Endoglucanase gene of M42 aminopeptidase/endoglucanase family from thermophilic Bacillus sp. PW1 and PW2 isolated from Tattapani hot spring, Himachal Pradesh, India

Background Thermostable cellulases are in constant demand for several biotechnological applications. Two thermophilic bacterial strains PW1 and PW2 isolated from Tattapani hot spring were found to have cellulolytic activity. Subsequently, PW1 and PW2 were identified and mined for genes encoding cellulase activity. Results Sequencing of the 16S rDNA of PW1 and PW2 identified them as Bacillus sp. PW1 (Acc no. KU711837) and Bacillus sp. PW2 (Acc no. KU711838), respectively, which clustered in the clades containing thermophilic members of Bacillus sp. and Geobacillus species. Phylogenetic analysis revealed that despite the morphological and sequence identities, Bacillus sp. PW1 and Bacillus sp. PW2 are different at the genetic level. The cellulase genes (~ 1.1 kb) of the two bacterial strains were amplified using primers designed against related thermophilic cellulases. Sequencing of the cellulase gene amplicons of PW1 and PW2 revealed that they encode proteins of 280 and 206 amino acid residues, respectively. Sequence and domain analysis of the protein products of PW1 and PW2 revealed that they belong to M42 family of aminopeptidase/endoglucanase. The PW2 endoglucanase coding sequence was submitted to Genbank under accession no. MH049504. The structures of putative endoglucanases of PW1 and PW2 were generated using 1VHE.A as template, which showed the presence of vast proportion of random coils. Molecular docking of the modeled endoglucanase proteins with various substrates and products of cellulases showed that carboxymethyl cellulose and maltose exhibit the highest binding affinity, while xylan and glucose the least. Conclusions The two thermophilic bacteria PW1 and PW2 and their endoglucanase gene can be further utilized for recombinant production of thermostable cellulases for their application in industries.

In vivocloning of β 1-4 endoglucanase gene ofSerratia liquefaciensusing Muduction and itsin silicoanalysis

Cellulose is the major structural component in the plant cell wall. The bio-degradation of cellulose molecules is facilitated by cellulase. In the present study,in vivocloning of cellulase (β-1, 4 endoglucanase) gene from a cellulolytic bacteriumSerratia liquefaciensintoE. coliDH5α has been performed using mini-Mu phage transduction. The enzyme activity of cloned endoglucanase was 81.2U/mg at optimum temperature (40°C) and 80.2U/mg at optimum pH7, while the wildtype has 65.9U/mg and 64.9U/mg respectively. The conserved domain analysis shows thatS. liquefaciensendoglucanase belongs to GH8 family. The nucleotide sequence analysis of wildtype and cloned endoglucanase shows that mutations were found at residues 51(Lys - Asn), 203(Trp-Cys), 246(Thr-Iso), 260(Gly-Ala) and 288(Phe-Leu). The structural analysis shows the active site of wildtype endoglucanase is a narrow groove which lies parallel to the central axis, whereas cloned endoglucanase is broad and tilted to ∼70° from the central axis. The increased enzyme activity in the cloned endoglucanase is due to the structural modification conferred by changes in amino acid resulting in widening of the cleft in the active site.