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A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85.

ABSTRACT: We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenes S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25 degrees C, and retains about 70% of its maximum activity at 0 degrees C, while EGD has a temperature optimum of 35 degrees C and retains only about 18% of its maximal activity at 0 degrees C. When assayed at 4 degrees C, CelG exhibits a 33-fold-higher kcat value and a 73-fold-higher physiological efficiency (kcat/Km) than EGD. CelG has a low thermal stability, as indicated by the effect of temperature on its activity and secondary structure. The presence of small amino acids around the putative catalytic residues may add to the flexibility of the enzyme, thereby increasing its activity at cold temperatures. Its activity is modulated by sodium chloride, with an increase of over 1.8-fold at an ionic strength of 0.03. Possible explanations for the presence of a cold-active enzyme in a mesophile are that cold-active enzymes are more broadly distributed than previously expected, that lateral transfer of the gene from a psychrophile occurred, or that F. succinogenes originated from the marine environment.

SUBMITTER: Iyo AH

PROVIDER: S-EPMC91134 | biostudies-literature | 1999 Mar

REPOSITORIES: biostudies-literature

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A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85.

Iyo A H AH Forsberg C W CW

Applied and environmental microbiology 19990301 3

We previously characterized two endoglucanases, CelG and EGD, from the mesophilic ruminal anaerobe Fibrobacter succinogenes S85. Further comparative experiments have shown that CelG is a cold-active enzyme whose catalytic properties are superior to those of several other intensively studied cold-active enzymes. It has a lower temperature optimum, of 25 degrees C, and retains about 70% of its maximum activity at 0 degrees C, while EGD has a temperature optimum of 35 degrees C and retains only abo ...[more]

PMID: 10049853

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Project description:Fibrobacter succinogenes is a cellulolytic bacterium that plays an essential role in the degradation of plant fibers in the rumen ecosystem. It converts cellulose polymers into intracellular glycogen and the fermentation metabolites succinate, acetate, and formate. We developed dynamic models of F. succinogenes S85 metabolism on glucose, cellobiose, and cellulose on the basis of a network reconstruction done with the automatic reconstruction of metabolic model workspace. The reconstruction was based on genome annotation, five template-based orthology methods, gap filling, and manual curation. The metabolic network of F. succinogenes S85 comprises 1,565 reactions with 77% linked to 1,317 genes, 1,586 unique metabolites, and 931 pathways. The network was reduced using the NetRed algorithm and analyzed for the computation of elementary flux modes. A yield analysis was further performed to select a minimal set of macroscopic reactions for each substrate. The accuracy of the models was acceptable in simulating F. succinogenes carbohydrate metabolism with an average coefficient of variation of the root mean squared error of 19%. The resulting models are useful resources for investigating the metabolic capabilities of F. succinogenes S85, including the dynamics of metabolite production. Such an approach is a key step toward the integration of omics microbial information into predictive models of rumen metabolism. IMPORTANCE F. succinogenes S85 is a cellulose-degrading and succinate-producing bacterium. Such functions are central for the rumen ecosystem and are of special interest for several industrial applications. This work illustrates how information of the genome of F. succinogenes can be translated to develop predictive dynamic models of rumen fermentation processes. We expect this approach can be applied to other rumen microbes for producing a model of rumen microbiome that can be used for studying microbial manipulation strategies aimed at enhancing feed utilization and mitigating enteric emissions.

Project description:Although Fibrobacter succinogenes S85 is one of the most proficient cellulose degrading bacteria among all mesophilic organisms in the rumen of herbivores, the molecular mechanism behind cellulose degradation by this bacterium is not fully elucidated. Previous studies have indicated that cell surface proteins might play a role in adhesion to and subsequent degradation of cellulose in this bacterium. It has also been suggested that cellulose degradation machinery on the surface may be selectively expressed in response to the presence of cellulose. Based on the genome sequence, several models of cellulose degradation have been suggested. The aim of this study is to evaluate the role of the cell envelope proteins in adhesion to cellulose and to gain a better understanding of the subsequent cellulose degradation mechanism in this bacterium. Comparative analysis of the surface (exposed outer membrane) chemistry of the cells grown in glucose, acid-swollen cellulose and microcrystalline cellulose using physico-chemical characterisation techniques such as electrophoretic mobility analysis, microbial adhesion to hydrocarbons assay and Fourier transform infra-red spectroscopy, suggest that adhesion to cellulose is a consequence of an increase in protein display and a concomitant reduction in the cell surface polysaccharides in the presence of cellulose. In order to gain further understanding of the molecular mechanism of cellulose degradation in this bacterium, the cell envelope-associated proteins were enriched using affinity purification and identified by tandem mass spectrometry. In total, 185 cell envelope-associated proteins were confidently identified. Of these, 25 proteins are predicted to be involved in cellulose adhesion and degradation, and 43 proteins are involved in solute transport and energy generation. Our results supports the model that cellulose degradation in F. succinogenes occurs at the outer membrane with active transport of cellodextrins across for further metabolism of cellodextrins to glucose in the periplasmic space and inner cytoplasmic membrane.

Dataset Information

A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85.

Publications

A cold-active glucanase from the ruminal bacterium Fibrobacter succinogenes S85.

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