linked glucose, was also cleaved by both ß-glucosidases. The relative activity toward cardran of 110 K and 210 K ß-glucosidase was 12% and 32%, respectively, of the activity against laminarin. These results suggest that the primary role of 110 K and 210 K ß-glucosidase is the decomposition of cello-oligosaccharides and laminarin to glucose.

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