The non-catalytic cellulose-binding domain of a novel cellulase from Pseudomonas fluorescens subsp. cellulosa is important for the efficient hydrolysis of Avicel.
A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA, constructed in lambda ZAPII, was screened for carboxymethyl-cellulase activity. The pseudomonad insert from a recombinant phage which displayed elevated cellulase activity in comparison with other cellulase-positive clones present in the library, was excised into pBluescript SK- to generate the plasmid pC48. The nucleotide sequence of the cellulase gene, designated celE, revealed a single open reading frame of 1710 bp that encoded a polypeptide, defined as endoglucanase E (CelE), of M(r) 59663. The deduced primary structure of CelE revealed an N-terminal signal peptide followed by a 300-amino-acid sequence that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase Family 5. Adjacent to the catalytic domain was a 40-residue region that exhibited strong sequence identity to non-catalytic domains located in two other endoglucanases and a xylanase from P. fluorescens. The C-terminal 100 residues of CelE were similar to Type-I cellulose-binding domains (CBDs). The three domains of the cellulase were joined by linker sequences rich in serine residues. Analysis of the biochemical properties of full-length and truncated derivatives of CelE confirmed that the enzyme comprised an N-terminal catalytic domain and a C-terminal CBD. Analysis of purified CelE revealed that the enzyme had an M(r) of 56000 and an experimentally determined N-terminal sequence identical to residues 40-54 of the deduced primary structure of full-length CelE. The enzyme exhibited an endo mode of action in hydrolysing a range of cellulosic substrates including Avicel and acid-swollen cellulose, but did not attack xylan or any other hemicelluloses. A truncated form of the enzyme, which lacked the C-terminal CBD, displayed the same activity as full-length CelE against soluble cellulose and acid-swollen cellulose, but exhibited substantially lower activity than the full-length cellulase against Avicel. The significance of these data in relation to the role of the CBD is discussed.
Hall
J
J
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, UK.
Black
G W
GW
Ferreira
L M
LM
Millward-Sadler
S J
SJ
Ali
B R
BR
Hazlewood
G P
GP
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D002850:Chromatography, Gel; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D004269:DNA, Bacterial; D002482:Cellulose; D002480:Cellulase