Specific adhesion and hydrolysis of cellulose by intact Escherichia coli expressing surface anchored cellulase or cellulose binding domains.
The entire Cex exoglucanase from Cellulomonas fimi and the Cex Cellulose Binding Domain (CBDCex) were expressed in Escherichia coli as fusions to an Lpp-OmpA hybrid which had been shown earlier to direct a heterologous protein to the cell surface. Both Cex and CBDCex were readily localized on the cell surface and could be detected by immunofluorescence microscopy, whole cell ELISAs and functional assays. In cells expressing the entire Cex, about 90% of the total cellobiose hydrolase activity was anchored on the external side of the outer membrane and was susceptible to protease (papain) added in the extracellular fluid. Cells expressing either Cex or CBDCex bound tightly and rapidly to cellulosic materials such as cotton fibers. This property can be exploited for the preparation of immobilized microbial biocatalysts via adsorption to cellulose and for cell separation through specific agglutination on inexpensive cellulosic materials. In addition, our results demonstrate the general utility of fusions to lpp-ompA for the efficient display of proteins and the engineering of the surface topology of Gram-negative bacteria.
Francisco
J A
JA
Department of Chemical Engineering, University of Texas, Austin 78712.
Stathopoulos
C
C
Warren
R A
RA
Kilburn
D G
DG
Georgiou
G
G
D000192:Actinomycetales / Q000201:enzymology; D000595:Amino Acid Sequence; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002462:Cell Membrane / Q000201:enzymology; D002480:Cellulase / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D043326:Glucan 1,3-beta-Glucosidase; D006868:Hydrolysis; D008074:Lipoproteins / Q000235:genetics; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010206:Papain / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics / Q000378:metabolism*
D001425:Bacterial Outer Membrane Proteins; D008074:Lipoproteins; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D001617:beta-Glucosidase; D002480:Cellulase; D043326:Glucan 1,3-beta-Glucosidase; D010206:Papain