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

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.

Figure 1. 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.

Figure 2. Mode of action of purified cellulases and ß-glucosidases from sea hare.

(A) Effect of treatment with purified cellulases or 210K ß-glucosidase on CMC viscosity. CMC (2 mL, 40 mg/mL in 50 mM acetate, pH 5.5) was incubated at 37°C for 4, 8, 10, 12, and 24 h in the absence or presence of the enzyme (0.2 µg) and the viscosity of the CMC solution was then measured as described in Materials and Methods. (B) Degradation of cello-oligosaccharides by purified cellulase. Cello-oligosaccharides (50 mL, 20 mg/mL in 20 mM acetate buffer, pH 5.5) were incubated with the enzyme (0.1 µg) at 37°C for 24 h and then subjected to TLC as described in Materials and Methods. G1, glucose; G2, cellobiose; G3, cellotriose; G4, cellotetraose; G5, cellopentaose; G6, cellohexaose. (C) Degradation of filter paper and CMC by purified cellulase and ß-glucosidases. Filter paper (60 mg) was incubated with 10 µg of the purified enzyme at 37°C for 15 h in 1 mL of 50 mM acetate buffer (pH 5.5). Furthermore, 1% CMC in 1 mL of 50 mM acetate buffer (pH 5.5) was incubated with the purified enzymes (2 µg) at 37°C for 1 h. The reaction mixture (2 mL) was subjected to TLC. (D) Digestion of cello-oligosaccharides with 210K or 110K ß-glucosidase. Cello-oligosaccharides (50 µL, 2 mg/mL in 10 mM acetate buffer, pH 5.5) were incubated with the enzyme (0.2 µg) at 37°C for 4 h and then subjected to TLC. (E) Time-course of hydrolysis of cellohexaose by the enzyme. Purified ß-glucosidase (BGL, 0.2 µg) was incubated with cellohexaose (50 µL, 20 mg/mL in 20 mM acetate, pH 5.5) for the time indicated. (F) Hydrolysis of lactose with 210K or 110K ß-glucosidase. Lactose (50 µL, 20 mg/mL in 20 mM acetate, pH 5.5) was incubated with the enzyme (0.2 µg) at 37°C for 4 h.

Figure 3. 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.

Figure 4. 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.