| Abstract | The C-terminal boundary of primary sequence of the Bacillus subtilis PAP115 endo-β-1,4-glucanase (EG) required for stable catalytic activity has been mapped by site-directed mutagenesis using Escherichia coli as host. The 52 kDa cel gene product, EG470 and a 33 kDa mutant (EG300), lacking 170 residues through a nonsense mutation at the leucine-330 codon of the gene, exhibited similar patterns of enzymatic activity and pH optima using cellooligopentaose as substrate.CD spectra indicated that the bulk of the α-helical secondary structure in EG470 was contained within EG300. However, relative to EG470, the specific activity of EG300 was 3- to 4-fold lower with amorphous cellulose as substrate and ˜4-to5-fold higher with carboxymethylcellulose (soluble cellulose).These results along with data which show that EG470 binding capacity to mirocrystalline cellulose is ˜ 11 times more than that of EG300, demonstrate the importance of residues 330–499 for non-catalytic binding of cellulose. A construct of the cel gene carrying a deletion of codons 330–499 and an insertion of a nonsense codon at leucine-330, was further used to make mutants EG296 and EG291 with nonsense codon substitutions at arginine and serine-321, respectively.Western analysis using EG-specific antiserum revealed that relative losses in enzymatic activity of EG296 (50%) and EG291 (95%) could be accounted for by the extent of their proteolysis, signifying a marked destabilization of these enzymes by removal of only a few amino acids. |
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