Limits...
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.

Show MeSH

Related in: MedlinePlus

SDS-PAGE and amino acid sequence of purified enzymes.(A) SDS-PAGE of purified enzymes (2 µg protein). The marker proteins were as follows: myosin heavy chain (200 kDa), ß-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (67 kDa), ovalbumin (45 kDa), and glyceraldehyde-3-phosphate dehydrogenase (36 kDa). (B) Alignment of N-terminal and internal sequences of purified enzymes with other endo-ß-1,4-glucanases from freshwater snail (UniProt: A7KMF0, A0SGK2), brackish water clam (B9X0W1), abalone (B6RB06, Q86M37), and scallop (C6L866) and ß-glucosidases from brackish water clam (B5U9B3) and termite (D0VYS0). The molecular mass of A7KMF0, B6RB06, B9X0W1, Q86M37, C6L866, A0SGK2, B5U9B3, and D0VYS0 is 19 kDa, 21 kDa, 22.6 kDa, 66 kDa, 64 kDa, 66 kDa, 110 kDa, and 55 kDa, respectively. The internal sequences of fragments (LEP#37 from 21K cellulase, LEP#30 from 45K cellulase, LEP#5 from 65K cellulase, LEP#59 from 110K ß-glucosidase, LEP#33 and 43 from 210K ß-glucosidase) generated by lysyl endopeptidase digestion of purified enzymes were determined as described in Materials and Methods. The amino acid residue numbers of other endo-ß-1,4-glucanases and ß-glucosidases are indicated on both sides of the corresponding sequences.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3675134&req=5

pone-0065418-g001: SDS-PAGE and amino acid sequence of purified enzymes.(A) SDS-PAGE of purified enzymes (2 µg protein). The marker proteins were as follows: myosin heavy chain (200 kDa), ß-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (67 kDa), ovalbumin (45 kDa), and glyceraldehyde-3-phosphate dehydrogenase (36 kDa). (B) Alignment of N-terminal and internal sequences of purified enzymes with other endo-ß-1,4-glucanases from freshwater snail (UniProt: A7KMF0, A0SGK2), brackish water clam (B9X0W1), abalone (B6RB06, Q86M37), and scallop (C6L866) and ß-glucosidases from brackish water clam (B5U9B3) and termite (D0VYS0). The molecular mass of A7KMF0, B6RB06, B9X0W1, Q86M37, C6L866, A0SGK2, B5U9B3, and D0VYS0 is 19 kDa, 21 kDa, 22.6 kDa, 66 kDa, 64 kDa, 66 kDa, 110 kDa, and 55 kDa, respectively. The internal sequences of fragments (LEP#37 from 21K cellulase, LEP#30 from 45K cellulase, LEP#5 from 65K cellulase, LEP#59 from 110K ß-glucosidase, LEP#33 and 43 from 210K ß-glucosidase) generated by lysyl endopeptidase digestion of purified enzymes were determined as described in Materials and Methods. The amino acid residue numbers of other endo-ß-1,4-glucanases and ß-glucosidases are indicated on both sides of the corresponding sequences.

Mentions: Four cellulases (21K, 45K, 65K and 95K) and 2 ß-glucosidases (110K and 210K) were purified from digestive fluid of the sea hare, as described in Materials and Methods. The procedures used for purification of the 4 cellulases and 2 ß-glucosidases are shown in Figure S1. The 21K, 45K, and 65K cellulase found in the CM-Sepharose bound fraction were purified using phenyl-Sepharose (Figure S2, upper) and gel filtration (Figure S2 lower). 95K cellulase and 210K and 110K ß-glucosidase found in the CM-Sepharose unbound fraction were purified using DEAE-Sepharose (Figure S3A), phenyl-Sepharose, and gel filtration (Figure S3B, 3C). The 210K and 110K ß-glucosidase were further purified using hydroxyapatite (Figure S4A) and Mono-Q (Figure S4B), respectively. As shown in Figure S4B, 210K ß-glucosidase is active against 4MU-ß-glucoside and laminarin. Final preparations yielded a single protein band on SDS-PAGE [26] (Figure 1A), and the molecular mass of these enzymes was estimated as 21 kDa, 45 kDa, 65 kDa, 95 kDa, 110 kDa, and 210 kDa by SDS-PAGE. Specific activities of the purified 21K, 45K, 65K, and 95K cellulase toward CMC were 0.72, 17.1, 5.21, and 0.316 µmol/min/mg, respectively. Specific activities of 210K and 110K ß-glucosidase toward 4-MU-ß-glucoside were 69.9 and 15.5 µmol/min/mg, respectively. Considering the protein content and enzyme activity, 45K cellulase may be the most abundant cellulase in the digestive fluid of the sea hare (Figure S1).


