Cloning, heterologous expression and purification of the novel thermo-alkalistable cellulase from Geobacillus sp. TP-3 and its molecular characterisation

Background

Thermophilic cellulases are essential for effectively degrading cellulose, which is a significant part of lignocellulosic waste. In this study, we focused on a cellulase gene (~ 1.2 kb) obtained from Geobacillus sp. TP-3, a thermo-alkalophilic bacterium isolated from the hot springs of Tapovan (Uttarakhand, India). Cellulase gene (~ 1.2 kb) was amplified via PCR, cloned into pET-28a (+) vector, transferred to Escherichia coli DH5α cells and expressed in Escherichia coli BL21 (DE3). The recombinant cellulase (rCel_TP) was purified using Ni²⁺-NTA affinity chromatography.

Results

The purified rCel_TP enzyme exhibited optimal activity at 50 ºC and pH 8, displaying stability even after 3 h of incubation at 50 ºC. The molecular weight of the purified 6 × His-tagged rCel_TP was determined to be ~ 40.2 kDa. Under conditions of 50 ºC and pH 8, the kinetic parameters of the purified enzyme were determined, with K_m and V_max values of 116.78 mg/mL and 44.05 µmolmg⁻¹ min⁻¹, respectively. The activity of the rCel_TP cellulase was significantly improved by Hg²⁺, Cu²⁺ and Co²⁺. However, it was suppressed by dithiothreitol and β-mercaptoethanol. Ethylenediaminetetraacetic acid and solvents also had a slight inhibitory effect.

Conclusion

These results suggest the potential applications of the recombinant cellulase in biomass conversion processes for the production of fuels and other industrial operations. The study contributes valuable insights into the properties and applicability of cellulases derived from extremophilic microorganisms.

Graphical abstract