Activity enhancement of Cel5Z from Pectobacterium chrysanthemi PY35 by removing C-terminal region.
The phytopathogenic bacterium Pectobacteium chrysanthemi PY35 secretes Cel5Z endoglucanase belonging to the glycoside hydrolase family 5 of EC 3.2.1.4. The mutation of cel5Z::Omega gene was constructed by cloning the 2.0-kb SmaI fragment containing the streptomycin/spectinomycin-resistance gene of pHP45(Omega) into the BalI site of pPY100. The insertion of Omega fragment generated a new stop codon, removing the Ser/Thr-rich linker region and the cellulose binding domain (CBD) in the C-terminal region of cel5Z gene. By subsequent subcloning from this 4.9-kb fragment (pPY1001), a 1.0-kb (pPY1002) fragment was obtained and designated as cel5Z::Omega. The cel5Z::Omega gene had an open reading frame (ORF) of 1011 bp, encoding 336 amino acids, starting with an ATG codon and ending with a new TGA stop codon. The molecular mass of the Cel5Z::Omega protein in E. coli transformant appeared to be 32 kDa by SDS-PAGE analysis in the presence of carboxymethyl-cellulose (CMC). The Cel5Z::Omega protein hydrolyzed CMC with 1.7-fold higher activity than the intact Cel5Z cellulase.
Park
Sang Ryeol
SR
Research Institute of Life Science, Gyeongsang National University, Chinju 660-701, Korea.
Cho
Soo Jeong
SJ
Kim
Min Keun
MK
Ryu
Sung Kee
SK
Lim
Woo Jin
WJ
An
Chang Long
CL
Hong
Su Young
SY
Kim
Jeong Hwan
JH
Kim
Hoon
H
Yun
Han Dae
HD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016974:Dickeya chrysanthemi / Q000201:enzymology*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D015183:Restriction Mapping; D017384:Sequence Deletion; D013696:Temperature
D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; D002480:Cellulase; C449655:cel5Z protein, Erwinia chrysanthemi