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.
The celH gene from Clostridium thermocellum encodes a protein containing 900 residues and three components, including Cel5E, Lic26a, and carbohydrate-binding domains. Cel5E is a member of the glycoside hydrolase-5 family and is a bifunctional xylanase/cellulase enzyme. We targeted a semi-hydrophobic pocket near the Cel5E active site and theoretically screened mutated variants for enhanced levels of thermal stability. Cel5E mutations were inserted into celH by overlapping polymerase chain reaction, followed by expression of wild-type and mutant enzymes in Escherichia coli BL21 (DE3) and purification by affinity chromatography. Thermal-stabilizing mutations were subjected to molecular dynamics simulation, and measurement of the in vacuo potential energy, van der Waals forces, electrostatic interactions, and net nonbonded potential energies obtained an overall binding affinity of - 64.964 KJ/mol for wild-type Cel5E and - 176.148, - 200.921, and - 120.038 KJ/mol for the N94F, N94W, and E133F mutants, respectively. Additionally, the N94W, N94F, E133F, and N94A variants exhibited 1.92-, 1.29-, 1.1-, and 1.15-fold better carboxymethyl cellulase (CMCase) and 1.46-, 1.29-, 1.11-, and 1.12-fold better β-glucanase activity on barley β-glucan relative to the wild-type enzyme. Interestingly, the optimal temperature for CMCase activity by the N94W variant was shifted 2 °C higher than that for the wild-type enzyme. Mutated variants showed improved CMCase and β-glucanase activity and shifted toward higher temperature of maximum activity.
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