Pre and post cloning characterization of a beta-1,4-endoglucanase from Bacillus sp.
Consistent with its precloning characterization from the cellulolytic Bacillus sp., beta-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60 degrees C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70 degrees C and over a pH range of 6.0-8.0. The K(m) and V(max) values for CMCase activity of the enzyme were 4.1 mg/ml and 25 micromole/ml min(-1), respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.
Afzal
Sumra
S
Institute of Biochemistry and Biotechnology, University of the Punjab, Lahore, 54590, Pakistan.
Saleem
Mahjabeen
M
Yasmin
Riffat
R
Naz
Mamoona
M
Imran
Muhammad
M
D001407:Bacillus / Q000201:enzymology*; D002480:Cellulase / Q000235:genetics* / Q000302:isolation & purification; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011995:Recombination, Genetic; D013696:Temperature; D014170:Transformation, Genetic
D002480:Cellulase