Cloning of the cel8Y gene from Pectobacterium chrysanthemi PY35 and its comparison to cel genes of soft-rot Pectobacterium.
The phytopathogenic Pectobacterium chrysanthemi (Pch) PY35 secretes multiple isozymes of plant cell wall degrading enzyme cellulases. We cloned a second cel gene that encodes cellulase in Pch PY35. The inserted 2 kb fragment was subcloned in order to geneate pPY710 (cel8Y). The structural organization of the cel8Y gene consists of an open reading frame (ORF) of 999 bp that encodes 332 amino acid residues with a signal peptide of 23 amino acids. The predicted amino acid sequence of Cel8Y was very similar to that of Cellulomonas uda, but completely different from that of the Cel5Z of Pch PY35. It belonged to the glycoside hydrolase family 8, based on amino acid sequence similarities in contrast to Cel5Z of Pch PY35, which was confirmed as family 5. Cel8Y was not closely related to the known cellulases of Pectobacterium. It had the conserved region of the glycoside hydrolase family 8, ASDGDVLIAWALLKAGNKW. The apparent molecular mass of the Cel8Y protein was calculated to be approximately 34 kDa by a carboxymethylcellulosesodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE). The Cel8Y had a calculated pl of 6.49. It was optimally active at pH 7 with an approximate optimal temperature around 40 degrees C. The cellulase activity of Cel8Y was lower than that of Cel5Z.
Cho
Soo Jeong
SJ
Division of Applied Life Science, Gyeongsang National University, Chinju, Korea.
Par
Sang Ryeol
SR
Kim
Min Keun
MK
Lim
Woo Jin
WJ
Ryu
Sung Kee
SK
An
Chang Long
CL
Hong
Su Young
SY
Kim
Hoon
H
Cho
Yong Un
YU
Yun
Han Dae
HD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004269:DNA, Bacterial / Q000235:genetics; D016974:Dickeya chrysanthemi / Q000201:enzymology* / Q000235:genetics*; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000235:genetics; D007526:Isoelectric Point; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D010802:Phylogeny; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D007527:Isoenzymes; D006026:Glycoside Hydrolases; D002480:Cellulase; C449655:cel5Z protein, Erwinia chrysanthemi