Cold‐adapted enzymes, elaborated by psychrophiles and psychrotrophs, seem to have potential for current and future applications. In the current study, pullulanase production by the cold‐adapted Exiguobacterium sp. SH3 was investigated. The Plackett–Burman design and the response surface methodology were applied to identify and optimize the significant variables affecting the pullulanase production of Exiguobacterium sp. SH3. The results showed that temperature, time, and CaCl2 concentration were significant variables while shaking, starch, yeast extract, tryptone, pH, MnCl2, MgCl2, and KH2PO4 were not significant. Using statistical analyses and optimizations, the pullulanase production was significantly elevated from 200 ± 18 to 950 ± 27 U/mL (4.75 times) as compared to non‐optimized conditions. A pullulanase of about 70 kDa, designated as Pul‐SH3, was purified 16.2‐folds from the optimized culture, and identified to be an amylopullulanase. The Km and Vmax of the enzyme were 0.069 mg/mL and 967 U/mL, respectively. The optimum pH and temperature for maximum activity of Pul‐SH3 were 7.5 and 30°C, respectively. As a cold‐adapted enzyme, Pul‐SH3 retained 23% of the maximum activity at 0°C. The biochemical characteristics and N‐terminal amino acid sequence of Pul‐SH3 suggest that the enzyme is a novel cold‐adapted amylopullulanase with remarkably high specific activity at moderate ambient temperature.
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.
Lipases are one of the highest value commercial enzymes as they have broad applications in detergent, food, pharmaceutical, and dairy industries. To provide chimeric Bacillus thermocatenulatus lipase (BTL2), the completely conserved pentapeptide (¹¹²Ala-His-Ser-Gln-Gly¹¹⁶) was replaced with similar sequences (²⁰⁷Gly-Glu-Ser-Ala-Gly²¹¹) of Candida rugosa lipase (CLR) at the nucleophilic elbow region. For this purpose, three mutations including A112G, H113E, and Q115A were inserted in the conserved pentapeptide sequence of btl2 gene. Based on the crystal structures of 2W22, the best structure of opened form of the chimeric lipases were garnered using the MODELLER v9.10 software. The native and chimeric lipases were docked to a set of ligands, and a trial version of Molegro Virtual Docker (MVD) software was used to obtain the energy values. Docking results confirmed chimeric lipase to be better than the native lipase. Following the in silico study, cloning experiments were conducted and expression of native and chimeric btl2 gene in Pichia pastoris was performed. The native and chimeric lipases were purified, and the effect of these mutations on characteristics of chimeric lipase studied and then compared with those of native lipase. Chimeric lipase exhibited 1.6-fold higher activity than the native lipase at 55 °C. The highest percentage of both lipases activity was observed at 60 °C and pH of 8.0. The ion Ca²⁺ slightly inhibited the activity of both lipases, whereas the organic solvent enhanced the lipase stability of chimeric lipase as compared with the native lipase. According to the results, the presence of two glycine residues at the conserved pentapeptide region of this chimeric lipase (¹¹²Gly-Glu-Ser-Ala-Gly¹¹⁶) may increase the flexibility of the nucleophilic elbow region and affect the enzyme activity level.
Desulfurization protein named DszC from Rhodococcus erythropolis is the key enzyme for biodesulforization of dibenzothiophene (DBT) in 4S pathway, which is a pathway with four enzymes. DszC enzyme biodesulfurizes DBT and its derivatives in oil components and biphasic systems. It functions well at the oil- water interface. In this study point mutation performed in DszC enzyme regarding to increase protein hydrophobicity and stability for application in immobilized form. 3D model of DszC predicted using Phyre2, SAM-T08 and M4t servers. I-Mutant 2 server used to determine potential spots for point mutation, and Molegro Virtual Docker (MVD) used for performing point mutation on 3D model. Hydrophobicity plots generated by Bioedit version 7.0.8.0 in Kyte-Doolittle scale indicated that protein hydrophobicity is increased after mutation. Also protein stability increased 26.11 units in scale of DDC2.
Purpose The most common intraocular tumor in childhood, retinoblastoma, is largely associated with mutations in the RB1 gene. In the most comprehensive RB1 screening in Iran, we evaluated the RB1 mutations in 106 patients with retinoblastoma, including 73 bilateral (heritable) and 33 unilateral (sporadic) cases. Patients and methods Mutations were identified using amplification refractory mutation system (ARMS) PCR and direct sequencing of the 27 coding exons of RB1 and multiplex ligation-dependent probe amplification (MLPA). Results and ConclusionWe found 33 (31%) and 64 (60%) patients with sporadic unilateral and bilateral retinoblastoma, respectively as well as 9 (8.5%) cases with hereditary bilateral retinoblastoma. In total, we identified 52 causative RB1 mutations in 106 patients (global mutation rate of 49%). Of the 52 patients, 48 (92%) had sporadic and familial bilateral and 4 (8%) had sporadic unilateral RB. Therefore, the detection rate of RB1 mutations was 66% (48/73) and 12% (4/33) in bilateral and unilateral cases, respectively. Mutations were classified as nonsense in 31 (60%), missense in 1 (2%), large deletion in 11 (21%), small deletion in the 7 novel (15%) and splice site mutation in 2 (4%) patients with RB. Of 31 nonsense mutations, 23 (74%) occurred in the 11 Arginine codons of the RB1. Seven mutations (13%) were novel, and 45 (87%) had been previously reported. Thirty-three mutations were single-base substitutions leading to 31 nonsense amino acid changes and 2 splice site mutations in introns 12 and 16 of RB1. The altered 3D model structures of the RB1 novel mutant proteins are also predicted in this study.
The ToxT transcription factor mediates the transcription of the two major virulence factors in Vibrio cholerae. It has a DNA binding domain made of α4, α5, α6, α7, α8, α9 and α10 helices that is responsible for the transcription of virulence genes. Therefore, it is of interest to screen ToxT against the ZINC ligand database containing data for a million compounds. The QSAR model identified 40 top hits for ToxT. Two target protein complexes with ligands Lig N1 and Lig N2 with high score were selected for molecular dynamics simulation. Simulation data shows that ligands are stable in the DNA binding domain of ToxT. Moreover, Lig N1 and Lig N2 passed pharmacological as well as ADME filters along with g-mmpbsa analysis with binding affinity of -199.831 kJ/mol for Lig N1 and - 286.951 kJ/mol for Lig N2. Moreover, no Lipinski and PhysChem violations were identified. It is further observed that these compounds were not inhibitors of P-glycoprotein, CYP450 and renal organic cation transporters. The LD50 of 2.5804 mol/kg for Lig N1 and 2.7788 mol/kg for Lig N2 was noted with acceptable toxicity index.
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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