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Abstract 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 Km and Vmax 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

The geothermal springs are said to contain the greatest diversity of undiscovered microorganisms, making them the best source for enzymes with economic significance. The untapped microbial diversity living in the geothermal springs can be mined for novel genes, bioactive substances, and industrially significant biocatalysts using the metagenomics technique. Metagenome was extracted from soil samples of various geothermal springs of India. Metagenome was screened for various carbohydrate degrading enzymes (amylase, cellulase, xylanase, amylopullulanase) using degenerate primers-based Polymerase chain reaction amplifications. Further amplicons were cloned, sequenced and analysis of data was done using various bioinformatics tools, e.g., Blast analysis, Protparam and phre2 program. We have isolated numerous enzymes, including cellulase, amylase, amylopullulanase, and xylanase, from diverse geothermal spring in different parts of India using sequence and function-based metagenomics. In this study, we describe the metagenomics-based isolation of a thermostable amylase from the geothermal spring of Odisha. The amylase gene (1503 bp) was amplified using the metagenome as a template using degenerate primers and cloned into the linearized T vector. The putative gene was likely to encode a protein of 469 amino acids with a molecular weight of 53895.05 Da with pI-7.78. Sequence analysis showed its maximum identity of 98.95% with Bacillus licheniformis alpha-amylase gene. Homology modeling of the amylase protein was done using the phyre2 program, which shows it belongs to the (trans) glycosidase superfamily and contains the catalytic TIM alpha/beta-barrel fold. Hence, we can conclude that geothermal springs are hotspots for the mining of industrially robust biocatalysts.