Showing papers by "Neil R. Gilkes published in 1992"

The binding of Cellulomonas fimi endoglucanase C (CenC) to cellulose and Sephadex is mediated by the N-terminal repeats.

Abstract: Endoglucanase C (CenC) from Cellulomonas fimi binds to cellulose and to Sephadex. The enzyme has two contiguous 150-amino-acid repeats (N1 and N2) at its N-terminus and two unrelated contiguous 100-amino-acid repeats (C1 and C2) at its C-terminus. Polypeptides corresponding to N1, N1N2, C1, and C1C2 were produced by expression of appropriate cenC gene fragments in Escherichia coli. N1N2, but not N1 alone, binds to Sephadex; both polypeptides bind to Avicel, (a heterogeneous cellulose preparation containing both crystalline and non-crystalline components). Neither C1 nor C1C2 binds to Avicel or Sephadex. N1N2 and N1 bind to regenerated ('amorphous') cellulose but not to bacterial crystalline cellulose; the cellulose-binding domain of C. fimi exoglucanase Cex binds to both of these forms of cellulose. Amino acid sequence comparison reveals that N1 and N2 are distantly related to the cellulose-binding domains of Cex and C. fimi endoglucanases A and B.

Cellulose-binding domains: potential for purification of complex proteins

Abstract: The endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi each contain a discrete cellulose-binding domain (CBD), at the amino-terminus or carboxyl-terminus respectively. The gene fragment encoding the CBD can be fused to the gene of a protein of interest. Using this approach hybrid proteins can be engineered which bind reversibly to cellulose and exhibit the biological activity of the protein partner. Alkaline phosphatase (PhoA) from Escherichia coli, and a beta-glucosidase (Abg) from an Agrobacterium sp. are dimeric proteins. The fusion polypeptides CenA-PhoA and Abg-CBC(Cex) are sensitive to proteolysis at the junctions between the fusion partners. Proteolysis results in a mixture of homo- and heterodimers; these bind to cellulose if one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoA and CenA-PhoA/PhoA. CBD fusion polypeptides could be used in this way to purify polypeptides which associate with the fusion partner.

Method of preparing a B-1,4 glycan matrix containing a bound fusion protein

Abstract: A fusion protein that can function as a removable label is prepared containing a polypetide such as an enzyme and an amino acid sequence having a substrate binding region of a polysaccharidase such as cellulase that has essentially no polysaccharidase activity. By contacting the fusion protein with a β-1,4 glycan matrix such as cellulose, the substrate binding region binds to the matrix to immobilize the polypeptide. The polypetide or fusion protein can be removed from the matrix with a protease capable of cleaving a specific protease cleavage site, or with a solution having a low ionic strength or a high pH. The fusion protein can be prepared by recombinant DNA technology.

Method for modification of polysaccharide fibres

Abstract: Methods and compositions are provided for the modification of polysaccharide structures using polysaccharidase binding or catalytic domains either alone or in tandem to modify the structure of polysaccharides. These methods and compositions are exemplified by the use of cellulase binding and catalytic domains to polish cotton, and to alter dying characteristics, texture and porosity of cellulose fibers.

The tertiary structure of endo-beta-1,4-glucanase B (CenB), a multidomain cellulase from the bacterium Cellulomonas fimi.

Abstract: Endo-beta-1,4-glucanase B (CenB) is a large (110 kDa) extracellular enzyme from the cellulolytic bacterium Cellulomonas fimi. CenB contains five domains, including a typical C.fimi cellulose-binding domain, separated by distinctive linker polypeptides (Meinke et al., 1991b). X-ray scattering analyses show that CenB has a highly elongated shape resembling beads on a string. The sizes of the polypeptides produced by treatment of CenB with proteases, together with their N-terminal amino acid sequences, show that at least two of the four linkers connecting the five domains of CenB are more sensitive to proteolysis than the domains themselves. It is concluded that the beads represent the domains of CenB, the string represents the linkers.

Endoglucanase A from Cellulomonas fimi in which the hinge sequence of human IgA1 is substituted for the linker connecting its two domains is hydrolyzed by IgA proteases from Neisseria gonorrhoeae.

Abstract: The hinge in IgA1 and the linker in endoglucanase A (CenA) are quite similar. The IgA1 hinge is 18 amino acids long and contains only proline, threonine and serine. The linker in CenA is 27 amino acids long and contains only proline, threonine and a single serine. IgA proteases from Neisseria gonorrhoeae cleave Pro-Ser and Pro-Thr bonds within the IgA1 hinge sequence, but they do not attack CenA. When the linker sequence of CenA is replaced with the hinge sequence of IgA1, the hybrid polypeptide is susceptible to the N. gonorrhoeae proteases. It is cleaved within the hinge sequence at the same sites as IgA1.

The Adsorption of a Bacterial Cellulase to Crystalline Cellulose

Abstract: CenA is a bacterial cellulase @-1,4-glucanase) comprised of a globular catalytic domain joined to an extended cellulose-binding domain (CBD) by a short linker peptide. The adsorption of CenA and its two isolated domains to crystalline cellulose was analyzed. CenA and CBD*PTcenA (the CBD plus linker) adsorbed rapidly to cellulose at 30 O C , and no net desorption of protein was observed during the following 16.7 h. There was no detectable adsorption of the catalytic domain. Scatchard plots of adsorption data for CenA and for CBD*PTcenA were nonlinear (concave upward). The adsorption of CenA and CBD*PTcen~ exceeded 7 and 8 pmol/g cellulose, respectively, but saturation was not attained at the highest total protein concentrations employed. A new model for adsorption was developed to describe the interaction of a large ligand (protein) with a lattice of overlapping potential binding sites (cellobiose residues). A relative equilibrium association constant (Kr) of 40.6 and 45.3 liter-g cellulose” was estimated for CenA and CBD*PTcenA, respectively, according to this model. A similar K , value (33.3 liter-g”) was also obtained for Cex, a Cellulomonas fimi enzyme which contains a related CBD but which hydrolyzes both B1,4-xylosidic and ~-1,4-glucosidic bonds. It was estimated that the CBD occupies approximately 39 cellobiose residues on the cellulose surface.