endo-Beta-1,4- glucanase belonging to GH family 5 from a rhizosphere metagenomic library. rhizosphere metagenomic library

Cellulases have a broad range of different industrial applications, ranging from food and beverages to pulp and paper and the biofuels area. Here a metagenomics based strategy was used to identify the cellulolytic enzyme CelRH5 from the rhizosphere. CelRH5 is a novel monospecific endo‐β‐1,4‐glucanase belonging to the glycosyl hydrolase family 5 (GH5). Structural based modelling analysis indicated that CelRH5 is related to endo‐β‐1,4‐glucanases derived from thermophilic microorganisms such as Thermotoga maritima, Fervidobacterium nodosum and Ruminiclostridium thermocellum sharing 30-40% amino acid sequence identity. The molecular weight of the enzyme was determined as 40.5 kDa. Biochemical analyses revealed that the enzyme displayed good activity with soluble forms of cellulose as a substrate such as ostazin brilliant red hydroxyethyl cellulose (OBR-HEC), carboxymethylcellulase (CMC), hydorxylethyl cellulose (HEC) and insoluble azurine cross-linked hydorxylethyl cellulose (AZCL-HEC). The enzyme shows highest enzymatic activity at pH 6.5 with a high pH tolerance, remaining stable in the pH range 4.5 – 8.5. Highest activity was observed at 40˚C, but CelRH5 is psychrotolerant being active and stable at temperatures below 30 ˚C. The presence of final products of cellulose hydrolysis (glucose and cellobiose) or metal ions such as Na+, K+, Li+ and Mg2+, as well as ethylenediaminetetraacetic acid (EDTA), urea, dithiothreitol (DTT), dimethyl sulfoxide (DMSO), 2-mercaptoethanol (2-ME) or glycerol, did not have a marked effect on CelRH5 activity. However, the enzyme is quite sensitive to the presence of 10mM ions Zn2+, Ni2+, Co2+, Fe3+ and reagents such as 1 M guanidine HCl, 0.1% sodium dodecyl sulphate (SDS) and 20% ethanol. Given that it is psychrotolerant and retains activity in the presence of final cellulose degradation products, metal ions and various reagents, whichare common in many technological processes; CelRH5 may be potentially suitability for a variety of different biotechnologicalapplications.