Optimization of Monochloroacetic Acid Biodegradation by Recombinant E. coli BL21 (DE3)/pET-bcfd1 Carrying Haloacid Dehalogenase Gene from Bacillus cereus IndB1

2021 ◽  
Vol 10 (4) ◽  
pp. 857-863
Author(s):  
Enny Ratnaningsih ◽  
Rachmad Ade ◽  
Rindia Maharani Putri ◽  
Idris Idris
Keyword(s):  
Bacillus Cereus ◽  
Chloride Ion ◽  
Local Strain ◽  
Luria Bertani ◽  
Chloride Ions ◽  
Monochloroacetic Acid ◽  
Iptg Induction ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Dehalogenase Gene

In recent years, attention to microbial dehalogenase has continually increased due to its potential application, both in bioremediation and in the biosynthesis of fine chemicals. Many microbial recombinant strains carrying dehalogenase gene have been developed, particularly to increase the dehalogenase production and its quality. In this study, we aimed to find the optimum condition for the production of active haloacid dehalogenase by E. coli BL21 (DE3) harboring recombinant plasmid pET-bcfd1 that carried haloacid dehalogenase gene from Bacillus cereus IndB1 local strain. This would be examined by assessing the ability of whole cell life culture to degrade monochloroacetic acid (MCA) and quantifying the chloride ion released into the medium. Several variables were evaluated to find this optimal condition. We found that the best condition for MCA biodegradation using this recombinant clone was at 0.2 mM MCA, 10 μM of isopropyl β-D-1-thiogalactopyranoside (IPTG), 6 hours of pre-induction incubation at 37ºC with shaking, 2 hours IPTG induction at 30ºC with shaking, at pH 7 in Luria Bertani (LB) liquid medium without NaCl, which produced about 0.056 mM chloride ions. Inducer concentration, pre-induction incubation time and temperature, as well as induction time and temperature were apparent to be associated with the expression of the protein, while the MCA concentration and the pH of the medium influenced the ability of the recombinant E. coli BL21 (DE3)/pET-bcfd1 to grow in toxic environment. Our findings laid the foundation for exploration of dehalogenases from local Bacillus strains through genetic engineering for MCA biodegradation

scholarly journals Effect of Chloride Ions on the Point-of-Use Drinking Water Disinfection Performance of Porous Ceramic Media Embedded with Metallic Silver and Copper

Water ◽  
2020 ◽  
Vol 12 (6) ◽  
pp. 1625
Author(s):  
Rekha Singh ◽  
Woohang Kim ◽  
James A. Smith
Keyword(s):  
Drinking Water ◽  
Porous Ceramic ◽  
Chloride Ion ◽  
Silver Ions ◽  
Chloride Ions ◽  
Ion Concentration ◽  
Metallic Silver ◽  
E Coli ◽  
Ion Concentrations ◽  
Copper Release

This study quantifies the effects of chloride ions on silver and copper release from porous ceramic cubes embedded with silver and copper and its effect on E. coli disinfection in drinking water. Log-reduction of E. coli by silver ions decreased after 4 h of contact time as the chloride ion concentration increased from 0 to 250 mg/L but, it was not changed by copper ions under the same conditions. For silver addition by silver-ceramic cubes, log reductions of E. coli decreased sharply from 7.2 to 1.6 after 12 h as the chloride concentration increased from 0 to 250 mg/L. For the silver-ceramic cube experiments, chloride ion also reduced the total silver concentration in solution. After 24 h, total silver concentrations in solution decreased from 61 µg/L to 20 µg/L for corresponding chloride ion concentrations. According to the MINTEQ equilibrium model analysis, the decrease in disinfection ability with silver embedded ceramic cubes could be the result of precipitation of silver ions as silver chloride. This suggests that AgCl was precipitating within the pore space of the ceramic. These results indicate that, although ionic silver is a highly effective disinfectant for E. coli, the presence of chloride ions can significantly reduce disinfection efficacy. For copper-ceramic cubes, log reductions of E. coli by copper embedded cubes increased from 1.2 to 1.5 when chloride ion concentration increased from 0 to 250 mg/L. Total copper concentrations in solution increased from 4 µg/L to 14 µg/L for corresponding chloride ion concentrations. These results point towards the synergistic effect of chloride ions on copper oxidation as an increased concentration of chloride enhances copper release.

scholarly journals Recombinant Production and One-Pot Purification for Enhancing Activity of Haloacid Dehalogenase from Bacillus cereus IndB1

