Cloning and sequencing of the celA gene encoding CMCase of Erwinia carotovora subsp. carotovora LY34.

The phytopathogenic Erwinia carotovora subsp. carotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases. Genomic DNA from Ecc LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript II SK+. One of the E. coli clones containing a Sau3AI fragment of Ecc genomic DNA hydrolyzed carboxymethyl cellulose and was shown to contain the 2.2 kb BamHI restriction fragment, which was subcloned to generate pLYCA100 named as celA. The structural organization of a celA gene encoding 387 amino acids consists of an open reading frame (ORF) of 1161 bp starting with an ATG start codon and followed by a TAA stop codon. CelA protein contained a typical catalytic domain, interdomain, cellulose binding domain, and prokaryotic signal peptide of 32 amino acids. Since the deduced amino acid sequences of CelA protein was very similar to those of CelV of Erwinia carotovora subsp. carotovora SCC3193 enzyme and to those of CelN of Erwinia atroceptica enzyme, it belongs to the cellulase family 5. The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). Activity staining of carboxymethyl cellulase (CMCase) in sodium dodecyl sulfate polyacrylamide gel containing 0.1% CMC revealed that the cloned isozyme comigrated with a corresponding isozyme produced by Ecc LY34. The CelA had a calculated pI of 5.42. The optimum pH was 7 and the optimum temperature was about 45 degrees C.