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)

SDS-PAGE and amino acid sequence of purified enzymes.(A) SDS-PAGE of purified enzymes (2 µg protein). The marker proteins were as follows: myosin heavy chain (200 kDa), ß-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (67 kDa), ovalbumin (45 kDa), and glyceraldehyde-3-phosphate dehydrogenase (36 kDa). (B) Alignment of N-terminal and internal sequences of purified enzymes with other endo-ß-1,4-glucanases from freshwater snail (UniProt: A7KMF0, A0SGK2), brackish water clam (B9X0W1), abalone (B6RB06, Q86M37), and scallop (C6L866) and ß-glucosidases from brackish water clam (B5U9B3) and termite (D0VYS0). The molecular mass of A7KMF0, B6RB06, B9X0W1, Q86M37, C6L866, A0SGK2, B5U9B3, and D0VYS0 is 19 kDa, 21 kDa, 22.6 kDa, 66 kDa, 64 kDa, 66 kDa, 110 kDa, and 55 kDa, respectively. The internal sequences of fragments (LEP#37 from 21K cellulase, LEP#30 from 45K cellulase, LEP#5 from 65K cellulase, LEP#59 from 110K ß-glucosidase, LEP#33 and 43 from 210K ß-glucosidase) generated by lysyl endopeptidase digestion of purified enzymes were determined as described in Materials and Methods. The amino acid residue numbers of other endo-ß-1,4-glucanases and ß-glucosidases are indicated on both sides of the corresponding sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675134&req=5

pone-0065418-g001: SDS-PAGE and amino acid sequence of purified enzymes.(A) SDS-PAGE of purified enzymes (2 µg protein). The marker proteins were as follows: myosin heavy chain (200 kDa), ß-galactosidase (116 kDa), phosphorylase b (97 kDa), BSA (67 kDa), ovalbumin (45 kDa), and glyceraldehyde-3-phosphate dehydrogenase (36 kDa). (B) Alignment of N-terminal and internal sequences of purified enzymes with other endo-ß-1,4-glucanases from freshwater snail (UniProt: A7KMF0, A0SGK2), brackish water clam (B9X0W1), abalone (B6RB06, Q86M37), and scallop (C6L866) and ß-glucosidases from brackish water clam (B5U9B3) and termite (D0VYS0). The molecular mass of A7KMF0, B6RB06, B9X0W1, Q86M37, C6L866, A0SGK2, B5U9B3, and D0VYS0 is 19 kDa, 21 kDa, 22.6 kDa, 66 kDa, 64 kDa, 66 kDa, 110 kDa, and 55 kDa, respectively. The internal sequences of fragments (LEP#37 from 21K cellulase, LEP#30 from 45K cellulase, LEP#5 from 65K cellulase, LEP#59 from 110K ß-glucosidase, LEP#33 and 43 from 210K ß-glucosidase) generated by lysyl endopeptidase digestion of purified enzymes were determined as described in Materials and Methods. The amino acid residue numbers of other endo-ß-1,4-glucanases and ß-glucosidases are indicated on both sides of the corresponding sequences.
Mentions: Four cellulases (21K, 45K, 65K and 95K) and 2 ß-glucosidases (110K and 210K) were purified from digestive fluid of the sea hare, as described in Materials and Methods. The procedures used for purification of the 4 cellulases and 2 ß-glucosidases are shown in Figure S1. The 21K, 45K, and 65K cellulase found in the CM-Sepharose bound fraction were purified using phenyl-Sepharose (Figure S2, upper) and gel filtration (Figure S2 lower). 95K cellulase and 210K and 110K ß-glucosidase found in the CM-Sepharose unbound fraction were purified using DEAE-Sepharose (Figure S3A), phenyl-Sepharose, and gel filtration (Figure S3B, 3C). The 210K and 110K ß-glucosidase were further purified using hydroxyapatite (Figure S4A) and Mono-Q (Figure S4B), respectively. As shown in Figure S4B, 210K ß-glucosidase is active against 4MU-ß-glucoside and laminarin. Final preparations yielded a single protein band on SDS-PAGE [26] (Figure 1A), and the molecular mass of these enzymes was estimated as 21 kDa, 45 kDa, 65 kDa, 95 kDa, 110 kDa, and 210 kDa by SDS-PAGE. Specific activities of the purified 21K, 45K, 65K, and 95K cellulase toward CMC were 0.72, 17.1, 5.21, and 0.316 µmol/min/mg, respectively. Specific activities of 210K and 110K ß-glucosidase toward 4-MU-ß-glucoside were 69.9 and 15.5 µmol/min/mg, respectively. Considering the protein content and enzyme activity, 45K cellulase may be the most abundant cellulase in the digestive fluid of the sea hare (Figure S1).

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