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Enhancement of Cellulose Degradation by Cattle Saliva.

Seki Y, Kikuchi Y, Kimura Y, Yoshimoto R, Takahashi M, Aburai K, Kanai Y, Ruike T, Iwabata K, Sugawara F, Sakai H, Abe M, Sakaguchi K - PLoS ONE (2015)

Bottom Line: Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose.Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva.We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.

ABSTRACT
Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale.

No MeSH data available.


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).
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pone.0138902.g002: 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).

Mentions: We also characterized enhancing effect of cattle saliva for the real biomass substrate (Fig 2). First, approximately 0.15 mg/mL of glucose was detected in the non-reacted mixture. Timothy hey originally contained slight glucose. At 50 μg/mL celluase loading, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were 0.537 mg/mL (16.34%) and 0.448 mg/mL (13.38%). At 250 μg/mL celluase loading, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were 0.960 mg/mL (33.97%) and 0.968 mg/mL (35.07%). The enhancement effect occurred at a low enzyme loading (less than 100 μg/mL), paralleled with the result in pure cellulose experiment, whereas the enhancement effect did not occurred at a high enzyme loading (250 μg/mL) (Fig 2A). The maximum enhancement effect was 1.2-fold at 50 μg/mL celluase loading. In addition, the cellulose conversion rates produced with and without cattle saliva were higher for the real biomass substrate (16.34% and 13.38%) than that for pure cellulose (11.99% and 5.35%) at 50 μg/mL celluase loading (Fig 1B). We next measured the effect of reaction time for cellulose conversion at 50 μg/mL celluase loading (Fig 2B). The amount of glucose produced higher in the mixture with cattle saliva than in the mixture without cattle saliva. After incubation for 12 hours, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were 0.377 mg/mL (8.85%) and 0.296 mg/mL (6.06%). After incubation for 72 hours, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were respectively 0.737 mg/mL (23.84%) and 0.620 mg/mL (19.54%). The enhancement effect was approximately 1.2-fold when the incubation times were from 12 to 72 hours. When the incubation time was 12 hours, the rate of sugar production in the Saliva (+) mixture was higher than that in the Saliva (-) mixture. However, when incubation time was more than 12 hours, the rate of sugar production in the Saliva (+) mixture was almost same as that in the Saliva (-) mixture. Initial acceleration for the real biomass substrate degradation occurred until at least 12 hours in addition of cattle saliva. Cattle saliva also accelerated the degradation reaction of pure cellulose until 24 hours (Fig 1C). The changes of glucose production depended on the incubation time between the real biomass substrate and pure cellulose were similar. These results also showed that the enhancement rate for the real biomass substrate (1.2-fold) was lower than for pure cellulose (2.9-fold) (Figs 1A and 2A). The real biomass substrate, such as timothy hay, contains various types of organic substances and minerals, including protein, pectin, lignin, fat, calcium and phosphorus. The enhancement effect of cattle saliva may compete with some kind of substance in real biomass. Indeed, the cellulose conversion produced without cattle saliva for the real biomass (13.38%) was much higher than that for pure cellulose (5.35%). We identified the substance in cattle saliva responsible for enhancement effect in the next section in order to delineate the mechanism of enhancement effect, including the low enhancement effect for the real biomass substrate.


Enhancement of Cellulose Degradation by Cattle Saliva.

Seki Y, Kikuchi Y, Kimura Y, Yoshimoto R, Takahashi M, Aburai K, Kanai Y, Ruike T, Iwabata K, Sugawara F, Sakai H, Abe M, Sakaguchi K - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581621&req=5

