Molecular Characterization of an Endo-β-1,4-Glucanase, CelAJ93, from the Recently Isolated Marine Bacterium, Cellulophaga sp. J9-3

Featured Application CelA(J93) is a novel thermostable endo-beta -1,4-glucanase that can be used to process cellulosic material for various purposes. Abstract A novel cellulase was characterized from a newly isolated marine bacterium, strain J9-3. Phylogenetic analysis based on the 16S rRNA gene revealed that strain J9-3 belonged to the genus Cellulophaga, and thus, it was named Cellulophaga sp. J9-3. An extracellular cellulase was purified from cell-free culture broth of J9-3 cultured in Marine Broth containing 0.2% carboxymethylcellulose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein revealed a single band with an apparent molecular weight (Mw) of 35 kDa. Based on the NH2-terminal amino acid sequence (N-N-T-E-Q-T-V-V-D-A-Y-G), the gene (named celA(J93)) encoding the protein was identified from J9-3 genomic sequencing data. CelA(J93) was expected to be translated into a premature protein (359 amino acids) and then processed to a mature protein (307 amino acids, Mw = 34,951 Da), which is consistent with our results. CelA(J93) had high homology with many uncharacterized putative glycosyl hydrolases of the genus Cellulophaga and it was highly specific for carboxymethylcellulose and cellooligosaccharides under optimum conditions (pH 7.5, 60 degrees C). Co2+ completely recovered CelA(J93) activity that was severely inhibited by ethylenediaminetetraacetic acid (EDTA), indicating that CelA(J93) required Co2+ as a cofactor. Thus, CelA(J93) is a Co2+-dependent endo-beta -1,4-glucanase that can hydrolyze carboxymethylcellulose and cellooligosaccharides into cellobiose at a relatively high temperature.