REAKTOR ◽  
2021 ◽  
Vol 21 (2) ◽  
pp. 59-64
Author(s):  
Enny Ratnaningsih ◽  
Sulistiya Nirta Sunaryo ◽  
Idris Idris ◽  
Rindia Maharani Putri
Keyword(s):  
Bacillus Cereus ◽  
Recombinant Expression ◽  
Host Cells ◽  
Monochloroacetic Acid ◽  
Affinity Column ◽  
One Pot ◽  
Crude Enzyme Extract ◽  
Local Strains ◽  
Haloacid Dehalogenase ◽  
Purification System

In recent years we have witnessed the emergence of organohalogen utilization in various chemical-based industries, particularly polymer-based, agricultural, and pharmaceutical sectors. Despite this, organohalogen compounds are actually very dangerous to the environment, as they are difficult to be naturally degraded and generally toxic to organisms. A green and biocompatible method to overcome this issue is by employing enzymes that could convert organohalogens into non-toxic compounds, such as the class of enzymes known as haloacid dehalogenases. To enhance the activity of haloacid dehalogenase isolated from local strains of Bacillus cereus IndB1, we have developed a recombinant expression system using pET-bcfd1 plasmid in E. coli BL21 (DE3) host cells. Following enzyme production, we also demonstrated a one-pot purification system for the expressed dehalogenase, harnessing the presence of His-tag in the recombinant clones. Purification was carried out using Ni-NTA affinity column chromatography, using imidazole eluent with a concentration gradient of 10 mM to 500 mM. The enzyme activity was tested against the monochloroacetic acid (MCA) substrate according to the Bergmann and Sanik method, and the protein content in the solution was measured using the Bradford method. The purity of the enzyme after one-pot purification was confirmed by SDS-PAGE analyses, showing a single band of 40 kDa in size. Remarkably, the purified haloacid dehalogenase specific activity was increased by 12-fold compared to its crude enzyme extract. Therefore, the expression and purification system developed in this study allow further exploration of dehalogenases from local strains as an efficient catalyst for MCA biodegradation.Keywords: recombinant expression, haloacid dehalogenase, monochloroacetic acid, enzyme purification

scholarly journals Heat shock transformation and expression of the plasmid containing cytolethal distending toxin of Campylobacter fetus subsp venerealis in Escherichia coli

2021 ◽  
Vol 888 (1) ◽  
pp. 012021
Author(s):  
N Herlina ◽  
N D Yanthi ◽  
R D Pratiwi ◽  
K S Dewi ◽  
F Setiyoningrum ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Luria Bertani ◽  
Protein Characterization ◽  
Bacterial Cells ◽  
Iptg Induction ◽  
E Coli ◽  
Sds Page ◽  
Fetus Subsp ◽  
Campylobacter Infection

Abstract The cytolethal distending toxins (cdt) is a multi-subunit toxin consisted of three subunit encoded cdtA, cdtB and cdtC. The cdt played an important role as a virulence factor of Campylobacter infection, including C. fetus subsp venerealis. The cdtA which responsible for binding the cdt to cell membrane, was cloned in plasmid expression and inserted into bacterial cells of Escherichia coli BL21(DE3). The research was conducted to evaluate the transformation using the heat shock method of a plasmid containing cdtA3 gene and the protein expression induced by various concentration of IPTG. Transformation was done using the heat shock method at 42oC for 90 second. Evaluation of the transformation was observed on the presence of E. coli BL21(DE3) colonies on Luria Bertani agar containing Ampicillin antibiotic with 100 µg/mL dosage. The recombinant protein was expressed using IPTG-induction with various concentration (0.1mM, 0.25mM, 0.5mM, 0.75mM and 1 mM). The result showed that the transformation and IPTG-induction 0.1 mM produced higher concentration of protein than other concentration applied. The protein characterization was observed with SDS PAGE and cdtA3 protein was detected on 23,4 kDa.

scholarly journals Molecular Cloning and Expression of Haloacid Dehalogenase Gene from a Local Pseudomonas aeruginosa ITB1 Strain and Tertiary Structure Prediction of the Produced Enzyme

2021 ◽  
Vol 24 (5) ◽  
pp. 161-169
Author(s):  
Enny Ratnaningsih ◽  
Lousiana Dwinta Utami ◽  
Nurlaida Nurlaida ◽  
Rindia Maharani Putri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Tertiary Structure ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Local Strains ◽  
Haloacid Dehalogenase ◽  
E Coli ◽  
Cloned Gene ◽  
Dehalogenase Gene

