Summary auto-generated
This study reports the molecular characterization of the celY gene from the thermophilic bacterium Clostridium stercorarium, which encodes Avicelase II, an exo-1,4-β-glucanase enzyme. The celY gene consists of a 2,742 base pair open reading frame encoding a 914 amino acid preprotein with a molecular mass of 103 kDa. The gene is located adjacent to celZ, which encodes a complementary endoglucanase. Using PCR amplification and molecular cloning techniques, the researchers successfully expressed the gene in Escherichia coli as a fusion protein with LacZ sequences and purified the recombinant enzyme through heat treatment and anion-exchange chromatography. The purified recombinant CelY enzyme exhibited properties identical to the native Avicelase II previously isolated from C. stercorarium cultures. Sequence analysis revealed that CelY contains an N-terminal catalytic domain, an unknown function domain of 95 amino acids, and a type III cellulose-binding domain at the C-terminus. The catalytic domain belongs to cellulase family L (family 48 of glycosyl hydrolases), a recently identified family comprising only bacterial enzymes. The recombinant enzyme demonstrated optimal activity at pH 5.0 and 75°C, showed pronounced thermostability, and exhibited substrate specificity preferring amorphous cellulose over crystalline forms.
Key findings
- The celY gene encodes a 914 amino acid exo-1,4-β-glucanase that acts as a cellodextrinohydrolase, releasing cellobiose and other oligosaccharides from cellulose chains
- The CelY enzyme belongs to cellulase family L (family 48 of glycosyl hydrolases) and shares 54-93% sequence identity with other family members
- The recombinant CelY enzyme produced in E. coli has biochemical properties identical to native Avicelase II from C. stercorarium, with optimal activity at pH 5.0 and 75°C
- The celY gene is located immediately upstream of the celZ gene in the C. stercorarium genome, suggesting functional coordination between the exo- and endoglucanase enzymes in cellulose degradation
- The CelY protein contains three functional domains: an N-terminal catalytic domain, a middle domain of unknown function, and a C-terminal type III cellulose-binding domain
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Abstract
The nucleotide sequence of the celY gene coding for the thermostable exo-1,4-beta-glucanase Avicelase II of Clostridium stercorarium was determined. The gene consists of an ORF of 274Z bp which encodes a preprotein of 914 amino acids with a molecular mass of 103 kDa. The signal-peptide cleavage site was identified by comparison with the N- terminal amino acid sequence of Avicelase II purified from C stercorarium. The celY gene is located in close vicinity to the celZ gene coding for the endo-1,4-beta-glucanase Avicelase I. The CelY- encoding sequence was isolated from genomic DNA of C. stercorarium with the PCR technique. The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion protein could be purified using a simple two- step procedure. The properties of CelY proved to be consistent with those of Avicelase II purified from C. stercorarium. Sequence comparison revealed that CelY consists of an N-terminal catalytic domain flanked by a domain of 95 amino acids with unknown function joined to a type III cellulose-binding domain. The catalytic domain belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).