PMID:9581291

Citation	Halldórsdóttir, S, Thórólfsdóttir, ET, Spilliaert, R, Johansson, M, Thorbjarnardóttir, SH, Palsdottir, A, Hreggvidsson, GO, Kristjánsson, JK, Holst, O and Eggertsson, G (1998) Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12. Appl. Microbiol. Biotechnol. 49:277-84
Abstract	A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6-7 and its highest measured initial activity at 100 degrees C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 degrees C.
Links	PubMed
Keywords	Bacterial Proteins/chemistry; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Base Sequence; Cellulase/chemistry; Cellulase/genetics; Cellulase/metabolism; Electrophoresis, Polyacrylamide Gel; Genes, Bacterial/genetics; Gram-Negative Aerobic Bacteria/enzymology; Gram-Negative Aerobic Bacteria/genetics; Gram-Negative Aerobic Bacteria/growth & development; Molecular Sequence Data; Recombinant Proteins/biosynthesis; Recombinant Proteins/metabolism; Sequence Alignment
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RHOMR:O33897		GO:0008810: cellulase activity	ECO:0000314:		F		Figures 2,3, and 4 show SDS page gels for the native CelA enzyme, the recombinant form of the enzyme, and a gel comparing the activity and protein of both. Figure 2 shows the native CelA molecular mass corresponds to the expected value. This information is important for future comparisons to recombinant types. Figure 3 shows the recombinant CelA molecular mass and activity after boiling with proteinase K. The control (lane 2) showed activity bands at higher molecular weights than the native enzyme. After boiling, activity began to disappear and a protein band of 26 kDa appeared in lanes 3, 4, and 5. Figure 4 shows an SDS gel with CMC for both enzymes. The native activity band appeared in a corresponding position to the smallest activity band of the recombinant enzyme. The same activity bands were seen regardless of heating with protease.	complete CACAO 4434
RHOMR:O33897	enables	GO:0008810: cellulase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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