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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.


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).
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pone.0138902.g008: 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).

Mentions: In order to delineate the mechanism of cattle saliva mediated enhancement of cellulase activity, we thought that it was necessary to determine whether cattle saliva interacts with cellulase, cellulose or both. Thus, we carried out an addition order experiment performed according to the strategy shown in Fig 7A. Results obtained from this addition order experiment were shown in Fig 7B. First, in the absence of cattle saliva (Saliva (-)), the amount of glucose produced under the experimental conditions ‘Simultaneous’, ‘Added with cellulase’, ‘Added with saliva’ and ‘Simultaneous (25 h)’ was approximately 0.070 mg/mL, whereas the amount of glucose produced under the ‘Added with cellulose’ condition was 0.049 mg/mL. The decrease in cellulase activity under the ‘Added with cellulose’ condition was probably because cellulase was diluted and it became partly denatured as a result of incubation at 50°C for 1 h in the absence of any cellulose molecule to interact with. Second, in the presence of cattle saliva (Saliva (+)), the amount of glucose produced under experimental conditions ‘Simultaneous’, ‘Added with cellulase’, ‘Added with cellulose’, and ‘Simultaneous (25 h)’ was 0.150 mg/mL, whereas the amount of glucose produced under the ‘Added with saliva’ condition was 0.129 mg/mL, indicating that the enhancement effect of cattle saliva was slightly decreased when the cattle saliva was added at a later stage. Thus, the production of reducing sugar, to some extent, was dependent on the addition order by which the components were mixed. The observed result also suggested that cattle saliva needed to interact with cellulose before it was degraded by cellulase. In order to confirm that cattle saliva interacts with cellulose, we used SDS-PAGE analysis and Bradford protein assay to determine whether cattle saliva proteins would get adsorbed to cellulose. As shown in Fig 8, proteins were detected in the Supernatant, Wash 1, Wash 2 and Wash 3 fractions, but the amount of proteins in these fractions decreased with the number of wash; however, more proteins were eluted off the cellulose after it was mixed with SDS and the mixture was incubated at 96°C for 1 h. Thus, several cattle saliva proteins were adsorbed to cellulose. Taken together, these results suggested that the enhancement effect was caused by the adsorbed non-enzymatic proteins in cattle saliva.


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)

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).
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Related In: Results  -  Collection

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pone.0138902.g008: 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).
Mentions: In order to delineate the mechanism of cattle saliva mediated enhancement of cellulase activity, we thought that it was necessary to determine whether cattle saliva interacts with cellulase, cellulose or both. Thus, we carried out an addition order experiment performed according to the strategy shown in Fig 7A. Results obtained from this addition order experiment were shown in Fig 7B. First, in the absence of cattle saliva (Saliva (-)), the amount of glucose produced under the experimental conditions ‘Simultaneous’, ‘Added with cellulase’, ‘Added with saliva’ and ‘Simultaneous (25 h)’ was approximately 0.070 mg/mL, whereas the amount of glucose produced under the ‘Added with cellulose’ condition was 0.049 mg/mL. The decrease in cellulase activity under the ‘Added with cellulose’ condition was probably because cellulase was diluted and it became partly denatured as a result of incubation at 50°C for 1 h in the absence of any cellulose molecule to interact with. Second, in the presence of cattle saliva (Saliva (+)), the amount of glucose produced under experimental conditions ‘Simultaneous’, ‘Added with cellulase’, ‘Added with cellulose’, and ‘Simultaneous (25 h)’ was 0.150 mg/mL, whereas the amount of glucose produced under the ‘Added with saliva’ condition was 0.129 mg/mL, indicating that the enhancement effect of cattle saliva was slightly decreased when the cattle saliva was added at a later stage. Thus, the production of reducing sugar, to some extent, was dependent on the addition order by which the components were mixed. The observed result also suggested that cattle saliva needed to interact with cellulose before it was degraded by cellulase. In order to confirm that cattle saliva interacts with cellulose, we used SDS-PAGE analysis and Bradford protein assay to determine whether cattle saliva proteins would get adsorbed to cellulose. As shown in Fig 8, proteins were detected in the Supernatant, Wash 1, Wash 2 and Wash 3 fractions, but the amount of proteins in these fractions decreased with the number of wash; however, more proteins were eluted off the cellulose after it was mixed with SDS and the mixture was incubated at 96°C for 1 h. Thus, several cattle saliva proteins were adsorbed to cellulose. Taken together, these results suggested that the enhancement effect was caused by the adsorbed non-enzymatic proteins in cattle saliva.

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.