pone.0138902.g002: 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).
Mentions: We also characterized enhancing effect of cattle saliva for the real biomass substrate (Fig 2). First, approximately 0.15 mg/mL of glucose was detected in the non-reacted mixture. Timothy hey originally contained slight glucose. At 50 μg/mL celluase loading, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were 0.537 mg/mL (16.34%) and 0.448 mg/mL (13.38%). At 250 μg/mL celluase loading, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were 0.960 mg/mL (33.97%) and 0.968 mg/mL (35.07%). The enhancement effect occurred at a low enzyme loading (less than 100 μg/mL), paralleled with the result in pure cellulose experiment, whereas the enhancement effect did not occurred at a high enzyme loading (250 μg/mL) (Fig 2A). The maximum enhancement effect was 1.2-fold at 50 μg/mL celluase loading. In addition, the cellulose conversion rates produced with and without cattle saliva were higher for the real biomass substrate (16.34% and 13.38%) than that for pure cellulose (11.99% and 5.35%) at 50 μg/mL celluase loading (Fig 1B). We next measured the effect of reaction time for cellulose conversion at 50 μg/mL celluase loading (Fig 2B). The amount of glucose produced higher in the mixture with cattle saliva than in the mixture without cattle saliva. After incubation for 12 hours, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were 0.377 mg/mL (8.85%) and 0.296 mg/mL (6.06%). After incubation for 72 hours, the glucose concentrations (and cellulose conversion rates to glucose) in the mixtures with and without cattle saliva were respectively 0.737 mg/mL (23.84%) and 0.620 mg/mL (19.54%). The enhancement effect was approximately 1.2-fold when the incubation times were from 12 to 72 hours. When the incubation time was 12 hours, the rate of sugar production in the Saliva (+) mixture was higher than that in the Saliva (-) mixture. However, when incubation time was more than 12 hours, the rate of sugar production in the Saliva (+) mixture was almost same as that in the Saliva (-) mixture. Initial acceleration for the real biomass substrate degradation occurred until at least 12 hours in addition of cattle saliva. Cattle saliva also accelerated the degradation reaction of pure cellulose until 24 hours (Fig 1C). The changes of glucose production depended on the incubation time between the real biomass substrate and pure cellulose were similar. These results also showed that the enhancement rate for the real biomass substrate (1.2-fold) was lower than for pure cellulose (2.9-fold) (Figs 1A and 2A). The real biomass substrate, such as timothy hay, contains various types of organic substances and minerals, including protein, pectin, lignin, fat, calcium and phosphorus. The enhancement effect of cattle saliva may compete with some kind of substance in real biomass. Indeed, the cellulose conversion produced without cattle saliva for the real biomass (13.38%) was much higher than that for pure cellulose (5.35%). We identified the substance in cattle saliva responsible for enhancement effect in the next section in order to delineate the mechanism of enhancement effect, including the low enhancement effect for the real biomass substrate.

Bottom Line: Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose.Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva.We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect.

View Article: PubMed Central - PubMed

Affiliation: Research Institute for Science and Technology, Tokyo University of Science, Noda, Chiba, Japan.

ABSTRACT
Saccharification of cellulose is a promising technique for producing alternative source of energy. However, the efficiency of conversion of cellulose into soluble sugar using any currently available methodology is too low for industrial application. Many additives, such as surfactants, have been shown to enhance the efficiency of cellulose-to-sugar conversion. In this study, we have examined first whether cattle saliva, as an additive, would enhance the cellulase-catalyzed hydrolysis of cellulose, and subsequently elucidated the mechanism by which cattle saliva enhanced this conversion. Although cattle saliva, by itself, did not degrade cellulose, it enhanced the cellulase-catalyzed degradation of cellulose. Thus, the amount of reducing sugar produced increased approximately 2.9-fold by the addition of cattle saliva. We also found that non-enzymatic proteins, which were present in cattle saliva, were responsible for causing the enhancement effect. Third, the mechanism of cattle saliva mediated enhancement of cellulase activity was probably similar to that of the canonical surfactants. Cattle saliva is available in large amounts easily and cheaply, and it can be used without further purification. Thus, cattle saliva could be a promising additive for efficient saccharification of cellulose on an industrial scale.

No MeSH data available.