Organohalogens are widely utilized as pesticides, herbicides, solvents, and for many other industrial purposes. However, the use of these compounds caused some negative impacts to the environment due to their toxicity and persistency. In the light of this, some microbes have been identified and employed to perform dehalogenation, converting halogenated organic compounds to non-toxic materials. In this research, we successfully cloned and sequenced the haloacid dehalogenase gene from a local Pseudomonas aeruginosa ITB1 strain, which is involved in the degradation of monochloroacetate. First, the haloacid dehalogenase gene was amplified by PCR using a pair of primers designed from the same gene sequences of other P. aeruginosa strains available in the GenBank. The cloned gene in pGEM-T in E. coli TOP10 was sequenced, analyzed, and then sub-cloned into pET-30a(+) for expression in E. coli BL21 (DE3). To facilitate direct sub-cloning, restriction sequences of EcoRI (G/AATTC) and HindIII (A/AGCTT) were added to the forward and reversed primers, respectively. The expressed protein in E. coli BL21 (DE3) appeared as a 26-kDa protein in SDS-PAGE analysis, which is in good agreement with the size predicted by ExPASy Protparam. We obtained that the best expression in LB liquid medium was achieved with 0.01 mM IPTG induction at 30°C incubation for 3 hours. We also found that the enzyme is more concentrated in the pellet cells as inclusion bodies. Furthermore, the in-silico analysis revealed that this enzyme consists of 233 amino acid residues. This enzyme’s predicted tertiary structure shows six β-sheets flanked by α-helixes and thus belongs to Group II haloacid dehalogenase. Based on the structural prediction, amino acid residues of Asp7, Ser121, and Asn122 are present in the active site and might play essential roles in catalysis. The presented study laid the foundation for recombinant haloacid dehalogenase production from P. aeruginosa local strains. It provided an insight into the utilization of recombinant local strains to remediate environmental problems caused by organohalogens.

scholarly journals MOLECULAR EXPRESSION OF WINGLESS-TYPE MMTV INTEGRATION SITE FAMILY MEMBER 4 GENE USING Escherichia coli BL21

2017 ◽  
Vol 11 (1) ◽  
pp. 11-14
Author(s):  
Agung Janika Sitasiwi ◽  
Wayan Tunas Artama ◽  
Agung Budiyanto ◽  
Edy Dharmana
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Recombinant Dna ◽  
Integration Site ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Negative Control ◽  
Luria Bertani ◽  
Iptg Induction ◽  
E Coli

This research was conducted to find out the Wnt4 recombinant proteins which expressed by Escherichia coli (E. coli) BL21 carrying the recombinant DNA wnt4 (E. coli transformation). Research materials were E. coli BL21 transformation and E. coli BL21 non-transformation (negative control). The expression of recombinant protein was conducted by culturing E. coli for 24 hours in Luria-Bertani (LB) media with isopropyl β-D-1-thiogalactopyranoside (IPTG) induction. Recombinant protein was isolated by sonication of pellet bacteria. Protein analysis performed by 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that recombinant protein with a molecular weight of 33 kDa has been expressed by E. coli BL21 transformation successfully. 

scholarly journals Experimental Study and Simulation Calculation of the Chloride Resistance of Concrete under Multiple Factors

2021 ◽  
Vol 11 (12) ◽  
pp. 5322
Author(s):  
Yang Ding ◽  
Tong-Lin Yang ◽  
Hui Liu ◽  
Zhen Han ◽  
Shuang-Xi Zhou ◽  
...  
Keyword(s):  
Hydrostatic Pressure ◽  
Penetration Depth ◽  
Chloride Ion ◽  
Cementitious Materials ◽  
Chloride Ions ◽  
Finite Element Software ◽  
Chloride Resistance ◽  
Simulation Calculation ◽  
Marine Concrete ◽  
Resistance Performance

