Studies on some aspects of gene organization and cellulolysis in Fibrobacter succinogenes S85

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Iyo, Abiye Herbert
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University of Guelph

Fibrobacter succinogenes is a predominant cellulolytic rumen bacterium. It possesses multiple cellulase activities as evidenced from the number of cellulase genes that have been cloned and expressed. The cellodextrinase (cedA) and two endoglucanase genes (celD and celG) were characterized. The cellodextrinase gene was sequenced and found to encode a single catalytic domain of 357 amino acids with a calculated molecular mass of 41.9 kDa. The nucleotide sequence of the endoglucanase gene celG, was found to encode a 519 amino acid polypeptide of approximately 55 kDa. The N-terminal region which contained the catalytic domain was linked to the C-terminal basic domain by an array of serine-rich periodic sequences (SRPS). Purified recombinant CelG had pH and temperature optima of 5.5 and 25\sp∘C, respectively, with a specific activity of 16.5 μmol.min\sp−1mg\sp−1 on barley-β-glucan. The enzyme produces a mixture of cellooligosaccharides from the hydrolysis of acid swollen cellulose. The distribution of the celG gene amongst Fibrobacter subspecies was restricted primarily to the type strain S85. Endoglucanase D (EGD) like CelG contains a N-terminal catalytic domain linked to a C-terminal basic domain by an array of SRPS. Over-expressed EGD and its truncated form lacking the SRPS and basic terminal domain (BTD) were compared. Both enzymes had similar pH and temperature optima of 6.0 and 35\sp∘C, respectively, but were distinct in their kinetic properties. While K\sbm for lichenan were 5.41 and 1.63 mg/ml for EGD\sbCAT and EGD, respectively, barley-β-glucan presented much different values of $>10mg/mlforEGD\rm\sb{CAT}$ and 0.22 mg/ml for EGD. Endoglucanase D had a unique localization pattern, it was detected only in the culture fluid of cellulose grown cells. The enzyme was not present in glucose grown cells, indicating it was cellulose induced. This location would mean that the EGD associated BTD had no role in cell adhesion. Instead it appears to have a specific involvement in modulating the K\sbm for hydrolysis of selected glucan substrates.

Fibrobacter succinogenes, cellulolytic rumen bacterium, cellulase genes, cellodextrinase gene, cedA, endoglucanase genes, celD, celG