The expression in E. coli of a polymeric gene coding for an esterase mimic catalyzing the hydrolysis of p -nitrophenyl esters

FEBS Letters ◽  
1987 ◽  
Vol 210 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Leif Bülow ◽  
Klaus Mosbach
Keyword(s):  
E Coli ◽  
Gene Coding ◽  
Hydrolysis Of

scholarly journals Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465

2022 ◽  
Vol 82 ◽  
Author(s):  
A. Al-Amri ◽  
M. A. Al-Ghamdi ◽  
J. A. Khan ◽  
H. N. Altayeb ◽  
H. Alsulami ◽  
...  
Keyword(s):  
Pilot Scale ◽  
Docking Studies ◽  
Industrial Applications ◽  
Geobacillus Thermodenitrificans ◽  
E Coli ◽  
Gene Coding ◽  
Km Value ◽  
Escherichia Coli Expression ◽  
Hydrolysis Of

Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

scholarly journals Public-good driven release of heterogeneous resources leads to genotypic diversification of an isogenic yeast population in melibiose.

2021 ◽  
Author(s):  
Anjali Mahilkar ◽  
Phaniendra Alugoju ◽  
Vijendra Kavatalkar ◽  
Rajeshkannan E. ◽  
Jayadeva Bhat ◽  
...  
Keyword(s):  
Carbon Source ◽  
Public Good ◽  
Molecular Basis ◽  
Model System ◽  
Microbial Populations ◽  
Adaptive Diversification ◽  
E Coli ◽  
Hydrolysis Of ◽  
Convenient Model ◽  
Heterogeneous Resources

Adaptive diversification of an isogenic population, and its molecular basis has been a subject of a number of studies in the last few years. Microbial populations offer a relatively convenient model system to study this question. In this context, an isogenic population of bacteria (E. coli, B. subtilis, and Pseudomonas) has been shown to lead to genetic diversification in the population, when propagated for a number of generations. This diversification is known to occur when the individuals in the population have access to two or more resources/environments, which are separated either temporally or spatially. Here, we report adaptive diversification in an isogenic population of yeast, S. cerevisiae, when propagated in an environment containing melibiose as the carbon source. The diversification is driven due to a public good, enzyme α-galactosidase, leading to hydrolysis of melibiose into two distinct resources, glucose and galactose. The diversification is driven by a mutations at a single locus, in the GAL3 gene in the GAL/MEL regulon in the yeast.

scholarly journals 1D TiO2 Nanostructures Prepared from Seeds Presenting Tailored TiO2 Crystalline Phases and Their Photocatalytic Activity for Escherichia coli in Water

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Julieta Cabrera ◽  
Dwight Acosta ◽  
Alcides López ◽  
Roberto J. Candal ◽  
Claudia Marchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Photocatalytic Activity ◽  
Bactericidal Activity ◽  
Sol Gel ◽  
Titanium Isopropoxide ◽  
E Coli ◽  
1D Nanostructures ◽  
Sol Gel Synthesis ◽  
Hydrolysis Of ◽  
Tio2 Nanostructures

TiO2 nanotubes were synthesized by alkaline hydrothermal treatment of TiO2 nanoparticles with a controlled proportion of anatase and rutile. Tailoring of TiO2 phases was achieved by adjusting the pH and type of acid used in the hydrolysis of titanium isopropoxide (first step in the sol-gel synthesis). The anatase proportion in the precursor nanoparticles was in the 3–100% range. Tube-like nanostructures were obtained with an anatase percentage of 18 or higher while flake-like shapes were obtained when rutile was dominant in the seed. After annealing at 400°C for 2 h, a fraction of nanotubes was conserved in all the samples but, depending on the anatase/rutile ratio in the starting material, spherical and rod-shaped structures were also observed. The photocatalytic activity of 1D nanostructures was evaluated by measuring the deactivation of E. coli in stirred water in the dark and under UV-A/B irradiation. Results show that in addition to the bactericidal activity of TiO2 under UV-A illumination, under dark conditions, the decrease in bacteria viability is ascribed to mechanical stress due to stirring.

scholarly journals Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
María-Efigenia Álvarez-Cao ◽  
María-Esperanza Cerdán ◽  
María-Isabel González-Siso ◽  
Manuel Becerra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Food Industry ◽  
Culture Conditions ◽  
Raffinose Family Oligosaccharides ◽  
Recombinant Production ◽  
Gene Coding ◽  
Productive Process ◽  
Hydrolysis Of ◽  
Degree Of Purification