Cement is widely used in marine concrete, and its resistance to chloride ion corrosion has been widely considered. In this paper, based on a laboratory test, the influence of different hydrostatic pressures, coarse aggregate contents and w/c ratios on the chloride resistance performance is analyzed. Based on COMSOL finite element software, a two-dimensional cementitious materials model is established, and the simulation results are compared with the experimental results. The results show that the penetration depth of chloride ions in cement increases with the increase of the w/c ratio. Under the hydrostatic pressure of 0 MPa, when the w/c ratio is 0.35, the penetration depth of chloride ions is 7.4 mm, and the simulation result is 8.0 mm. When the w/c ratio is 0.45, the penetration depth of chloride ions is 9.3 mm, and the simulation result is 9.9 mm. When the w/c ratio is 0.55, the penetration depth of chloride ions is 12.9 mm, and the simulation result is 12.1 mm. Under different hydrostatic pressures, the penetration depth of chloride ions obviously changes, and with the increase in hydrostatic pressure, the penetration depth of chloride ions deepens. Under the w/c ratio of 0.35, when the hydrostatic pressure is 0.5 MPa, the penetration depth of chloride ions is 11.3 mm, and the simulation result is 12.1 mm. When the hydrostatic pressure is 1.0 MPa, the penetration depth of chloride ions is 16.2 mm, and the simulation result is 17.5 mm.

scholarly journals Broadband dielectric spectroscopy for monitoring temperature-dependent chloride ion motion in BiOCl plates

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Adrian Radoń ◽  
Dariusz Łukowiec ◽  
Patryk Włodarczyk
Keyword(s):  
Electrical Conductivity ◽  
Dielectric Spectroscopy ◽  
Low Frequency ◽  
Chloride Ion ◽  
Ion Source ◽  
Chloride Ions ◽  
Ion Concentration ◽  
Broadband Dielectric Spectroscopy ◽  
Free Chloride ◽  
Structure Rebuilding

AbstractThe dielectric properties and electrical conduction mechanism of bismuth oxychloride (BiOCl) plates synthesized using chloramine-T as the chloride ion source were investigated. Thermally-activated structure rebuilding was monitored using broadband dielectric spectroscopy, which showed that the onset temperature of this process was 283 K. This rebuilding was related to the introduction of free chloride ions into [Bi2O2]2+ layers and their growth, which increased the intensity of the (101) diffraction peak. The electrical conductivity and dielectric permittivity were related to the movement of chloride ions between plates (in the low-frequency region), the interplanar motion of Cl− ions at higher frequencies, vibrations of these ions, and charge carrier hopping at frequencies above 10 kHz. The influence of the free chloride ion concentration on the electrical conductivity was also described. Structure rebuilding was associated with a lower concentration of free chloride ions, which significantly decreased the conductivity. According to the analysis, the BiOCl plate conductivity was related to the movement of Cl− ions, not electrons.

scholarly journals degS Is Necessary for Virulence and Is among Extraintestinal Escherichia coli Genes Induced in Murine Peritonitis

2003 ◽  
Vol 71 (6) ◽  
pp. 3088-3096 ◽  
Author(s):  
Peter Redford ◽  
Paula L. Roesch ◽  
Rodney A. Welch
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Luria Bertani ◽  
Broth Culture ◽  
Wild Type ◽  
E Coli ◽  
Virulence Attenuation

ABSTRACT Extraintestinal Escherichia coli strains cause meningitis, sepsis, urinary tract infection, and other infections outside the bowel. We examined here extraintestinal E. coli strain CFT073 by differential fluorescence induction. Pools of CFT073 clones carrying a CFT073 genomic fragment library in a promoterless gfp vector were inoculated intraperitoneally into mice; bacteria were recovered by lavage 6 h later and then subjected to fluorescence-activated cell sorting. Eleven promoters were found to be active in the mouse but not in Luria-Bertani (LB) broth culture. Three are linked to genes for enterobactin, aerobactin, and yersiniabactin. Three others are linked to the metabolic genes metA, gltB, and sucA, and another was linked to iha, a possible adhesin. Three lie before open reading frames of unknown function. One promoter is associated with degS, an inner membrane protease. Mutants of the in vivo-induced loci were tested in competition with the wild type in mouse peritonitis. Of the mutants tested, only CFT073 degS was found to be attenuated in peritoneal and in urinary tract infection, with virulence restored by complementation. CFT073 degS shows growth similar to that of the wild type at 37°C but is impaired at 43°C or in 3% ethanol LB broth at 37°C. Compared to the wild type, the mutant shows similar serum survival, motility, hemolysis, erythrocyte agglutination, and tolerance to oxidative stress. It also has the same lipopolysaccharide appearance on a silver-stained gel. The basis for the virulence attenuation is unclear, but because DegS is needed for σE activity, our findings implicate σE and its regulon in E. coli extraintestinal pathogenesis.

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