THE NONCATALYTIC 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, con structed in lambda ZAPII, was screened for carboxymethylcellulase acti vity. The pseudomonad insert from a recombinant phage which displayed elevated cellulase activity in comparison with other cellulase-positiv e clones present in the library, was excised into pBluescript SK- to g enerate the plasmid pC48. The nucleotide sequence of the cellulase gen e, designated celE, revealed a single open reading frame of 1710 bp th at encoded a polypeptide, defined as endoglucanase E (CelE), of M(r) 5 9663. The deduced primary structure of CelE revealed an N-terminal sig nal peptide followed by a 300-amino-acid sequence that exhibited signi ficant 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-catalyt ic 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 doma in and a C-terminal CBD. Analysis of purified CelE revealed that the e nzyme 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 h ydrolysing a range of cellulosic substrates including Avicel and acid- swollen cellulose, but did not attack xylan or any other hemicellulose s. A truncated form of the enzyme, which lacked the C-terminal CBD, di splayed the same activity as full-length CelE against soluble cellulos e and acid-swollen cellulose, but exhibited substantially lower activi ty than the full-length cellulase against Avicel. The significance of these data in relation to the role of the CBD is discussed.