Abstract Background α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. Results The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-d-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with β-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. Conclusions ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Inhibition of Escherichia coli O157:H7 in Ripening Dry Fermented Sausage by Ground Yellow Mustard

2008 ◽  
Vol 71 (3) ◽  
pp. 486-493 ◽  
Author(s):  
GARY H. GRAUMANN ◽  
RICHARD A. HOLLEY
Keyword(s):  
Escherichia Coli ◽  
Starter Culture ◽  
Escherichia Coli O157 ◽  
Fermented Sausage ◽  
Yellow Mustard ◽  
E Coli ◽  
Dry Fermented Sausage ◽  
Hydrolysis Of ◽  
Antimicrobial Effectiveness ◽  
Dry Sausage

Compounds generated by the enzymatic hydrolysis of glucosinolates naturally present in mustard powder are potently bactericidal against Escherichia coli O157:H7. Because E. coli O157:H7 can survive the dry fermented sausage manufacturing process, 2, 4, and 6% (wt/wt) nondeheated (hot) mustard powder or 6% (wt/wt) deheated (cold) mustard powder were added to dry sausage batter inoculated with E. coli O157:H7 at about 7 log CFU/g to evaluate the antimicrobial effectiveness of the powders. Reductions in E. coli O157:H7 populations, changes in pH and water activity (aw), effects on starter culture (Pediococcus pentosaceus and Staphylococcus carnosus) populations, and effects of mustard powder on sausage texture (shear) were monitored during ripening. Nondeheated mustard powder at 2, 4, and 6% in dry sausage (0.90 aw) resulted in significant reductions in E. coli O157:H7 (P < 0.05) of 3.4, 4.4, and 6.9 log CFU/g, respectively, within 30 days of drying. During fermentation and drying, mustard powder did not affect P. pentosaceus and S. carnosus activity in any of the treatments. Extension of drying to 36 and 48 days reduced E. coli O157:H7 by >5 log CFU/g in the 4 and 2% mustard powder treatments, respectively. The 6% deheated mustard powder treatment provided the most rapid reductions of E. coli O157:H7 (yielding <0.20 log CFU/g after 24 days) by an unknown mechanism and was the least detrimental (P < 0.05) to sausage texture.

scholarly journals NB2001, a Novel Antibacterial Agent with Broad-Spectrum Activity and Enhanced Potency against β-Lactamase-Producing Strains

2002 ◽  
Vol 46 (5) ◽  
pp. 1262-1268 ◽  
Author(s):  
Qing Li ◽  
Jean Y. Lee ◽  
Rosario Castillo ◽  
Mark S. Hixon ◽  
Catherine Pujol ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Antibacterial Agent ◽  
Bacterial Resistance ◽  
Enterobacter Aerogenes ◽  
Order Rate Constant ◽  
Common Mechanism ◽  
E Coli ◽  
Lactam Ring ◽  
Spectrum Activity ◽  
Hydrolysis Of

ABSTRACT Enzyme-catalyzed therapeutic activation (ECTA) is a novel prodrug strategy to overcome drug resistance resulting from enzyme overexpression. β-Lactamase overexpression is a common mechanism of bacterial resistance to β-lactam antibiotics. We present here the results for one of the β-lactamase ECTA compounds, NB2001, which consists of the antibacterial agent triclosan in a prodrug form with a cephalosporin scaffold. Unlike conventional β-lactam antibiotics, where hydrolysis of the β-lactam ring inactivates the antibiotic, hydrolysis of NB2001 by β-lactamase releases triclosan. Evidence supporting the proposed mechanism is as follows. (i) NB2001 is a substrate for TEM-1 β-lactamase, forming triclosan with a second-order rate constant (k cat/Km ) of greater than 77,000 M−1 s−1. (ii) Triclosan is detected in NB2001-treated, β-lactamase-producing Escherichia coli but not in E. coli that does not express β-lactamase. (iii) NB2001 activity against β-lactamase-producing E. coli is decreased in the presence of the β-lactamase inhibitor clavulanic acid. NB2001 was similar to or more potent than reference antibiotics against clinical isolates of Staphylococcus aureus (including MRSA), Staphylococcus epidermidis, Streptococcus pneumoniae, vancomycin-resistant Enterococcus faecalis, Moraxella catarrhalis and Haemophilus influenzae. NB2001 is also active against Klebsiella pneumoniae, Enterobacter aerogenes, and Enterobacter cloacae. The results indicate that NB2001 is a potent, broad-spectrum antibacterial agent and demonstrate the potential of ECTA in overcoming β-lactamase-mediated resistance.

