Insights into the metagenomic approach : identification and characterization of cellulases involved in bacterial cellulose synthesis

the mining of an Antarctic soil sample by functional metagenomics allowed the
isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new
enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri
(Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and
purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and
cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal
dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained
significantly active when temperature decreased (18% of activity at 10 1C). It is interesting that
RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover,
by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1
protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as
the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and
Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.