Cloning of cellulase gene using metagenomic approach of soils collected from Wadi El Natrun, an extremophilic desert valley in Egypt.

Abstract

Background: Wadi El Natrun microorganisms have been considered as a new resource for natural products due to its extreme condition of salinity and alkalinity. Therefore, this study was devoted to generate metagemic library from soils collected from such an extreme environment in order to clone a novel cellulase for physique industrial applications.

Results: Total soil-DNA was successfully extracted, and then digested by different restriction enzymes. Purified fragments ranged ~ 200-6500 bp were ligated and were cloned into plasmid cloning vector (pUC19) by using Escherichia coli DH5α (E. coli) host cells. A constructed metagenomic library composed of 270 clones was screened on carboxymethylcellulose (CMC) agar plate where the active clones had been characterized by the formation of the yellowish halo zone. Thereafter, clone 1 was selected as the most active as being based on cellulase activity quantification (19 μ/ml). Plasmid related to clone 1 encoded cellSNSY gene of approximately 1.5 kb was subjected to molecular characterization; the obtained partial sequence of 861 bps encoded 287 amino acids showing 76% similarity to the endoglucanase gene of Bacillus amyloliquefaciens. The recombinant cellSNSY was expressed under lacz promoter at 1 mM of isopropyl β-d-1-thiogalactopyranoside (IPTG), giving 21 μ/ml cellulase after ~ 27 h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and an activity staining of the recombinant cellSNSY which revealed an active band with a molecular mass ~ 59 kDa appeared in the induced sample. The maximum enzyme activity of crude cellSNSY was observed at 45 °C and for a pH of 8.5. Interestingly, the enzyme activity was slightly inhibited by ethylenediamine tetraacetic acid (EDTA) and methanol. It showed high resistance to the tested heavy metals and the surfactant which ordered Zn> (SDS,Fe)>Mn>Cu.

Conclusions: This study established an easy and a skillful way to clone/express a new found cellulase gene(s) under lacZ promoter. The isolated recombinant cellSNSY showed 76% similarity to endoglucanase gene, and the enzyme showed tolerance to the mostly tested agents including heavy metals, surfactant, solvents, and EDTA. Additionally, the studied recombinant showed a high stability up to 55 °C and for alkaline pH 8.5. These features make it an ample and viable for many applications.