scholarly journals Principles of RNA and nucleotide discrimination by the RNA processing enzyme RppH

2020 ◽  
Vol 48 (7) ◽  
pp. 3776-3788 ◽  
Author(s):  
Ang Gao ◽  
Nikita Vasilyev ◽  
Abhishek Kaushik ◽  
Wenqian Duan ◽  
Alexander Serganov
Keyword(s):  
Rna Processing ◽  
Reaction Rates ◽  
Growth Conditions ◽  
Rna Degradation ◽  
Nudix Hydrolase ◽  
X Ray ◽  
E Coli ◽  
Cellular Processes ◽  
Protein Factor ◽  
Hydrolysis Of

Abstract All enzymes face a challenge of discriminating cognate substrates from similar cellular compounds. Finding a correct substrate is especially difficult for the Escherichia coli Nudix hydrolase RppH, which triggers 5′-end-dependent RNA degradation by removing orthophosphate from the 5′-diphosphorylated transcripts. Here we show that RppH binds and slowly hydrolyzes NTPs, NDPs and (p)ppGpp, which each resemble the 5′-end of RNA. A series of X-ray crystal structures of RppH-nucleotide complexes, trapped in conformations either compatible or incompatible with hydrolysis, explain the low reaction rates of mononucleotides and suggest two distinct mechanisms for their hydrolysis. While RppH adopts the same catalytic arrangement with 5′-diphosphorylated nucleotides as with RNA, the enzyme hydrolyzes 5′-triphosphorylated nucleotides by extending the active site with an additional Mg2+ cation, which coordinates another reactive nucleophile. Although the average intracellular pH minimizes the hydrolysis of nucleotides by slowing their reaction with RppH, they nevertheless compete with RNA for binding and differentially inhibit the reactivity of RppH with triphosphorylated and diphosphorylated RNAs. Thus, E. coli RppH integrates various signals, such as competing non-cognate substrates and a stimulatory protein factor DapF, to achieve the differential degradation of transcripts involved in cellular processes important for the adaptation of bacteria to different growth conditions.

scholarly journals Optimization of the apolipoprotein B mRNA editing enzyme catalytic polypeptidelike-3G (APOBEC3G) gene to enhance its expression in Escherichia coli

2020 ◽  
Vol 29 (2) ◽  
pp. 120-8
Author(s):  
Rizkyana Avissa ◽  
Silvia Tri Widyaningtyas ◽  
Budiman Bela
Keyword(s):  
Escherichia Coli ◽  
Apolipoprotein B ◽  
Plasmid Vector ◽  
Nitrilotriacetic Acid ◽  
Mrna Editing ◽  
Specific Domain ◽  
Iptg Induction ◽  
E Coli ◽  
Gene Coding ◽  
Editing Enzyme

BACKGROUND Apolipoprotein B mRNA editing enzyme catalytic polypeptide-like-3G (APOBEC3G) can abolish HIV infection by inducing lethal mutations in the HIV genome. The HIV protein virion infectivity factor (Vif) can interact with APOBEC3G protein and cause its degradation. Development of a method that can screen substances inhibiting the APOBEC3G-Vif interaction is necessary for identification of substances that potentially used in anti-HIV drug development. In order to increase expression of recombinant APOBEC3G protein that will be used in APOBEC3G-Vif interaction assay, we developed an optimized APOBEC3G gene for expression in Escherichia coli.  METHODS The gene coding APOBEC3G was codon-optimized in accordance with prokaryotic codon using DNA 2.0 software to avoid bias codons that could inhibit its expression. The APOBEC3G gene was synthesized and sub-cloned into pQE80L plasmid vector. pQE80L containing APOBEC3G was screened by polymerase chain reaction, enzyme restriction, and sequencing to verify its DNA sequence. The recombinant APOBEC3G was expressed in E. coli under isopropyl-β-D-thiogalactoside (IPTG) induction and purified by using nickel-nitrilotriacetic acid (Ni-NTA) resin.  RESULTS The synthetic gene coding APOBEC3G was successfully cloned into the pQE80L vector and could be expressed abundantly in E. coli BL21 in the presence of IPTG.  CONCLUSIONS Recombinant APOBEC3G is robustly expressed in E. coli BL21, and the APOBEC3G protein could be purified by using Ni-NTA. The molecular weight of the recombinant APOBEC3G produced is smaller than the expected value. However, the protein is predicted to be able to interact with Vif because this interaction is determined by a specific domain located on the N-terminal of APOBEC3G. 

