Fig 1.
Effects of cattle saliva on cellulose degradation.
(a) Enhancement effect of cattle saliva. Effect of cattle saliva addition on the production of reducing sugar from micro-crystalline cellulose. Reaction mixtures containing 10 μg/mL cellulase and 0.8% (wt%) cellulose were incubated in the presence or absence of 10% cattle saliva at 50°C for 24 h. Effects of (b) cellulase concentration, (c) incubation time and (d) cattle saliva concentration on reducing sugar production. In (b), concentrations of cellulase used were 0, 1, 5, 10, 50, 100, 500 and 1000 μg/mL, while the concentration of cellulose was kept same as in (a) above and the reaction mixtures were incubated at 50°C for 24 h. In (c), different incubation times were used (0, 1, 3, 6, 12, 24, 48 and 72 h) while keeping the composition of the reaction mixture same as in (a) above. In (d), different concentrations of cattle saliva were used here: 0, 0.5, 1, 2, 3, 4, 7 and 10%; concentrations of cellulase and cellulose and reaction conditions were same as in (a) above. All experiments were performed in triplicate and results are expressed as average means. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).
Fig 2.
Properties of cattle saliva on real biomass degradation.
Effects of (a) cellulase concentration and (b) incubation time on cellulose conversion. (a) Cellulase concentrations were 0, 10, 50, 100 and 250 μg/mL, while concentration of cattle saliva was constantly 10%. The reaction mixtures were incubated at 50°C for 24 h. (b) Different incubation times were tested (0, 12, 24, 48 and 72 h), while concentrations of cellulase and cattle saliva were constantly 50 μg/mL and 10%, respectively. The reaction mixtures were incubated at 50°C. All experiments were performed in triplicate and average mean values were plotted. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).
Fig 3.
Effect of various treatments on the enhancement effect of cattle saliva.
(a) Denatured and dialyzed cattle saliva. Denaturation of cattle saliva: Cattle saliva was autoclaved for 13 minites at 121°C to denature proteins. After that, the saliva was centrifuged at 20,400 x g for 10 min. The supernatant (called ‘Autoclaved saliva’) was collected and subsequently used in experiments. Dialysis of cattle saliva: Cattle saliva was dialyzed against distilled water for 72 h at room temperture. The water was exchanged every other day. (b) Proteinase K treatment. Twenty microliters cattle saliva was mixed with 20 μL proteinase K (20 mg/mL) and the mixture was incubated at 50°C for 12 h. After the incubation, the mixture was incubated at 96°C for 10 min to denature proteinase K. This mixture was called ‘Proteinase K Saliva’ and used in the cellulose degradation assay. The concentration of cattle saliva in the reaction mixture was 5%. All experiments were performed in triplicate and average mean values were plotted. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).
Fig 4.
Removal of protein in cattle saliva.
(a) Protein concentration in cattle saliva treated with methanol or acetone. Protein concentration was measured using Bradford protein assay. (b) Enhancement effect of cattle saliva treated with methanol or acetone. Cattle saliva treated with methanol or acetone used in the cellulose degradation assay. The reaction condition follows the basic experimental protocol. All experiments were performed in triplicate and average mean values were plotted. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).
Fig 5.
Fractionation of cattle saliva proteins.
(a) SDS-PAGE analysis of fractionated cattle saliva proteins. Proteins present in the cattle saliva were fractionated by gel filtration chromatography. An aliquot of each fraction was used for SDS-PAGE analysis, following which the acrylamide gel was stained with silver stain. Collected fractions were then divided as indicated (based on molecular weight distribution of proteins) into five sample groups (called here as sample A, B, C, D and E) for further use. (b) Enhancement effect of fractionated cattle saliva. Cellulase-catalyzed cellulose degradation assay (in triplicate) was performed using one of the fractionated saliva samples (A, B, C, D, or E) or Mix sample as an additive, and the amount of reducing sugar produced in each case was measured. Error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).
Fig 6.
Crystal structure analysis of cellulose.
The crystal structure of cellulose in the presence of cattle saliva (Saliva(+)) or in the absence of cattle saliva (Saliva(-)) was analyzed by (a) X-ray diffraction and (b) FT-IR.
Fig 7.
(a) Schematic representation of the experimental design. (b) Effect on the production of reducing sugar. The amount of reducing sugar produced at each addition order experimental condition, shown schematically in (a), was measured. Simultaneous: A mixture in which cellulose, cellulase and cattle saliva were added simultaneously. Added with cellulase: Cellulase was added to a mixture containing cellulose and cattle saliva. Added with cellulose: Cellulose was added to a mixture containing cellulase and cattle saliva. Added with saliva: Cattle saliva was added to a mixture containing cellulose and cellulase. Simultaneous (25 hours): A mixture in which cellulose, cellulase and cattle saliva were added simultaneously and incubated for 25 h. Error bars indicate ± standard deviations (n = 9). Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).
Fig 8.
Adsorption analysis of cattle saliva proteins.
Adsorption of cattle saliva proteins to cellulose was analyzed using (a) SDS-PAGE and (b) Bradford protein assay. (a) SDS-PAGE analysis. Lane 1: Cattle saliva solution (80%). Lane 2: Supernatant, supernatant after the mixture was incubated at 50°C for an hour. Lane 3: Wash 1, supernatant of Wash buffer 1. Lane 4: Wash 2, supernatant of Wash buffer 2. Lane 5: Wash 3, supernatant of Wash buffer 3. Lane 6: Elute, eluted fraction after the cellulose pellet was mixed with 0.5% SDS and incubated at 96°C for 1 h. (b) Amount of protein in each sample used for SDS-PAGE analysis was quantified by Bradford protein assay. Error bars indicated ± deviations (n = 3).
Fig 9.
Comparison and competition between cattle saliva and canonical additives.
(a) Comparison between the enhancing effects of cattle saliva and canonical additives. The amount of reducing glucose produced was measured using optimal concentration one of the following additives: PEG 4000 (50 mg/mL), Tween 20 (2.5 mg/mL) BSA (0.01 mg/mL), cattle saliva (7.75%) or none. (b) Competition between cattle saliva and Tween 20. Concentrations of cattle saliva and Tween 20 required for saturating the cellulase catalyzed production of reducing sugar were 7.75% and 2.5 mg/mL, respectively. All experiments were performed in triplicate and error bars indicate ± standard deviations. Values labeled with asterisk are statistically different as established by Student's t-test (P < 0.05).