Characterization, structural, and evolutionary analysis of an extremophilic GH5 endoglucanase from Bacillus sp. G131: Insights from ancestral sequence reconstruction.

Nature has developed extremozymes that catalyze complex reaction processes in extreme environmental conditions. Accordingly, a combined approach consisting of extremozyme screening, ancestral sequence resurrection (ASR), and molecular dynamic simulation was utilized to construct a developed endoglucanase. The primary experimental and in-silico data led to the prediction of a hypothetical sequence of endoglucanase (EG5-G131) using Bacillus sp. G131 confirmed by amplification and sequencing. EG5-G131 exhibited noticeable stability in a broad-pH range, several detergents, organic solvents, and temperatures up to 80 °C. The molecular weight, Vmax, and Km of the purified endoglucanase were estimated to be 36 kDa, 4.32 µmol/min, and 23.62 mg/ml, respectively. The calculated thermodynamic parameters for EG5-G131 confirmed its intrinsic thermostability. Computational analysis revealed Glu142 and Glu230 as active-site residues of the enzyme. Furthermore, the enzyme remained bound to cellotetraose at 298 K, 333 K, 343 K, and 353 K for 300 ns, consistent with our experimental data. ASR of EG5-G131 led to the introduction of ancestral ANC204 and ANC205, which show similar thermodynamic characteristics with the last Firmicute common ancestor. Finally, truncating loops from the N-terminal of two sequences created two variants with desirable thermal stability, suggesting the evolutionary deciphering of the functional domain of the GH5 family in Bacillus sp. G131.

Characterization of a thermostable endoglucanase produced by Isoptericola variabilis sp. IDAH9

Abstract This study aimed to isolate and evaluate the cellulase activity of cellulolytic bacteria in hot springs of Dehloran, Ilam province, Iran. Water and sludge samples were collected from the hot springs and the bacterial enrichment was performed in a medium containing rice barn and carboxymethyl cellulose (CMC). The cultures were incubated at 50 °C in aerobic conditions. The bacteria were isolated on CMC agar (1%) medium. Cellulase assay of the isolates was measured by the evaluation of endoglucanase enzyme activity, which is also called as carboxymethyl cellulase (CMCase). The isolated thermotolerant bacteria were then identified and optimized for the production of CMCase. Moreover, stabilizing elements of the enzyme were identified with in silico approach. The chosen isolate was identified as Isoptericola variabilis sp. IDAH9. The identified strain produced the most thermostable CMCase at a concentration of 5.6 g/L of ammonium sulfate, 9 g/L CMCase or 12 g/L rice bran, 0/6% Tween-80, and 0.2% sucrose. The produced enzyme showed 80% of the residual activity after 1 h of incubation at 65 °C. In silico data indicated that the remaining residual activity was due to the redundant stabilizing elements in the protein structure. Consequently, I. variabilis can be isolated from the extreme environment and has a thermostable endoglucanase which may be used for various applications after studying them.