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 Ta...
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Veröffentlicht in: | Beni-Suef University journal of basic and applied sciences 2024-12, Vol.13 (1), p.36-15, Article 36 |
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Sprache: | eng |
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Zusammenfassung: | 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 Ni
2+
-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 Hg
2+
, Cu
2+
and Co
2+
. 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 |
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ISSN: | 2314-8543 2314-8535 2314-8543 |
DOI: | 10.1186/s43088-024-00495-9 |