scholarly journals Development of a Coupled VanA/VanX Assay: Screening for Inhibitors of Glycopeptide Resistance

1997 ◽  
Vol 2 (4) ◽  
pp. 241-247 ◽  
Author(s):  
Steven D. Pratt ◽  
Xiaoling Xuei ◽  
Alexander C. Mackinnon ◽  
Angela M. Nilius ◽  
Dena M. Hensey-Rudloff ◽  
...  
Keyword(s):  
Enterococcus Faecium ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Glycopeptide Antibiotics ◽  
Glycopeptide Resistance ◽  
E Coli ◽  
Hydrolysis Of ◽  
Lac Promoter ◽  
Dipeptidase Activity ◽  
Coupled Assay

Resistance in Enterococcus faecium to the glycopeptide antibiotics vancomycin and teicoplanin is encoded by five genes: vanR, vanS, vanH, vanA, and vanX.1 The mechanism of resistance involves replacement of the dipeptide D-Ala-D-Ala, destined for the peptidoglycan layer with the depsipeptide D-Ala-D-lactate. This alteration lowers the binding affinity of vancomycin for the bacterial cell wall by a factor of 1000. The functions of VanA and VanX are the ligation of D-Ala and D-lactate, and the hydrolysis of D-Ala-D-Ala, respectively. We report here the overexpression of both genes as well as the D-Ala-D-Ala ligase (Ddl) from Enterococcus faecium, development of a coupled assay and several inhibitors obtained by high-throughput screening (HTS). All genes were expressed in E. coli by translational coupling to kdsB, the CMP-KDO synthetase gene, under control of a modified lac promoter. The coupled VanA/VanX assay employs colorimetric detection of inorganic phosphate (Pi) released in the VanA ligation reaction, with the VanX dipeptidase activity providing the D-Ala substrate for VanA. A secondary VanX assay uses cadmium-ninhydrin calorimetric detection of free amino acid released by the dipeptidase activity of the enzyme on D-Ala-D-Ala. We have also developed an assay using Ddl ligase. Over 250,000 compounds have been screened to date using the coupled assay.

scholarly journals A Recombinant Multiepitope Protein for Hepatitis B Diagnosis

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Marilen Queiroz de Souza ◽  
Alexsandro Sobreira Galdino ◽  
José Carlos dos Santos ◽  
Marcus Vinicius Soares ◽  
Yanna C. de Nóbrega ◽  
...  
Keyword(s):  
Hepatitis B ◽  
Time Course ◽  
Clinical Stage ◽  
Acute Infection ◽  
Single Step ◽  
Liver Inflammation ◽  
E Coli ◽  
Terminal Time ◽  
Gene Coding ◽  
B Virus

Hepatitis B is a liver inflammation caused by hepatitis B virus (HBV) and can be diagnosed in clinical stage by hepatitis B core antibody from IgM class (anti-HBcIgM). Hepatitis B core antibody from IgG class (Anti-HBcIgG) appears quickly after IgM, reaching high titers in chronic hepatitis, and remains even after cure. Since hepatitis B core antibody (anti-HBc) is the first antibody identified and sometimes the only marker detected during the course of infection, it can be used both to indicate HBV acute infection (anti-HBc-IgM) and to identify individuals who have come into contact with the virus (anti-HBc-IgG). In this work we propose a recombinant hepatitis B core multiepitope antigen (rMEHB) to be used for diagnosis of hepatitis B. For this purpose, a synthetic gene coding for rMEHB was designed and cloned into vector pET21a with a 6xHis tag at the C-terminal. Time course induction inE. colishowed an induced protein with an apparent molecular mass of ~21 kDa. Protein purification was performed by a single step with affinity chromatography Ni-NTA. Circular dichroism spectroscopy indicated rMEHB as a thermal stable protein at pH 7.0 and 8.0. In these conditions rMEHB was successfully used to perform an enzyme linked immuno sorbent assay (ELISA) with positive and negative sera.

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