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Organophosphates are becoming an emerging pollutant due to their various applications, particularly as pesticides. In this study, an improved Colony (Live-cell) PCR method was developed for the detection of opd genes from bacteria encoding the organophosphate hydrolase enzymes capable of degrading various organophosphates. The improved method does not require pre-heating or pre-lysis of bacterial cells as essential in the conventional colony PCR. The reaction volume was scaled down to 10 µl by optimizing the PCR buffer and amplification conditions. The improved method was used for Gram positive and negative bacteria, glycerol stocks, liquid cultures, recombinant and mutant strains. Also, 16S rRNA gene was amplified from unknown environmental isolates and known E. coli strains. The amplified opd and 16S rRNA genes from the improved colony PCR method and by conventional PCR were sequenced, and similar results were obtained from both techniques. Thus, the improved method can be further explored in molecular biology or during biomarker studies. KEY POINTS: • Improved colony PCR method was developed for screening of opd genes from bacteria. • Method was validated for Gram positive/negative bacteria from solid as well as liquid media. • The improved method was rapid, efficient, and can be applied under various conditions., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

L-Asparaginase (3.5.1.1) is an enzyme widely used to treat the acute lymphoblastic leukemia. Two genes coding for L-asparaginase (ansA1 and ansA3) from Bacillus licheniformis MTCC 429 were cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant proteins were purified to homogeneity by one-step purification process and further characterized for various biochemical parameters. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that both the enzymes are monomers of ∼37 kDa. Recombinant ansA1 was found to be highly unstable, and recombinant ansA3 was catalytically active and stable, which showed an optimum activity of 407.65 IU/mg at 37 °C and pH 8. Recombinant ansA3 showed higher substrate specificity for L-asparagine with negligible glutaminase activity. Kinetic parameters like K m , V max, k cat, and k cat/K m were calculated for recombinant ansA3.

Discovery of RNA elements, including riboswitches and regulatory RNAs, has revealed additional regulatory mechanisms for transcript stability, transcript termination, and translational initiation. These regulatory RNA molecules act through direct binding to cellular targets including other RNA molecules, proteins, and low molecular weight metabolites. RNA-RNA interactions based on complementarity can be identified through bioinformatic analysis. However, identification of novel interactions between these regulatory RNA molecules and their partners other than complementary sequences is more challenging. We have developed a technique called Differential Radial Capillary Action of Ligand Assay (DRaCALA) to facilitate the detection of direct binding between RNA elements to proteins or low molecular weight ligands. Previously, we have described the adaptation of this technique to detect the binding interaction between Vc2 riboswitch to a signaling cyclic dinucleotide called cyclic-di-GMP. Here, we describe the adaptation of DRaCALA for identifying sequence-specific RNA-binding proteins directly from E. coli cell lysates expressing the recombinant binding protein. DRaCALA can be used to qualitatively and quantitatively assess RNA-protein interaction in whole cell lysate, determine the kinetics of the binding, and test for competitors. Using DRaCALA in a high-throughput format has the potential to rapidly identify sequence-specific RNA-binding proteins.

Experimental evidence in vivo as to the functional roles and binding properties to cadmium (Cd) of type-2 plants metallothionein (MT) has been limited thus far. We investigated the biological role of metallothionein from Colocasia esculenta (CeMT2b) in Escherichia coli and tobacco, and developed a new model for the relationship between Cd tolerance and Cd-binding ability. Heterologous expression of CeMT2b in Escherichia coli greatly enhanced Cd tolerance and accumulated Cd content as compared to control cells. The molecular weight of CeMT2b increased with Cd, and CeMT2b bound up to 5.96±1 molar ratio (Cd/protein). Under Cd stress, transgenic tobacco plants displayed much better seedling growth and high Cd accumulation than the wild type. The presence of an extra CXC motif in CeMT2b contributed to the enhanced Cd-tolerance. The present study provides the first insight into the ability of type-2 plant MT to bind physiological Cd.

Two series of C‐Mannich base derivatives were synthesized and evaluated through the reaction of formaldehyde, two thiazolo‐pyrimidine compounds, and various 2°‐amines. The chemical structures and inherent properties of the synthesized compounds were authenticated using a variety of spectroscopic techniques. The aseptic bactericidal potential of the compounds was assessed alongside five common bacterial microbes, with Ampicillin employed as the reference drug. Compounds 9b and 9d demonstrated comparable antibacterial activity to ampicillin against Bacillus subtilis and Bacillus megaterium, respectively, at 100 μg/mL. Furthermore, compounds 9f and 10f exhibited noteworthy action against Staphylococcus aureus (MIC: 250 μg/mL). Compounds 10b and 10f displayed excellent efficacy versus Escherichia coli, boasting (MIC: 50 μg/mL). Molecular docking studies elucidated the necessary connections and energies of molecular entities with the E. coli DNA gyrase B enzyme, a pivotal target in bacterial DNA replication. Further thermodynamic stability of the ligand‐receptor complex of 10b and 10f were further validated though 200 ns molecular dynamics simulation. The findings highlight the potential of these synthesized derivatives as effective antibacterial agents and provide valuable insights into their mechanism of action. Highlights: Two series of C‐Mannich base derivatives were synthesized by reacting formaldehyde, two thiazolo[3,2‐a] pyrimidine compounds, and seven heterocyclic amines.The antibacterial potential of the compounds was evaluated against five common bacterial pathogens, with Ampicillin as the reference drug.Compounds 9b and 9d showed comparable antibacterial activity to Ampicillin against Bacillus subtilis and Bacillus megaterium, respectively, at 100 μg/mL.Compounds 9 f and 10 f exhibited notable activity against Staphylococcus aureus, with MIC values of 250 μg/mL.Compounds 10b and 10 f displayed excellent efficacy against Escherichia coli, with MIC values of 50 μg/mL.Molecular docking studies revealed the binding interactions and energies of the compounds with the E. coli DNA gyrase B enzyme, a crucial target in bacterial DNA replication.The binding stability of promising compounds 10b and 10 f was further validated through 200 ns molecular dynamics (MD) simulations. [ABSTRACT FROM AUTHOR]

