Molecular cloning and biochemical characterization of a family-9 endoglucanase with an unusual structure from the gliding bacteria Cytophaga hutchinsonii.
Cytophaga hutchinsonii was originally isolated from sugarcane piles. This microorganism therefore probably produces an array of enzymes allowing it to digest cellulosic substrates. C. hutchinsonii thus represents a rich source of potentially effective cellulase enzymes that can be harnessed for conversion of biomass to simple sugars. These sugars can then be used as feedstock for ethanol production or other chemical syntheses. In this study, we report the PCR cloning of an endoglucanase gene (Cel9A) from C. hutchinsonii using degenerated primers directed at the catalytic domain. Alignment of the amino acids sequence revealed that Cel9A has a gene structure totally different from the other known cellulose degraders. The most striking feature of this cloned protein is the absence of a cellulose-binding domain (CBD), which to date was believed to be imperative in cellulose hydrolysis. Consequently, the Cel9A gene, encoding beta-1,4 endoglucanase from C. hutchinsonii was overexpressed in Escherichia coli with a His-Tag based expression vector. The resulting polypeptide, with a molecular mass of 105 KDa, was purified from cell extracts by affinity chromatography on cellulose. Mature Cel9A was optimally active at pH 5.0 and 45 degrees C. The enzyme efficiently hydrolyzes carboxymethyl- cellulose (CMC). Analysis of CMC and filter paper hydrolysis suggests that Cel9A is a nonprocessive enzyme with endo-cellulase activities.
Louime
Clifford
C
Environmental Sciences Institute, FSH Science Research Center, Florida A&M University, Tallahassee, FL 32307, USA. CLouime@som.umaryland.edu
Abazinge
Michael
M
Johnson
Elijah
E
Latinwo
Lekan
L
Ikediobi
Christopher
C
Clark
Ann-Marie
AM
D000595:Amino Acid Sequence; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003589:Cytophaga / Q000201:enzymology* / Q000235:genetics*; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D002480:Cellulase