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Mol. Cells 1998; 8(1): 27-35

Published online February 28, 1998

© The Korean Society for Molecular and Cellular Biology

Cloning and Sequencing of the celA Gene Encoding Endoglucanase of Erwinia carotovora subsp. carotovora LY34

Yong Woo Park, Sun Tech Lim and Han Dae Yun

Abstract

The phytopathogenic Erwinia earotovora subsp. earotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases. Genomic DNA from Eee LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript n SK+. One of the E. coli clones containing a Sau3AI fragment of Eee 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 earotovora subsp. carotovora SCC3193 enzyme and to those of CelN of Erwinia atroeeptiea enzyme, it belongs to the cellulase family s. The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfatepolyacrylamide 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℃.

Article

Research Article

Mol. Cells 1998; 8(1): 27-35

Published online February 28, 1998

Copyright © The Korean Society for Molecular and Cellular Biology.

Cloning and Sequencing of the celA Gene Encoding Endoglucanase of Erwinia carotovora subsp. carotovora LY34

Yong Woo Park, Sun Tech Lim and Han Dae Yun

Abstract

The phytopathogenic Erwinia earotovora subsp. earotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases. Genomic DNA from Eee LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript n SK+. One of the E. coli clones containing a Sau3AI fragment of Eee 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 earotovora subsp. carotovora SCC3193 enzyme and to those of CelN of Erwinia atroeeptiea enzyme, it belongs to the cellulase family s. The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfatepolyacrylamide 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℃.

Mol. Cells
Dec 31, 2021 Vol.44 No.12, pp. 861~919
COVER PICTURE
Structure of the fly peripheral neurons in the fly head. Flies have basic sensory organs including eyes for vision, antennae and maxillary palps for olfaction, and proboscis (magenta) for gustation which can be labelled with monoclonal antibody 22C10. The figure is a 3D reconstructed image with 30 slices of confocal sections with 3 μm interval. It shows that the proboscis is required for sensing attractive carboxylic acids such as glycolic acid, citric acid, and lactic acid (Shrestha and Lee, pp. 900-910).

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