Cel12A , a gene that encodes an endocellulase of Bacillus licheniformis , was cloned and overexpressed in E. coli DE3. The rBLCel (wild-type recombinant cellulase) enzyme was assayed and characterized for its physical properties, including its optimum pH (9.0), temperature (50 °C), thermostability (175 h) and kinetic parameters ( K m 16.38 ± 0.550 mg ml −1 ; k cat 134.91 ± 0.377 min −1 and k cat /K m 8.23 ± 0.026 ml min −1  mg −1 ). To determine the role of conserved amino acids in the substrate binding pocket, five single mutations and one double mutation were studied for their effects on substrate specificity. All six purified engineered enzymes were assayed and compared to rBLCel regarding their catalytic activity with a specific substrate (CMC). All of the recombinant enzymes (wild type and engineered) were also assayed with non-specific substrates, such as xylan, avicel and filter paper (FP). Out of five point-mutant enzymes, three mutants (W197A, W98A and Y89A) showed reduced specific activity compared to rBLCel. However, the W139A and W53A mutants exhibited a 67% and 4% relative increase in their specific activity with a specific substrate (CMC), respectively. After replacing aromatic residues, the affinity towards the CMC increased in the case of the W139A and W53A mutants, indicating that the aromatic residues play a key role in substrate affinity and binding. One of the significant results that we obtained was that the increase in activity was additive in the double mutant enzyme, W139A_W53A, which showed 1216% higher activity than rBLCel on filter paper (FP). The activity of all of the mutated enzymes with non-specific substrates indicates that the substrate binding pocket is altered and has become flexible toward other substrates after mutation. Moreover, given the importance of the two enzymes in degrading lignocellulose, one bifunctional enzyme endowed with the two catalytic activities may be of great advantage. Despite the broad spectrum of cellulases being isolated, no single enzyme is completely suitable as it is, for the hydrolysis of lignocellulose. These enzymes have been used due to their biotechnological applications in several industries, including bio pulping wood, treatment of animal feed to increase digestibility, agro-processing, juice processing and baking. [ABSTRACT FROM AUTHOR]

Contrary to extensive researches on the roles of metallothioneins (MTs) in metal tolerance of animals, the roles of plant MTs in metal tolerance are largely under investigation. In this study, we evaluated the functional role of type 2 MT from Colocasia esculenta (CeMT2b) in Zn tolerance of tobacco and E. coli cells. Under Zn-stress conditions, transgenic tobacco overexpressing CeMT2b displayed much better seedling growth, a significant decrease in the levels of HO and an increase in Zn accumulation compared with the wild type. Overexpression of CeMT2b in E. coli greatly enhanced Zn tolerance and Zn accumulation under Zn stresses compared with control cells. CeMT2b bound 5.38 ± 0.29 atoms of Zn per protein. To identify a structural domain of CeMT2b for Zn binding, we investigated the growth of E. coli expressing each of the N-terminal, C-terminal, and central linker domains or a CNC motif deletion from the C-terminus of full-length CeMT2b. The results showed that the CNC motif is required for Zn tolerance, and the N-terminal domain is more effective in Zn tolerance than the C-terminal domain. Taken together, our results provide direct evidence for functional contributions of CeMT2b in Zn tolerance of tobacco and E. coli cells. [ABSTRACT FROM AUTHOR]

The article presents a study that investigates the biological role of metallothionein (MT) from Colocasia esculenta (CeMT2b) in Escherichia coli (E. coli) and tobacco while developing a new model for the association between cadmium (Cd) tolerance and its Cd-binding ability. Results have confirmed that under the Cd stress, transgenic tobacco plants have demonstrated better seedling growth and high Cd accumulation than the wild types.

Enzymatic processes are useful for industrially important sugar production, and in vitro two-step isomerization has proven to be an efficient process in utilizing readily available sugar sources. A hypothetical uncharacterized protein encoded by ydaE of Bacillus licheniformis was found to have broad substrate specificities and has shown high catalytic efficiency on D-lyxose, suggesting that the enzyme is D-lyxose isomerase. Escherichia coli BL21 expressing the recombinant protein, of 19.5 kDa, showed higher activity at 40 to 45°C and pH 7.5 to 8.0 in the presence of 1.0 mM Mn2+. The apparent Km values for D-lyxose and D-mannose were 30.4 ± 0.7 mM and 26 ± 0.8 mM, respectively. The catalytic efficiency (kcat/Km) for lyxose (3.2 ± 0.1 mM-1 s-1) was higher than that for D-mannose (1.6 mM-1 s-1). The purified protein was applied to the bioproduction of D-lyxose and D-glucose from D-xylose and D-mannose, respectively, along with the thermostable xylose isomerase of Thermus thermophilus HB08. From an initial concentration of 10 mM D-lyxose and D-mannose, 3.7 mM and 3.8 mM D-lyxose and D-glucose, respectively, were produced by two-step isomerization. This two-step isomerization is an easy method for in vitro catalysis and can be applied to industrial production. [ABSTRACT FROM AUTHOR]