Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains.
The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.
Poole
D M
DM
Department of Agricultural Biochemistry and Nutrition, University of Newcastle upon Tyne, U.K.
Durrant
A J
AJ
Hazlewood
G P
GP
Gilbert
H J
HJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002482:Cellulose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010459:Peptococcaceae / Q000201:enzymology* / Q000235:genetics; D011549:Pseudomonas / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D002482:Cellulose; D002480:Cellulase