| Summary: | The CenC gene of Cellulomonas fimi, encoding endoglucanase CenC had an open reading
frame of 1101 codons closely followed by a 9 bp inverted repeat. The amino acid sequence of
mature CenC, which was 1069 amino acids long, is very unusual in that it had a 150 amino acid-long
tandem repeat (N1N2) at the N-terminus and an unrelated 100 amino acid-long tandem repeat
(C1C2) at the C-terminus. Similarity of the central domain of CenC to the catalytic domains of
other endoglucanases placed CenC in subfamily El of the β-l,4-glycanases. CenC could be
affinity purified on cellulose or Sephadex. The catalytic properties of recombinant CenC from E.
coil, for the substrate carboxymethylcellulose were indistinguishable from those of native CenC
from C. fimi.
In order to determine which of the repeats N1N2 or C1C2 bind to cellulose or to
Sephadex, both repeats were cloned separately. N1N2 mediated binding to both cellulose and
Sephadex. Nl or N2 alone did not bind Sephadex but did bind cellulose. The C-terminal repeats,
alone or in combination, did not mediate binding to cellulose or to Sephadex. N1N2 bound to
regenerated cellulose (phosphoric acid swollen cellulose) but had negligible affinity for bacterial
microcrystalline cellulose.
To show that the N-terminal repeats could be used as an affinity tag for a polypeptide other
than CenC, N1N2 was fused to the catalytic domain of CenA (another endoglucanase from C.
fimi). The resulting fusion polypeptide C’ ‘A could be affinity purified on cellulose or Sephadex
and retained catalytic activity. C’ ‘A was also used to study the influence of the binding domain on
the hydrolysis of cellulosic substrates by comparison to CenA. C’ ‘A had higher activity on the
amorphous substrate cellulose azure when compared to CenA. C’ ‘A and CenA had similar
activities on regenerated cellulose with the most striking difference being the poor activity of C’ ‘A
on crystalline cellulose. The ability (or lack thereof) of the binding domain to adsorb to crystalline
cellulose correlated well with the ability of the enzyme to hydrolyze the substrate. === Science, Faculty of === Microbiology and Immunology, Department of === Graduate
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